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Cortical Stimulation to Enhance Motor Performance after Stroke

Permanent Link: http://ufdc.ufl.edu/UFE0041204/00001

Material Information

Title: Cortical Stimulation to Enhance Motor Performance after Stroke
Physical Description: 1 online resource (197 p.)
Language: english
Creator: Boychuk, Jeffery
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: rehabilitation, rodent, stimulation, stroke
Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Motor rehabilitation after cerebral ischemia can enhance motor performance and induce cortical plasticity. Electrical stimulation of the motor cortex (CS) during rehabilitative training (CS/RT) augments motor map plasticity and enhances motor improvements. CS/RT?s ability to enhance motor improvements after ischemic insult has only been tested in a limited number of situations that do not completely represent stroke conditions in the human population. Indeed, the lack of treatment effect in a recent phase III clinical of CS/RT reflects knowledge gaps in how to fully translate basic science findings of CS/RT into the clinical setting. The goal of this dissertation is to use rodent models of stroke to examine several parameters of CS/RT therapy that may account for its lack of benefit in human stroke patients. Study #1: It is unclear how the distribution of electrical stimulation across the cortex influences both motor map reorganization and improvements in motor performance. Here, we examined the behavioral and neurophysiological effects of delivering CS/RT through a distributed versus focal arrangement of electrical contacts. In this experiment, stroke was modeled by focal ischemic damage with motor cortex. Results showed that animals given CS/RT with a distributed contact configuration condition exhibited greater motor improvements than animals given CS/RT with a focused contact arrangement or given rehabilitative training alone (RT). All groups that received rehabilitation exhibited greater increases in motor map area and reaching accuracy than animals that received no rehabilitative training (NT). However, both CS/RT groups exhibited larger motor maps than the RT animals. The results indicate that although both focal and distributed forms of CS/RT promote motor map reorganization, only the distributed form of CS/RT enhances motor performance with rehabilitation. Study #2: While distributed CS/RT proved more effective than focal CS/RT in the first experiment, the number of distributed stimulation contacts sites that will induce the greatest motor improvements with CS/RT is not known. Here, we examined the behavioral effects of delivering CS/RT with either four or nine independent contact sites that were distributed across motor cortex following ischemic insult. In this experiment, stroke was modeled by temporary occlusion of the middle cerebral artery. The results showed that both types of distributed CS/RT enhanced motor improvements regardless of the number of independent sites. Our results indicate that the number of independent stimulation sites does not affect CS/RT with distributed stimulation. Study #3: Human stroke patients who are candidates for CS/RT will likely have received standard forms of rehabilitation prior to the onset of CS/RT. It is not known whether CS/RT can induce motor improvements in individuals who have received rehabilitative experience following ischemic insult. Further, the phase III clinical trial that failed to find an effect of CS/RT had recruited stroke patients who had already received standard rehabilitative therapy. Here, the behavioral effects of administering CS/RT after early application of RT alone were measured. In this experiment, stroke was modeled by temporary occlusion of the middle cerebral artery. The results showed that CS/RT magnifies behavioral improvements above and beyond those occurring from rehabilitative training alone. Our results suggest that CS/RT and RT have a complimentary rather than antagonizing relationship. Study #4: While animal studies of CS/RT have observed enhanced motor improvements in models of cortical stroke, they have not tested its effects in subcortical stroke models. Subcortical damage is relatively common in stroke patients. Further, the phase III clinical trial that found no benefit with CS/RT had enrolled patients with subcortical stroke damage. In this experiment, the behavioral effects of CS/RT in a rodent model of subcortical ischemia involving damage within the internal capsule were compared to model of cortical ischemia. The results showed that CS/RT confers no additional motor improvements compared to RT following subcortical white matter ischemia. These data suggest that CS/RT may not be as effective in treating human stroke patients with subcortical damage.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jeffery Boychuk.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Kleim, Jeffrey.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041204:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041204/00001

Material Information

Title: Cortical Stimulation to Enhance Motor Performance after Stroke
Physical Description: 1 online resource (197 p.)
Language: english
Creator: Boychuk, Jeffery
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: rehabilitation, rodent, stimulation, stroke
Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Motor rehabilitation after cerebral ischemia can enhance motor performance and induce cortical plasticity. Electrical stimulation of the motor cortex (CS) during rehabilitative training (CS/RT) augments motor map plasticity and enhances motor improvements. CS/RT?s ability to enhance motor improvements after ischemic insult has only been tested in a limited number of situations that do not completely represent stroke conditions in the human population. Indeed, the lack of treatment effect in a recent phase III clinical of CS/RT reflects knowledge gaps in how to fully translate basic science findings of CS/RT into the clinical setting. The goal of this dissertation is to use rodent models of stroke to examine several parameters of CS/RT therapy that may account for its lack of benefit in human stroke patients. Study #1: It is unclear how the distribution of electrical stimulation across the cortex influences both motor map reorganization and improvements in motor performance. Here, we examined the behavioral and neurophysiological effects of delivering CS/RT through a distributed versus focal arrangement of electrical contacts. In this experiment, stroke was modeled by focal ischemic damage with motor cortex. Results showed that animals given CS/RT with a distributed contact configuration condition exhibited greater motor improvements than animals given CS/RT with a focused contact arrangement or given rehabilitative training alone (RT). All groups that received rehabilitation exhibited greater increases in motor map area and reaching accuracy than animals that received no rehabilitative training (NT). However, both CS/RT groups exhibited larger motor maps than the RT animals. The results indicate that although both focal and distributed forms of CS/RT promote motor map reorganization, only the distributed form of CS/RT enhances motor performance with rehabilitation. Study #2: While distributed CS/RT proved more effective than focal CS/RT in the first experiment, the number of distributed stimulation contacts sites that will induce the greatest motor improvements with CS/RT is not known. Here, we examined the behavioral effects of delivering CS/RT with either four or nine independent contact sites that were distributed across motor cortex following ischemic insult. In this experiment, stroke was modeled by temporary occlusion of the middle cerebral artery. The results showed that both types of distributed CS/RT enhanced motor improvements regardless of the number of independent sites. Our results indicate that the number of independent stimulation sites does not affect CS/RT with distributed stimulation. Study #3: Human stroke patients who are candidates for CS/RT will likely have received standard forms of rehabilitation prior to the onset of CS/RT. It is not known whether CS/RT can induce motor improvements in individuals who have received rehabilitative experience following ischemic insult. Further, the phase III clinical trial that failed to find an effect of CS/RT had recruited stroke patients who had already received standard rehabilitative therapy. Here, the behavioral effects of administering CS/RT after early application of RT alone were measured. In this experiment, stroke was modeled by temporary occlusion of the middle cerebral artery. The results showed that CS/RT magnifies behavioral improvements above and beyond those occurring from rehabilitative training alone. Our results suggest that CS/RT and RT have a complimentary rather than antagonizing relationship. Study #4: While animal studies of CS/RT have observed enhanced motor improvements in models of cortical stroke, they have not tested its effects in subcortical stroke models. Subcortical damage is relatively common in stroke patients. Further, the phase III clinical trial that found no benefit with CS/RT had enrolled patients with subcortical stroke damage. In this experiment, the behavioral effects of CS/RT in a rodent model of subcortical ischemia involving damage within the internal capsule were compared to model of cortical ischemia. The results showed that CS/RT confers no additional motor improvements compared to RT following subcortical white matter ischemia. These data suggest that CS/RT may not be as effective in treating human stroke patients with subcortical damage.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jeffery Boychuk.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Kleim, Jeffrey.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041204:00001


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1 CORTICAL STIMULATION TO ENHA NCE MOTOR IMPROVEMENTS AFTER STROKE By JEFFERY ALLEN BOYCHUK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Jeffery Allen Boychuk

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3 This work is dedicated to my dad for his seemingly limitless support

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4 ACKNOWLEDGMENTS If I have a talent, it is that I have a lways been able to surround myself with good people. My graduate experience has been no exception so I owe gratitude to many people. I w ould like to thank my father Garry Boychuk, and my brother Shawn Boychuk, for their words of encouragement and help with problems big and small. I am grateful to have them in my life. I thank Dr. John Vokey and Dr. Scott Allen for getting me interested in science during my undergraduate education. I dont know what I would be doing right now if I hadnt been introduced to academ ic research. I am also grateful for my mentor Dr. Jeffrey Klei m. Dr. Kleim has been supporti ve in all aspects of my graduate experience and I cannot thank Dr. Jeffrey Kleim enough for his guidance. I would also like to thank my committee memb ers, Dr. Dena Howland, Dr. Paul Reier and Dr. Lorie Richards for their ment orship through this process.

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5 TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 9LIST OF FIGURES ........................................................................................................ 10LIST OF ABBR EVIATIONS ........................................................................................... 11ABSTRACT ................................................................................................................... 14 CHA PTER 1 NEURAL PLASTICITY, STROKE AND CORTICAL STIMULATION....................... 17General Intr oduction ............................................................................................... 17Neurobiology of Stroke ........................................................................................... 21Cell Death During Stro ke .................................................................................. 22Ionic Imbalance by Energy Fa ilure ................................................................... 24Excitotoxiticty .................................................................................................... 24Oxidative/Nitrosa tive St ress ............................................................................. 25Acidosis ............................................................................................................ 25Peri-Infarct Spreadi ng Depolari zations ............................................................. 26Inflamma tion ..................................................................................................... 26Stroke Tr eatment .................................................................................................... 28Cortical Plasticity and As A Neural Substrate for Motor Rehabilitation ............. 31Motor Map Plasticity and Motor Learning in the Intact CNS ............................. 32Motor Cortex Plasticity and Improv ement of Motor Function Following Stroke ............................................................................................................ 37Synaptic Plasticity Mediates Motor Map R eorganizati on .................................. 40Electric Stimulation of Motor Cortex Induces Synaptic Plasticity and Motor Map Reorgani zation ...................................................................................... 41Electrical Stimulation of Motor Cortex Enhance Motor Improvement After Stroke ............................................................................................................ 42Translation of CS/RT in to Clinical Practice ....................................................... 45Thesis Ou tline ......................................................................................................... 472 DISTRIBUTED VERSUS FOCAL CORT ICAL STIMULATION TO ENHANCE MOTOR FUNCTION AND MOTOR MAP PLASTICITY AFTER EXPERIMENTAL ISCHEMIA .................................................................................. 49Introduc tion ............................................................................................................. 49Methods .................................................................................................................. 51Subjects ............................................................................................................ 51Reach Trai ning ................................................................................................. 51

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6 Electrophysiologic al Mapping ........................................................................... 53Infarction ........................................................................................................... 54Cortical Electrode Implantation ......................................................................... 54Determining Moto r Thres holds ......................................................................... 55Cortical Stimulation and Rehab ilitation Trai ning (CS/ RT) ................................. 56Assessing Residual Moto r Map Area and Topography .................................... 56Histology and Lesion Verifica tion ..................................................................... 57Results .................................................................................................................... 58Reaching A ccuracy .......................................................................................... 58Movement Th resholds ...................................................................................... 59Residual Mo tor Maps ....................................................................................... 59Estimation of Remaining Tissue ....................................................................... 61Discuss ion .............................................................................................................. 613 CORTICAL STIMULATION PLUS REH ABI LITATIVE TRAINING ENHANCES MOTOR FUNCTION INDEPENDENT OF THE NUMBER OF STIMULATING CONTACTS AFTER EXPER IMENTAL IS CHEMIA ................................................ 73Introduc tion ............................................................................................................. 73Methods .................................................................................................................. 76Subjects ............................................................................................................ 76Reach Trai ning ................................................................................................. 77Focal Infa rction ................................................................................................. 78Cortical Electrode Implantation ......................................................................... 79Movement Th resholds ...................................................................................... 79Cortical Stimulation and Rehab ilitation Trai ning (CS/ RT) ................................. 80Histology and Lesion Verifica tion ..................................................................... 81Results .................................................................................................................... 82Reaching A ccuracy .......................................................................................... 82Reaching A ttempts ........................................................................................... 83Movement Th resholds ...................................................................................... 84Estimation of Remaining Tissue ....................................................................... 85Discuss ion .............................................................................................................. 854 CORTICAL STIMULATION WITH REHABI LITATIVE TRAINING CAN ENHANCE MOTOR IMPROVEMENTS AFTER EARLY APPLICATION OF REHABILITATION ALONE IN A RODENT MODEL OF ISCHE MIA ....................... 95Introduc tion ............................................................................................................. 95Methods .................................................................................................................. 98Subjects ............................................................................................................ 98Reach Trai ning ................................................................................................. 99Focal Infa rction ............................................................................................... 100Rehabilitative Training .................................................................................... 101Cortical Electrode Implantation ....................................................................... 102Movement Th resholds .................................................................................... 102

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7 Cortical Stimulation and Rehab ilitation Trai ning (CS/ RT) ............................... 103Histology and Lesion Verifica tion ................................................................... 103Results .................................................................................................................. 104Reaching A ccuracy ........................................................................................ 104Reaching A ttempts ......................................................................................... 105Movement Th resholds .................................................................................... 106Estimation of Remaining Tissue ..................................................................... 107Discuss ion ............................................................................................................ 1075 CORTICAL STIMULATION DOES NOT ENHANCE MOTOR FUNCTION AFTER SUBCORTI CAL STRO KE ........................................................................ 116Introduc tion ........................................................................................................... 116Methods ................................................................................................................ 120Subjects .......................................................................................................... 120Reach Trai ning ............................................................................................... 121Infarction ......................................................................................................... 122Cortical Electrode Implantation ....................................................................... 123Movement Th resholds .................................................................................... 123CS/RT and RT ................................................................................................ 124Histology and Lesion Verifica tion ................................................................... 125Results .................................................................................................................. 126Reaching A ccuracy ........................................................................................ 126Reaching A ttempts ......................................................................................... 127Movement Th resholds .................................................................................... 128Estimation of Remaining Tissue ..................................................................... 128Discuss ion ............................................................................................................ 1296 GENERAL DISCUSSION ..................................................................................... 138Summary .............................................................................................................. 138Study #1: Distributed Versus Focal Co rtical Stimulation To Enhance Motor Function And Motor Map Plasticity After Experiment al Ischemia. ............... 139Study #2: Cortical Stimulation Plus Rehabilitative Training Enhances Motor Function Independent of The Number of Stimulating Contacts After Experimental Ischemia. ............................................................................... 141Study #3: Cortical Stimulation With Rehabilitative Training Can Enhance Motor Improvements After Early Application of Rehabilitation Alone in a Rodent Model of Ischemia ........................................................................... 144Study #4: Cortical Stimulation Does Not Enhance Motor Function After Subcortical Stroke ....................................................................................... 145Possible Neural Bases for CS/R Ts Enhanced Moto r Outcomes .......................... 146Direct Effects of CS /RT Stimul ation ................................................................ 147Potential Neuroplastic Responses to CS/RT .................................................. 149Reorganization of Motor Cortex With CS/RT .................................................. 152Shifts in Excitabi lity With CS/RT ..................................................................... 154

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8 The Effect of CS/RT in New Lesion Models .......................................................... 156CS/RT Afte r MCAo ......................................................................................... 156CS/RT After Caps ular In farct .......................................................................... 157Translation of CS/RT From An imal to Clinic al Studies .......................................... 161Conclusi on ............................................................................................................ 167LIST OF RE FERENCES ............................................................................................. 169BIOGRAPHICAL SKETCH .......................................................................................... 169

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9 LIST OF TABLES Table page 2-1 Estimate of spared cortical and subcortical tissue from e xperiment 1. ............... 723-1 Estimate of spared cortical and subc ortical tissue from experiment 2. ............... 944-1 Estimate of spared cortical and su bcortical tissue from experiment 3. ............. 1155-1 Estimate of spared cortical and subc ortical tissue from experiment 4. ............. 137

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10 LIST OF FIGURES Figure page 2-1 Representative Nissl stained coronal section from t he MCAo lesion .................. 662-2 The three different electr ode configurations examined ....................................... 662-3 Reaching performance prestr oke and during r ehabilitati on. ............................... 672-4 Mean (SD) movement thresholds for animals in the E1 and E2 conditions. ..... 682-5 Changes in the amount of movement representations during rehabilitation ....... 692-6 Changes in the proportion of movement representations during rehabilitation ... 702-7 Relationship between behav ior and motor map changes ................................... 713-1 Representative Nissl stained coronal section from t he MCAo lesion .................. 913-2 The three different electr ode configurations examined ....................................... 913-3 Reaching performance prestroke and during 20 days of rehabilitation. .............. 923-4 Mean (SD) movement thresholds for animals in the RT and all CS/RT conditions during r ehabilitati on. ......................................................................... 934-1 Representative Nissl stained coronal section from t he MCAo lesion. ............... 1124-2 Reaching performance prestr oke and during rehabi litation. ............................ 1134-3 Movement thresholds during rehabili tation. ..................................................... 1145-1 Representative Nissl stained coronal section from t he MCAo lesion ................ 1345-2 Representative Nissl stained coronal section from the capsular lesion. ........... 1345-3 Reaching performance prestroke and during 20 days of rehabilitation. ............ 1355-4 Mean (SD) movement thresholds afte r cortical or subcortical ischemia. ........ 136

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11 LIST OF ABBREVIATIONS AIF Apoptosis-inducing factor ApoE Apolipoprotein E BBB Blood-brain barrier BDNF Brain-derived neurotrophic factor CFA Caudal forelimb area CNS Central nervous system CS Cortical stimulation (motor cortex) CS/RT Cortical stimulation combined with rehabilitative training/therapy CST Cortical spinal tract DNA Deoxyribonucleic acid E1-CS/RT E1 configuration cortical stimulation with rehabilitative training condition E2-CS/RT E2 configuration cortical stimulation with rehabilitative training condition E3-CS/RT E3 configuration cortical stimulation with rehabilitative training condition ET-1 Endothelin-1 FASr Apoptosis-stimulating factor receptor GABA Gamma-Aminobutyric acid GDNF Glial cell line-der ived neurotrophic factor HC Healthy control condition IC Internal capsule ICMS Intracortical microstimulation LTD Long-term depression LTP Long-term potentiation

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12 M1 Primary motor cortex MCA Middle cerebral artery MCAo Middle cerebral artery occlusion MCA-CS/RT MCAo plus cortical stimulation combined with rehabilitative training condition MCAo Middle cerebral artery occlusion MCA-RT MCAo plus rehabilitative training condition MEP Motor evoked potential MT Movement/motor threshold NMDA N-methyl D-aspartate NO Nitric oxide NT Non-trained condition O2 Oxygen PARP1 Poly-(ADP ri bose) polymerase 1 PMd Dorsal premotor cortex PMv Ventral premotor cortex RFA Rostral forelimb area RT Rehabilitative trai ning/therapy condition S1 Primary somatosensory cortex SMA Supplementary motor area Sub-CS/RT Subcortical ischemia plus cortical stimulation combined with rehabilitative training condition Sub-RT Subcortical ischemia plus rehabilitative training condition tDCS Transcranial direct cortical stimulation TIA Transient ischemic attack TMS Transcranial magnetic stimulation

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13 VEGF Vascular-endothelial growth factor

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14 Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy CORTICAL STIMULATION TO ENHA NCE MOTOR IMPROVEMENTS AFTER STROKE By Jeffery Allen Boychuk December 2009 Chair: Jeffrey Kleim Major: Medical Sciences-Neuroscience Motor rehabilitation after cerebral i schemia can enhance motor performance and induce cortical plasticity. Electrical stimulation of the motor cortex (CS) during rehabilitative training (CS/RT) augments motor map plasticity and enhances motor improvements. CS/RTs ability to enhance motor improvements after ischemic insult has only been tested in a limited number of situat ions that do not completely represent stroke conditions in the human population. Indeed, the lack of treatment effect in a recent phase III clinical of CS/RT reflects k nowledge gaps in how to fully translate basic science findings of CS/RT into the clinical se tting. The goal of this dissertation is to use rodent models of stroke to examine severa l parameters of CS/RT therapy that may account for its lack of benefit in human stroke patients. Study #1: It is unclear how the distribut ion of electrical stimulation across the cortex influences both motor map r eorganization and improvements in motor performance. Here, we examined the behav ioral and neurophysiological effects of delivering CS/RT through a distributed versus focal arrangement of electrical contacts. In this experiment, stroke was modeled by focal ischemic damage with motor cortex.

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15 Results showed that animals given CS/RT with a distributed contact configuration condition exhibited greater motor improvements than animals given CS/RT with a focused contact arrangement or given rehabilitative training alone (RT). All groups that received rehabilitation exhibi ted greater increases in motor map area and reaching accuracy than animals that received no rehabili tative training (NT). However, both CS/RT groups exhibited larger motor maps than the RT animals. The results indicate that although both focal and distributed forms of CS/RT promote motor map reorganization, only the distributed form of CS/RT enhances motor performance with rehabilitation. Study #2: While distributed CS/RT proved mo re effective than focal CS/RT in the first experiment, the number of distributed stimulation contacts sites that will induce the greatest motor improvements with CS/RT is not known. Here, we examined the behavioral effects of delivering CS/RT with eit her four or nine in dependent contact sites that were distributed across motor cortex follo wing ischemic insult. In this experiment, stroke was modeled by temporary occlusion of the middle cerebral artery. The results showed that both types of distributed CS /RT enhanced motor improvements regardless of the number of independent sites. Our result s indicate that the number of independent stimulation sites does not affect CS /RT with distributed stimulation. Study #3: Human stroke patients who ar e candidates for CS/RT will likely have received standard forms of rehabilitation prior to the onset of CS/RT. It is not known whether CS/RT can induce motor improvem ents in individuals who have received rehabilitative experience following ischemic insult. Further, the phase III clinical trial that failed to find an effect of CS/RT had recruit ed stroke patients who had already received

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16 standard rehabilitative t herapy. Here, the behavioral effe cts of administering CS/RT after early application of RT alone were measured. In this experiment, stroke was modeled by temporary occlusion of the middle cerebral artery. The results showed that CS/RT magnifies behavioral improvements above and beyond those occurring from rehabilitative training alone. Our resu lts suggest that CS/RT and RT have a complimentary rather than antagonizing relationship. Study #4: While animal studies of CS/RT have observed enhanced motor improvements in models of cortical stroke, they have not tested its effects in subcortical stroke models. Subcortical damage is relative ly common in stroke patients. Further, the phase III clinical trial that found no benefit with CS/RT had enrolled patients with subcortical stroke damage. In this experim ent, the behavioral effects of CS/RT in a rodent model of subcortical ischemia involv ing damage within the internal capsule were compared to model of cortical ischemia. The results showed that CS/RT confers no additional motor improvements compared to RT following subcortical white matter ischemia. These data suggest that CS/RT ma y not be as effective in treating human stroke patients with subcortical damage.

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17 CHAPTER 1 NEURAL PLASTICITY, STROKE AND CORTICAL STIMULATION General Introduction In the United States, approximately 795 000 new or recurrent strokes occur each year with an estimated 6.5 millio n individuals currently living with the effects of this disease (American Heart Association, 2009) The functional deficits following stroke involve a number of systems ranging from cognitive, affective, communicative, motor and sensory domains. Motor impairments are f ound in more than 75% of stroke victims (Lawrence et al., 2001) and are the primary factor contributing to serious long-term adult disability (McNeil and Binette, 2001) and decreas ed quality of life (Hacket et al., 2000; Sturm et al., 2002). For 2009, the projected fi nancial burden of stroke is an estimated 68.9 billion dollars when estimates of the di rect costs involving the treatment of the injury and the indirect costs involving loss of productivity are combi ned. More effectives treatments for motor impairments after stroke will alleviate so me of the financial burden and improve the lives of both stro ke survivors and care givers. Given the incidence of motor deficits in st roke patients, motor rehabilitation is the primary treatment. Unlike many other medical conditions, there is currently no single treatment intervention that is recognized by therapists for enhancing motor function. After stroke, some motor improvements o ccur within the first three months while substantially fewer improvements are obser ved at later time points (Duncan et al., 2000). Further, the degree of motor improvement after stroke is typi cally incomplete and highly variable across individuals (Gres ham et al., 1995). The extent of motor improvement after stroke depends on many factors such as t he initial impairments, age, sex, the nature of the training experience and pati ent genotype (Duncan et al., 2000).

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18 After stroke, the severity of motor impairments during t he initial hospital admission predicts the severity of motor impairments and the ability to perform activities of daily living at the time of discharge (Shelton et al., 2001). Moderate to severe hemiparesis within the first month post stroke strongly predicts poor functional outcomes at least up to three years following the injury (Samuelss on et al., 1996). The location of injury can also predict functional outcomes in stroke patients. Subcortical stroke is typically associated with more severe impairments and fewer behavioral gains when there is damage to white matter connections (Norrving, 2003; Arakawa et al., 2006). In particular, ischemic damage in the posterior region of the internal capsule has been associated with relatively severe impairment s and little restoration of motor function (Morecraft et al., 2002; Lie et al., 2004; Wenzelburger et al., 2005). Reduced axonal integrity as assessed by lower fractional anisotropy with MR imaging correlates with further loss of fractional anisotropy and motor function in patients with poor motor outcome (Mller et al., 2007). More symmetric al bilateral fractional anisotropy after stroke correlates with improved motor func tion at a three-mont h assessment (Jang et al., 2005). Functionally, the abs ence of TMS responses within the first forty-eight hours is associated with complete hand palsy (Pennisi et al., 1999). The nature of the training experience also affects motor improvement s after stroke. In animal studies, skilled motor training is associated wit h improvements in skilled moto r function that are absent during endurance or strength motor training or training on si mple motor tasks (Adkins et al., 2006; Kleim and Jones, 2008). Human stroke patients given one of two types of rehabilitative therapy in a single study can exhibit different degrees of motor improvements (Langhammer and Stanghelle, 2000). In a meta-analysis, Cifu and

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19 Steward (1999) observed ear ly initiation of rehabilit ative therapy had a strong relationship with improved functional outcome s. However, immediate therapy may be harmful as animal data have observed exac erbated tissue damage wit h extreme use of an impaired limb in the acute hours after injury (Kozlowski et al., 1996; Risedal et al., 1999). Many patient characterist ics that are independent of the stroke can influence the level of impairments as well as the degree of motor improvements over time. Analysis of motor improvements on the Barthel Index have been used in statistical modeling to demonstrate that older age, prestroke disab ility, and the female sex are associated with fewer behavioral gains at least up to 1 year following injury (Tilling et al., 2001). Increasing age at stroke onset also correlates with poorer performance on activities of daily living (Nakayama et al, 1994). Func tional status assessed by having fewer disabilities in activities of daily living before stroke is also strongly associated with fewer difficulties in activities of daily living a fter stroke (Colantonio et al., 1996). There is emerging evidence that the genotype of stroke patients affects their recovery. Given the complexity of the human genome there are like ly numerous genes that affect recovery from stroke. In the context of stroke reco very, two of the more characterized genes include brain-derived neurotrophic factor (BDNF) and apo lipoprotein E (ApoE). It is estimated that 30-40% of t he human population carries at least one copy of the BDNF gene with a val66met polymorphism that is a ssociated with diminished release of the active protein (Egan et al., 2003; Chen et al., 2004). BDNF is a highly expressed growth factor in the brain that affects the survival structure and function of neurons (Desai et al., 1999; Lu and Chow, 1999). Levels of BDNF are altered in response to variety of manipulations making it a put ative signal for encoding experience in the brain (Pearson-

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20 Fuhrhop et al., 2009). Disrupting BDNF levels in the brain limits synaptic plasticity (Ma et al., 1998; Gorski et al ., 2003; Genoud et al., 2004) and impairs many types of learning (Linnarsson et al., 1997; Minichiello et al., 1999; Mizuno et al., 2000). In the context of stroke rehabilitation, anima ls given infusions of antisense BDNF oligonucleotide that diminish BDNF signaling do not exhibit motor improvements during motor training after experiment al stroke (Ploughman et al ., 2009). Humans that carry the val66met polymorphism ex hibit attenuated responses of cortical plasticity as assessed by TMS motor mapping (Kleim et al., 2006) or fMRI (McHughen et al., 2009). These findings suggest the Met allele may li mit neuroplastic changes following injury that support motor improvem ents (Pearson-Fuhrhop et al., 2009). Indeed, the val66met polymorphism has been associated with poorer outcome after subarachnoid hemorrhage (Siironen et al., 2007). The ApoE gene has two common single nucleotide polymorphisms resulting in three distinct alle les termed ApoE2-4 or E2-4 (Mahley et al., 2000). E3/E3 is the most common genotype wit h estimated frequencies of 43%-74% in human populations depending on ethnicity (Eichner et al., 2002). Approximate frequencies of the other s include: 22% E3/E4, 12% E2/E3, 3% E4/E4, 2% E2E4, 1% E2/E2 (Eichner et al., 2002; Bersano et al., 2008) ApoE mediates lipid transport from one cell type to another as well as serves in neuronal repair, remodeling and protection (Mahley et al., 2000; Cedazo-Minguez, 2007). In a meta-analysis, ApoE genotype was associated with functional outcome following subarachnoid hemorrhage (MartnezGonzlez and Sudlow, 2006). In another study, ApoE genotype related to behavioral outcome measures at 1 and 3 months pos t stroke and ApoE4 was associated with poorer outcome (Pearson-Fuhrhop et al., 2009) Motor improvements after stroke must

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21 therefore be thought of as the product of numerous behavioral and neurobiological factors. The growing identific ation of stroke recovery fa ctors and their interactions indicate our present lack of understanding fo r how to facilitate brain repair and drive behavioral improvements. Neurobiology of Stroke Stroke is a heterogeneous injury involving di sruption of blood flow to the brain. The resulting dy sfunction or death of neural tissue creates neurological deficits that reflect loss of function by the compromised areas. The two general ways that blood flow is disrupted during stroke are through ischem ic and hemorrhagic processes. Hemorrhagic stroke occurs when a blood leaks from the cardiovascular system resulting in reduced blood flow as well as toxic effects of the misplaced blood. Hemorrhagic stroke has an intracerebral origin in 10% and a subarachnoid origin in 3% of all stroke cases (American Heart Association, 2009). Ischemic stroke accounts for approximately 87% of all stroke types and is characterized by compromised blood flow due to a blood vessel obstruction or an inability for the cardiova scular system to maintain adequate supply such as cardiac arrest (American Heart Association, 2009). The overall diminished ability of the cardiovascular system to supply blood to the brain is termed global ischemia. If reduced blood flow is due to obs truction of a specific vessel or set of vessels it is termed focal ischemia. Thro mbotic focal ischemia occurs when the blockage is formed at the site where it disrupts blood flow whereas embolic focal ischemia occurs when the blockage is formed di stant to the site where it disrupts blood flow and is then carried in the blood stream to the place of the bl ockage. An additional type of ischemic injury is a transient isc hemic attack (TIA). TIAs are not considered strokes but rather temporary reductions in the blood flow where neurological symptoms

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22 last less than 24 hours. The onset of a TI A is usually sudden and the duration brief, generally lasting between 2 and 30 minutes (Hankey, 1996). While TIAs are highly transient they tend to foreshadow upcoming ischemic embolism strokes that are typically more severe (Hankey, 1996). Cell Death During Stroke The pathophysiology of focal ischemia stro ke is reviewed here because it is most relevant to the stroke models used in this dissertation (for a review of hemorrhagic stroke see Carmichael et al., 2008; Quresh i et al., 2009). Cell death during isc hemia is thought to largely arise from excitotoxicity and ionic imbalance, oxidative/nitrosative stress and activation of apoptotic pathwa ys, however, emerging evidence also implicates other detrimental processes including inflammation, tissue acidosis and periinfarct spreading depolarizations (Mergenthaler et al., 2004). During ischemic exposure cells may die by rupture, lysis, phagocytos is or involution and shrinkage (Lo et al., 2003). These modes of cell destruction are oft en classified using the terms necrosis and apoptosis to discriminate whet her the cell actively participates in its destruction. Necrosis is the most prominent form of cell death during extreme ischemia and is the result of rapid disruption of the plasma membrane or organelle failure (Lo et al., 2003). Necrotic cells endanger neighboring cells due to the rel ease of toxic or damaging molecules (Lo et al., 2003). Apoptosis in contrast represents a programmed cell death requiring adenosine triphosphate and gene ex pression that involves an organized degradation of the cell such that there is minimal release of cellular contents into its surrounding environment (Johnson et al., 1995). Apoptosis is triggered by an extrinsic pathway involving the activation of the apt ly named death receptors such as the FASreceptor (Mattson et al., 2001). Intrinsic factors such as elevated calcium, reactive

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23 species, glutamate and deoxyr ibonucleic acid (DNA) dama ge also stimulate apoptosis (Lo et al., 2003). Both extrinsic and in trinsic factors can induce a mitochondrial dependent apoptotic signal involving the re lease of cytochrome c and subsequent caspase activation (Li et al., 1997). Activate d caspases are prot ein-cleaving enzymes that disrupt homeostasis and disassemble cells (Dirnagl et al., 1999). Caspaseindependent apoptosis occurs through the activation of the poly-(ADP ribose) polymerase 1 (PARP1) by stimulating N-meth yl D-aspartate (NMDA) receptors using the signaling of apoptosis -inducing factor (AIF) (Lo et al., 2003). AIF is released from the mitochondria and binds to DNA where it promotes chromatin condensation and cell death through unknown mechanisms (Lo et al ., 2003). Apoptosiss contribution to overall ischemic cell death remains subject to debate, how ever, it is likely milder ischemia preferentially induces apoptosis (Lo et al., 2003; Mergenthaler et al., 2004). Further, cross talk between necrotic and apoptotic pathways leads to combinatorial forms of cells death with hybrid morpholog ical features with several cross talkmolecules having suggested role s including calpains, cathepsin B, nitric oxide (NO) and PARP1 (Lo et al., 2003). Another important distinction in stroke pathophysiology is describing the endangered tissue by its location within the i schemic region. The ischemic zone most deprived of blood is termed the core. The core has the fastest and greatest overall loss of cells as well as exhibiting the greatest proportion of necrotic cell death (Martin et al., 1998; Banasiak et al., 2000). Tissue surroundi ng the ischemic core has an outward gradient of increasing blood flow and decreas ing toxic elements that is collectively referred to as the ischemic penumbra. Wh ile the precise boundaries of these regions

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24 are difficult to define each area exhibits unique patterns of cell death, gene expression, promotion/inhibition of neuronal rewiring, neurogenes is and neuronal activity that must be considered when reviewing stroke pathoph ysiology (Mergenthaler et al., 2004; Carmichael et al., 2005; Carmi chael 2006; Popp et al., 2009). Ionic Imbalance by Energy Failure When blood flow sufficiently decreases dur ing stroke, cells in the nervous system deplete their energy stores and lose their abi lity to maintain ionic concentration gradients. Cells experiencing low energy also fail to remove neurotra nsmitters from their synaptic spaces. Cells lacking sufficient energy depolarize allowing an influx of sodium, chloride and calcium ions as well as the ef flux of potassium (Siesjo et al., 1991). Increased intracellular concentration of sodium and chloride ions leads to an influx of water causing cell and tissue swelling (edema) t hat further disturbs ion homeostasis and causes cell death by osmotic lysis. Incr easing intracellular calcium concentrations promote lipolysis, proteolysis, nitric oxide production, endonuc lease-mediated DNA degradation in addition to the activation of kinases and phoshatases that alter the activation states of proteins and direct certain gene expression (Emsley et al., 2008). Excitotoxiticty The depolarization of neurons and glia induced by insufficient energy and ionic disturbances in the extracellular space leads to the release of excitatory neurotransmitters in the absence of presyn aptic and astrocytic reuptake mechanisms. The resulting excessive presence of excita tory neurotransmitter in the extracellular space results in uncontrolled postsynaptic rec eptor binding leading to an ion influx in postsynaptic cells. The primary excitatory neurotransmitter of the brain glutamate is thought to be a key player in this excitotoxi city. Postsynaptic binding of glutamate to

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25 ionotropic NMDA receptors results in incr eased intracellular calcium concentrations while glutamate binding to -amino-3-hydroxy-5-methyl-4-i soxazole propionic acid receptors and ionotropic glutam ate receptors leads to increases in intracellular sodium, and chloride concentrations (Mergenthaler et al., 2004). The resulting influx of ions from glutamate binding again leads to osmotic lysi s (Dirnagl et al., 1999; Lo et al., 2003). The influx of ions also results in apoptosis and inflammation (Dirnagl et al., 1999; Lo et al., 2003). Excess intracellular calcium promotes other detrimental effects to cell survival including the activation of phospholipases and proteases that degrade essential membranes and proteins (Lo et al., 2003). Oxidative/Nitrosative Stress Reactive oxygen and nitrogen species su ch as superoxide, hydrogen peroxide, hydroxyl and peroxy-nitrite r adicals are dramatically increased during ischemia. These reactive species have the potential to damage membranes, DNA and almost all organelle types (Dirnagl et al., 1999; Lo et al., 2003). In particular, mitochondrial damage from reactive species results in a diminished production of ATP production as well as release of additional damaging radicals and proapoptotic molecules (Mergenthaler et al., 2004). Acidosis The hypoxic conditions created by low blo od flow cause a switch from aerobic to anaerobic cellular metabolism leading to an accumulation of lactate via anaer obic glycolysis (Mergenthaler et al., 2004). Incr eased anaerobic metabolism promotes the formation of lactic acid causing intracellula r pH levels to decrease (acidosis) (Sapolsky et al., 1996). This acidosis causes cellu lar dysfunction, including disruption to mitochondrial and glycolytic enzyme activity (Rehncrona et al., 1981). In addition,

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26 acidosis disrupts intracellular protein syn thesis and promotes t he formation of damaging reactive species (Mergenthaler et al., 2004). Peri-Infarct Spreading Depolarizations Under conditions of low blood flow, excessiv e potassium ions and glutamate released in the extracellular space act to c ontinually depolarize ce lls in the local area (Dirnagl et al., 1999). While cells in the core region of the infarct never repolarize, cells in the peri-infarct region do at the expense of further energy consumption (Dirnagl et al., 1999). As neurons and glia depolarize they release additional potassium ions and glutamate into the extracellu lar compartment caus ing neighboring cells to depolarize in consecutive waves. This pheno menon is known as spreading depressions/depolarizations. In experimental m odels of focal cerebral ischemia, periinfarct depolarizations have been observed at a frequency of 1-4 events per hour (Busch et al., 1996; Wolf et al., 1997). T hese cells deplete energy reserves through continual cycles of repolar ization and depolarization (Hossmann, 1996). The depletion of energy in metabolically compromised ce lls increases the potent ial of cell death and the growth of the lesion core (Iijima et al., 1992; Mies et al., 1993; Back et al., 1994; Hoehn-Berlage et al., 1995). Inflammation The inflam matory response to ischemia within the nervous system contributes to tissue loss, however, the detrimental role of specific inflammatory cascades remains unclear. The uncertainty in the role of infl ammation during ischemia is highlighted by the fact that experimental blocka des or mice knockout model s of inflammatory elements may reduce or increase infarct size (Mergent haler et al., 2004). A complete description of the localization and molecular interacti ons of the ischemia-induced inflammatory

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27 response is beyond the scope of the present review (see Barone and Feuerstein, 1999; DAmbrosio et al., 2001; Emsley and Hopkins, 2008; Emsley et al., 2008). Inflammation may alter ischemic damage through hyperthermi a, activation of both the hypothalamicpituitary-adrenal axis and sym pathetic nervous system in addition to its classic acute phase response (for review see Emsley et al., 2008). Within the classic response, inflammation may promote cell damage by microv ascular failure or the release of toxic molecules (Back, 1998; Jean et al., 1998; del Zoppo et al., 2001). Ischemic damage promotes the activation a nd proliferation of cells derived from the mononuclear phagocytic system such as macrophages and microglia. The role of these mononuclear phagocytic cells was thought to be a repair mechanism because of their potential to restore blood flow, repai r the blood brain barrier and promote general homoestasis (OCallaghan, 1991; Norton et al., 1992). However, recent data suggest they may be detrimental to cell survival dur ing ischemia. The activation of intracellular second messenger systems by excessive intrac ellular calcium levels and the increase in free radicals trigger the initiation of t he inflammatory response (Ruscher et al., 1998; Emsley et al., 2008). Activated leukocyt es (granulocytes, monocyte/macrophages and lymphocytes) in addition to neurons and g lia (astrocytes and microglia) produce key pro-inflammatory molecules such as cytokines and chemokines (Barone and Feuerstein, 1999). Cytokines are polypeptide hormones that may have positive or detrimental effects on tissue survival through known activity in inflammation, immune activation, cell differentiation and apoptosis (Emsley et al., 2008). Chemokines are a sub-family of cytokines that direct migrati on and entry of inflammatory cells into tissues

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28 thus suggesting a role where t hey promote the detrimental invasion of inflammatory cells such as leukocytes (Emsley et al., 2008). Several patterns of inflammatory respons e are thought to prom ote cell death. The accumulation of leukocytes, red blood cells, fibrin deposits and platelets can lead to occlusion of microvessels resulting in the no flow phenomenon (del Zoppo et al., 1991; del Zoppo, 1997; Winquist and Kerr, 1997; del Zoppo, 1998; Jean et al., 1998). The inflammatory response following ischemia increases permeability of the blood-brain barrier (BBB) by endothelial components causi ng adhesion of leukocytes and platelets to the vascular endothelium and disruption of the basal lamina (del Zoppo and Hallenbeck, 2000). Interaction between leuk ocytes and adhesion molecules across the microvessel/BBB interface results in leukocyt e transmigration into the brain parenchyma where further inflammatory responses may occur (Mergenthaler et al., 2004). In addition, the secretion of t he enzyme inducible NO-synthase increases concentrations of NO that are cytot oxic due to their formation of per oxynitrite (Crow and Beckman, 1995) while secretion of the enzyme cycloo xygenase-2 likely causes tissue damage through the production of free oxygen radicals and prostanoids (del Zoppo et al., 2000; Emsley and Tyrrell, 2002). Colle ctively, the ischemic infl ammatory response should be viewed as complex process with positive an d negative effects on tissue survival that involves numerous targets fo r neuroprotective therapies. Stroke Treatment The three common approaches to treating stro ke are prevention, neuroprotection and rehabilitation. Primary prevention involv es measures that attempt to reduce the incidence of stroke in the population at lar ge, while secondary prevention attempts to limit stroke incidence in patients who have already dev eloped symptoms of TIA or

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29 stroke because of the increased risk of stroke in this subpopulation (Johnston et al., 2000). Stroke prevention involves the identification of risk factors for stroke followed by attempts to limit these factor s. Some risk factors are non-modifiable such as increasing age, male sex, nonwhite race, the presence of coronary heart disease or congestive heart failure and a positive family history for stroke or TIA (Sullivan and Katajamaki, 2009). Whether diabetes mellitus is a modifiabl e risk factor remains subject to debate (Calvin et al., 2009). Effective reductions in t he expected incidence of stroke have been associated with adequate blood pre ssure reduction, treatment of hyperlipidemia, use of antithrombotic therapy in pat ients with atrial fibrillation and antiplatelet therapy in patients with myocardial infarction in bot h primary and secondary levels (Sullivan and Katajamaki, 2009). For secondary prevention, carotid endarterectomy in patients with severe carotid artery stenosis also reduces (Straus et al., 2002). Stroke prevention can also reduce stroke by emphasizing lifestyle changes such as avoiding tobacco, increasing physical activity and consuming a healthy diet (Sullivan and Katajamaki, 2009). While effective, stroke prevention s success is limited by the difficulty in identifying risk factors and the capacity to reduce them. Neuroprotective strategies attempt to limit the severity of stroke by antagonizing the injurious biochemical and molecular events that results in infarction. Hemodynamic agents or techniques used to maintain circul atory patency such as thrombolytics are not typically described as neuroprotective agents although they do comprise an important line of stroke research (Cheng et al ., 2004). The therapeutic window for most neuroprotective agents is within the first 4-6 hours following the onset of stroke (Gorelick, 2000). Some of the most studied neuroprotective agents include calcium

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30 channel blockers, glutamate antagonists, GABA agonists, antioxidant/radical scavengers, phosholipid precursors, nitric oxide signaling down regulators, leukocyte inhibitors and hemodilution (Ginsberg, 2009). While some attenuation of tissue loss has been associated with the use of neuroprotective agents in human stroke patients, these strategies are limited by their narrow therapeutic window as well as difficulty in translating from animal models to human st roke patients (OCollins et al., 2006). Nonetheless, neuroprotective strategies su ch as the aforementioned list and many others have strong evidence from animal model s that they are c apable of limiting the severity of tissue loss during stroke (Lipton et al., 2007). Even with all of the opportunities to pr event and limit disease, healthy care systems worldwide are dominated by the treat ment of people with chronic disability (Hoffman et al., 1996). In the context of stroke recovery, despite preventative and neuroprotective strategies, more than 350 000 Americans suffer a stroke and exhibit persistent motor deficits each year (Luke et al., 2004; Urton, et al., 2007). Some motor improvements occur within the first thr ee months following t he injury but these improvements are rarely complete and subs tantially fewer improvements occur beyond the first year of injury (Duncan et al., 2000). Rehabilitation of stroke patients aims to reduce impairments to allow these patients to perform more of the activities and participation in life situations they were c apable of prior to stro ke (Barak and Duncan, 2006). Unfortunately, current re habilitative strategies only demonstrate benefit on a general level whereas individual patterns of mo tor improvements after stroke are highly variable and typically incomplete (Gresham et al., 1995). It is now being appreciated that parameters of rehabilitative motor training such as int ensity, duration, timing, and

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31 the nature of the training expe rience impact its efficacy. Further, measures of brain function after stroke are a llowing an understanding of the me chanisms underlying motor improvements and allowing for therapy to be more tailored to individual stroke patients (Milot and Cramer, 2008). The increased knowle dge of brain repair is also facilitating the development of adjuvant therapies that have the potential to m agnify the benefits of rehabilitation (Floel and Cohen, 2009). Cortical Plasticity and As A Neural Substrate for Motor Rehabilitation Improvements after stroke can arise from compensation or recovery at both neural and behavior levels. It is important to dist inguish between these terms because they likely represent different neurobiological or behavior processes, each with unique contributions to motor improvements (Kleim 2009). Historically, the terms recovery and compensation have been used to describe functional improvements in conflicting ways. Levin et al. (2009) recently distinguis hed the two terms based on the International Classification of Function (ICF) framework proposed by the World Health organization. The ICF model recognizes post injury impairments in three levels: the pathophysiology of the body, the loss of body output (im pairment) and the loss of task performance (disability) (Kleim, 2009). Neural recovery de scribes the return of function to nervous tissue whereas neural compensation is when residual tissue adopts function that was lost by injury (Levin et al., 2009). Behavio ral recovery can involve the return of premorbid movement or pr emorbid task performance wher eas behavioral compensation describes performing movement or activities in a diffe rent manner than the behavior prior to injury (Kleim, 2009) The closest version of neural recovery after permanent cell loss would be cell replacement therapy th rough the addition of exogenous precursor cells or the guided targeting of cells from endogenous adult stem cells to the injury

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32 (Lindvall and Kokaia, 2004 ). Neural recovery can also occur by dysfunctional cells regaining their normal function. Stroke injury can disrupt spared tissue by many of the same ways that it causes cell death such as deinnervation, disrupted blood flow, ionic imbalances and inflammation (Nguyen and Botez, 1998). Many of these disruptions can be alleviated over time resulting in the re storation of neural function. Although the specific neurobiological mechanisms cont ributing to improvements are not fully understood, plasticity within residual motor brain areas supporting neural compensation is believed to be the primary factor underly ing recovery. Noninvasive brain imaging studies have identified ipsilesi onal reorganization of primary motor and sensory areas in patients with good behavioral outco mes after stroke (Rossini et al., 1998; Cramer and Bastings, 2000; Cramer and Crafton, 2006). Motor mapping using TMS has found that motor improvements after stroke are associated with increases in the ipsilesional motor map area and motor-evoked potential (MEP) am plitudes (Wittenberg et al., 2003). In animal models, motor improvements after ex perimental ischemia are often associated with reorganization of movem ent representations within motor cortex (Nudo et al., 1996a; Kleim et al., 2003; MacDonald et al ., 2007). The reorganization patterns that parallel motor improvements following ischemic injury are highly similar to the patterns observed in healthy animals (Kleim and J ones, 2008) and people (Pascual-Leone et al., 1995) during skilled motor training. This rela tionship suggests that the cortex has the capacity to undergo a relearning process follo wing injury that follows principles observed during skilled motor learning. Motor Map Plasticity and Motor Learning in the Intact CNS The motor cortexs somatopic organi zation was described more than a one hundred years ago by crude stimul ation of the canine precentral gyrus (Fritsch and

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33 Hitzig, 1870; Jackson, 1874). More refined stimulation techniques such as intracortical microstimulation have more recently been used to accurately and reliably define the functional organization of the rat motor cortex (Neafsey et al., 1986; Kleim et al., 1998). This technique allows for the construction of a "map" of the topography of forelimb movement representations withi n the rat motor cortex. Usi ng standard ICMS techniques (Kleim et al., 1998), the rat is anesthetized and a craniotomy is performed over the motor cortex contralateral to the move ments of interest such as forelimb extension/flexion. Following this preparation, a microelectrode controlled by a hydraulic microdrive is positioned at various locations on the cortex and then lowered into cortical layer V. A small amount of current is t hen passed through layer V, which stimulates pools of pyramidal cells. The resulting move ment pattern can then be recorded. After numerous microelectrode penetrations are made, the final result is a "motor map" of the functional representations within the motor cortex. Studies in rodents using ICMS have provided a detailed de scription of the rodent motor m ap as a fractured somatotopic mosaic, i.e., individual movements are r epresented multiple times and are interspersed with adjacent movement representations across separate regions of cortex (Stoney et al., 1968; reviewed in Schieber, 2001). Motor cortex has been shown to reorganize in response to skilled motor training in healthy individuals. In non-human primates, training on a task that requires skilled digit manipulation caused an ex pansion of digit representations and this expansion is lost by the cessation of training or training on a skilled wrist movement task (Nudo et al., 1996a ). The subsequent training on a skilled wrist task resulted in an expansion of wrist representations where digit representations previously occupied (Nudo et al., 1996a).

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34 The motor cortex of non-human primates does not functionally reorganize when the task does not require difficult reaching move ments indicating the nature of training experience is an important factor (Plautz et al., 2000). In r odents, non-skilled training such as pressing a lever (Kleim et al., 1998) or reaching for unattain able pellets (Kleim et al., 2004) does not induce map reorganization. Exercise training with running wheels does not alter motor map topography (Kleim et al., 2002b; Maldonado et al., 2008) but does induce angiogenesis and increased blood flow to motor cortex (Swain et al., 2003). Strength training does not result in map reorganization but induces synaptogenesis in the spinal cord (Remple et al., 2001). In contrast, skilled training in rodents induces functional reorganization of the motor map such that movements biased by the training subsequently increase their number of responsive sites in motor cortex (Kleim et al., 1998; Kleim et al., 2002a; Conner et al., 2003; Kleim et al., 2004). The positive shift in the proportion of trained sites occurs after substantial changes have occurred in reaching performance. In rodents, skilled reaching behavior is significantly improved as early as the th ird day of training whereas robust map reorganization is not observed un til the tenth day of training (Kleim et al., 2004). The training specificity observed with motor ma p plasticity has also been observed by measures of dendritic hypertrophy. Training on object recognition (delayed nonmatch to sample) or visuomotor tasks does not induce dendritic plasticity within motor cortex (Kolb et al., 2008). In contrast, unilateral r each training increases dendritic length and branching in motor cortex cont ralateral to the training and this dendritic plasticity is observed in both motor cortices following bila teral reach training (Kolb et al., 2008). Similarly, only complex walking tasks such as acrobatic training and walking up and

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35 down a runway increase synapse numbers within motor cortex (Black et al., 1990; Kleim et al., 1998). Finally, skilled reach training is also associated with dendritic growth (Greenough et al., 1985; Withers and Greenough, 1989; Bury and Jones, 2002; Allred and Jones, 2004), synaptogenesis (Kleim et al., 2002a; Kleim et al, 2004) and enhanced synaptic responses (Rioult-Pedotti et al., 1998; Monfils and Teskey, 2004; Hodgson et al., 2005) wit hin motor cortex. The invasiveness of microstimulation techniques has prevented its use for extensive study of human motor cortex however, organized somotatopy has been identified in humans during medical procedures (Penfield and Boldrey, 1937; Woolsey et al., 1952). Reorganization of human maps has also been observed in response to skilled training or in individuals with hi ghly skilled talents with imaging or mapping techniques using noninvasive transcranial ma gnetic stimulation (TMS). For example, skilled racquetball players have larger hand representations than less skilled players and naive individuals (Pearce et al., 2000). The reading hand of pr oficient Braille readers (Pascual-Leone et al., 1 993) or five days of unilatera l digit training using a piano results are associated with incr eased digit representations c ontralateral to the skilled behavior that are not observed in the ipsilateral hemisphere or in the contralateral hemisphere of control subjects (PascualLeone et al., 1995). Cessation of the piano training resulted in the maps shrinking back to baseline sizes (Pa scual-Leone et al., 1995). Remarkably, both skilled tongue protrusion training (Svensson et al., 2003) and skilled ankle training (Perez et al., 2004) are associated with in creases in their respective representations within motor cortex. Imaging studies sup port the relationship between cortical plasticity and skilled motor learning. For example, amateur keyboard

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36 players exhibit a greater amount of gray volume within mo tor cortex compared to nonplayers and expert keyboard players exhibit a higher amount than either group (Gaser and Schlaug, 2003). Imaging studies have de monstrated a progression of brain activation during the stages of learning. Early motor learning appears to involve a greater activation of premotor (Grafton et al, 1992; Ghilard i et al., 2000) and cerebellar (Eliassen et al., 2001; Penhune and Doyon, 2002) cortices whereas late stages are marked with increased activation of primary motor cortex (Karni et al, 1995; Floyer-Lea and Mathews, 2005). It is becoming increasingly clear that t he brain is endowed with an exceptional capacity to reorganize itself in response to perturbation. This point is demonstrated by a study where healthy individuals were imaged using fMRI while engaging in a hand opening/closing task. Compared to a resting state, performi ng the task was associated with activation of contralateral motor cort ex and rostral supplementary motor cortex (Walsh and Pascual-Leone 2003). Increasing activi ty in the contralateral motor cortex by prior application of high fr equency repetitive TMS resulted in a decrease in activation of rostral SMA, i.e., more exclusive activa tion within motor cortex (Walsh and PascualLeone 2003). Interestingly, prior application of slow repetitive TMS to suppress neuronal activity in the motor cortex contralateral to the hand task resulted in increased activation of rostral SMA and motor cortex ipsilateral to the task with no decrement in behavioral performance (Walsh and Pascual-Leone 2003) Maintenance of task performance following a similar low frequen cy TMS procedure in healthy individuals has also been associated with an a increase in movement-related PET activity in the premotor cortex contralateral to the stimulated hemisphere (Lee et al., 2003).

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37 Motor Cortex Plasticity and Improvement of Motor Function Follow ing Stroke Deficits after stroke are as much a mani festation of the loss of brain regions as they are the limit to which remaining brai n structures can com pensate for the lost functions (Cramer, 2004; Takahashi et al., 2004). At least some of the abnormal patterns of brain activity observed during move ment tasks after stroke are the result of spared circuits attempting to es tablish alternate forms of in tracortical, subcortical and descending projections. If stroke results in damage/disruption to motor cortex, these parallel circuits may originate from remaining active portions of this region, contralesional motor cortex, or bilaterally from secondary motor area such as SMA, PMA, somatosensory areas, cerebellum, basal gang lia, etc. It is likely cortico-cortical and cortico-subcortico-cortical circuits will continue to shift interactions across the network in an attempt to establish suitable brain activation that will produce the desired behavioral result so long as some efferent cortico-spinal output pathways exist. It is likely initial network changes after stro ke aim at minimizing damage and rapid improvements in behavior arise from the amelio ration of dysfunction in spared tissue or the repair of partially damaged structures (P ascual-Leone et al., 2005). The process of reorganizing residual circuits begins as t he damage and these im mediate changes have stabilized (Pascual-Leone et al., 2005). Early after cortical stroke patients typica lly exhibit bilateral increases in fMRI activation patterns in primary and non-primar y motor areas that steadily reduce with time and the timing of these r eductions correlate with motor improvements (Ward et al., 2003). Acute stroke damage is associated with increased excitability in the unaffected hemisphere presumably resulting from its incr eased use as well as reduced intracortical inhibition and increased intracortical facili tation (Liepert et al., 2000). Transcallosal

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38 inhibition from the unaffected to affected hemisphere during voluntary movement is also abnormal (Murase et al., 2004). Several months following stroke a normalization of the over-activity in the unaffected hemisphere is observed (Shimizu et al., 2002). Long-term poor functional outcome is associated with larger areas of cortical activation in cortical networks spanning both the intact and damaged hemisphere (Carey et al., 2002; Rossini and Dal Forno, 2004). Tombari et al. (2004) observed a progressive shift from contralesional activity in motor and so matosensory cortices to the ipsilesional hemisphere by sampling twenty days, four months and one year after stroke onset. There is a negative correlation between amount of cortical activation during movement and transcranial magnetic stimulation-derived MEPs (War d, 2006). These MEPs are important because acute stroke patients with MEPs show better functional outcome than those without presumably be cause of greater CST integrity (Rapisarda et al., 1996; Escudero et al., 1998). Subjects with the best motor outcomes typica lly exhibit recruitment of ipsilesional sensory and motor cortices that is highly simila r to that of controls (Ward et al., 2003; Zemke et al., 2003; Cramer and Crafton, 2006). Strong acti vation of secondary motor areas such as PMd in the affected hemis phere was more prominent in patients with greater disability (Johansen-Berg et al., 2002; Ward et al., 2003). Chronic stroke patients with good behavioral outcomes demonstrate expanded and shifted sensorimotor fMRI activation patterns in the ipsilesional hemis phere (Cramer et al., 2000; Zemke et al., 2003). Greater motor improvements are associated with an enlargement in ipsilesional motor areas or a poster shift at the boundary between motor and sensory cortices (Rossini et al., 1998; Cramer and Bastings 2000). Stroke damage

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39 within the precentral gyrus is associated wit h a reorganization of finger and face fMRI activations after good behavioral outcome (C ramer and Crafton, 2006). Improvements in upper extremity motor performance after constraint-induced movement therapy are associated with increases in ipsilesi onal motor map area and MEP amplitudes (Wittenberg et al., 2003). Changes in map area and excitability are accompanied by shifts in map center of gravity indicating recruitment/reorganizati on of motor cortex adjacent to the original location (Liepert et al., 1998). In aphasics, greater return of language function is associated with a higher degree of left perilesional reorganization whereas contralesional reorganization was accompanied by poor language performance (Karbe et al., 1998; Cao et al., 1999; Rosen et al., 2000). Animal models of stroke have been developed to investigate the basis for motor cortex reorganization. Focal ischemic infa rcts within localized regions of squirrel monkey motor cortex result in a widespread reduction in the size of representations adjacent to the lesion (Nudo and Milliken, 1996). In primates motor retraining of the impaired limb restores the representations adjacent to the infarct and induces an expansion into regions that formerly disp layed other represent ations (Nudo et al., 1996a). Primates not given motor training do not show the same gains in motor function or map reorganization (Nudo et al., 1996b). Similarly, ischemic damage within the forelimb area of rodent motor cortex cause a loss of movement representations outside of the lesioned area within twent y-four hours of insult that can be restored with several weeks of rehabilitative training (Kleim et al., 2003; Monfils et al., 2005; MacDonald et al., 2007; Kleim and Jones, 2008). Map restoration is dependent upon skilled rehabilitation and does not occur in animals that experience unskilled rehabilitation

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40 involving extensive repetition of limb movem ent without acquisition of skill (Kleim et al., 2003; Kleim et al., 2004). Synaptic Plasticity Mediates Motor Map Reorganiz ation While the exact contribution of motor maps to movement in unclear, they appear to be surrogate markers for the capacity to prod uce and acquire skilled movement (Monfils et al., 2005). TMS studies in children bet ween 2 years of age and adolescence have observed a progressive decrease in the stim ulation threshold needed to produce MEPs (Eyre et al., 2001). Another study observed a decrease in TMS conduction times that was associated with the maturity of hand dexteri ty (Fietzek et al., 2000). Motor maps are typically absent in cats at postnatal day 45 but develop as the animals enter adulthood (Chakrabarty and Martin 2000). Ar mand and Kably (1993) observed that early maps contain proximal representations and then develop distal representations as well as progress from simple evoked respon ses to more complex responses involving multi-joint movements that co-associate with the em ergence of complex forelimb movements. The stimulation used in ICMS indirectly measures the synaptic connectivity and biasing of local cortical networks because its low levels of electrical current require the recruitment of many intracortical synapses onto corticospinal neurons. Understanding the cellular basis of reorganization will aid in the development of adjuvant therapies to enhance recovery. Stimulation experiments have revealed tw o important characteristics of motor cortex organization. First, the area of cortex devoted to a particular movement is related to the dexterity of the move ment. Second, the proportion of the cortex devoted to any one movement is not static and can be changed in response to a variety of manipulations including moto r training and/or damage. Neurons within motor cortex are

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41 aggregated such that small groups of cells appear to encode elementary movement representations in that nei ghboring cells have similar ou tput properties (Mountcastle, 1997). Further, these pools of neurons ar e interconnected by dense horizontal connections that can extend several millimet ers (Ghosh and Porter, 1988; Keller, 1993). Intracortical microstimulation (ICMS) evokes movement via direct (Stoney et al., 1968) and indirect (Jankowska et al., 1975) activation of pyramidal tract neurons. In fact, the majority of pyramidal tract neurons that driv e movement in response to stimulation are trans-synaptically activated (Cheney and Fe tz, 1985; Lemon et al., 1987), presumably through activation of horizontal afferents. Any alteration in mo tor map topography or loss of map area after damage must therefore involve c hanges in the pattern of intracortical connectivity through modifi cations in synaptic efficacy (Hess and Donoghue, 1994; Monfils et al., 2005). In su pport, synaptic potentiation that occurs in response to motor skill learning (Rioult-Pedo tti et al., 1998; Monfils and Teskey, 2004) is colocalized within regions of co rtex that exhibit motor map reorganization (Kleim et al., 1998). In addition, learning-dependent motor map plasticity is also co-localized with synaptogenesis (Kleim et al., 2002a; Kleim et al., 2004). Electric Stimulation of Mo tor Cortex Induces Synap tic Plasticity and Motor Map Reorganization Excitability in the central nervous system can also be altered by electrical stimulation (Blundon and Zakharenko, 2008). I ndeed, electrical stimulation can induce a phenomenon known as long-term potentiation (L TP). LTP is one of the cardinal examples of plasticity and fulfils many of th e criteria for a neural correlate of memory. The LTP phenomenon is a facilitation of c hemical transmission between neurons that results from coincident activity of preand post-synaptic cells. LTP experiments typically

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42 use high-frequency trains of electrical stimul ation in pre-synaptic cells that promote action potentials post-synaptically (Cooke and Bliss, 2006). Follo wing stimulation, enhanced postsynaptic responses are observ ed for hours in vitro (Bliss and GardnerMedwin, 1973). More recently, it was dem onstrated that LTPlike phenomena could be induced in vivo by repeated electrical condi tioning sessions (Racine, et al., 1995) or even one session (Trepel and Racine, 1998) over several days. Decreases in postsynaptic responses, termed long te rm depression (LTD) can be induced by electrical conditioning using low frequency stimulation in vitro (Dudek and Bear, 1992) and in vivo (Froc et al., 2000). Manipulations that induce changes in synaptic strength also induce map reorganization. For instance, cortical kindling that drives increases in cortical excitatory postsynaptic potentials al so increases motor map area (Teskey et al., 2002). Stimulation protocols that elicit LT P causes map expansion and synaptogenesis (Monfils et al., 2004) while protocols for long-term depression (LTD) induces map retraction and synaptic loss (Teskey et al., 2007). Jackson et al. (2006) used a neural implant to demonstrate that repeated conditi oning with a closed loop electronic device can alter the output of moto r cortex by creating stable connections between previously unconnected regions. The similarity in sensorim otor cortices response to both electrical stimulation and behavioral experience suggest el ectrical stimulation can be used to facilitate the encoding of information within this tissue. Electrical Stimulation of Motor Cortex Enhance Motor Improvement After Stroke There is a growing body of evidence that electrically stimulating th e motor cortex facilitates recovery of motor function after injury to the central nervous system (CNS). Stroke generally reduces activity in the ipsi lesional hemisphere (Desrosiers et al., 2006) while increasing activity in the contrale sional hemisphere (Cauraugh et al., 2000). The

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43 strategy of many noninvasive br ain stimulation therapies is to encourage activity in the ipsilesional hemisphere or discourage it in the contralesional hemisphere (Talelli and Rothwell, 2006). Transcranial magnetic brai n stimulation (TMS) works by passing a strong brief electrical current through an in sulated wire coil such that it generates a magnetic field that results in a secondary cu rrent within the brain (Bolognini et al., 2009). This secondary current is capable of transiently alte ring the membrane potential of neurons to suppress or activate cortical regions depending on the shape of the coil as well as the frequency and duration of st imulation (Pascual-Leone et al., 2002). Transcranial Direct Current St imulation (tDCS) causes weak polarizing direct current into cortex via an active electrode placed ov er the target area an d a reference electrode placed in the contralateral supraorbital ar ea or a non-cephalic region (Nitsche et al., 2007). Stimulation by tDCS results in sustained changes in neuronal membrane potentials with cathod al tDCS inducing hyperpolarization (inhibition) and anodal tDCS inducing depolarization (excitation) (Nitsche et al., 2003). Low frequency rTMS applied to the contralesional hemisphere in chr onic stroke patients reduces transcallosal inhibition from this hemisphere to the ipsilesional (Takeuchi et al., 2005) and increases excitability in the affected side (Fregni et al ., 2006). Intermittent theta burst stimulation with rTMS increases MEP amplitudes and transiently improves behavior (Talelli et al., 2007; Di Lazzaro et al., 2008). Continuous theta burst stimulation over the contralesional hemisphere increased excitabilit y in the affect motor cortex and resulted in motor improvements (Di Lazzaro et al., 2008). High frequency rTMS given ipsilesionally and paired with passive and acti ve motor therapy ca used greater motor improvements on a variety of scales compared to an rTMS sham group (Khedr et al.,

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44 2005). High frequency rTMS delivered ipsilesiona lly and paired with a finger motor task resulted in greater movement accuracy, less movement time and greater MEP amplitudes (Kim et al., 2006). Low-frequency rTMS deliver ed in the contralesional hemisphere, resulted in improved accelerati on and force of the affected hand (Takeuchi et al., 2008). Inhibiting the contralesional hemisphere with Low-frequency rTMS also results in other motor improvements (Fregni et al., 2006, Mansur et al., 2005). Transcranial direct cortical stimulation improves motor f unction in patients with chronic motor impairments when anodal current is delivered over lesioned motor cortex (Hummel and Cohen, 2005; Fregni et al., 2006). Similarly, cathodal transcranial direct current stimulation applied to contra lesional motor cortex induces behavioral improvements on various scales (Fregni et al., 2005; Fregni and Pascual-Leone, 2006). Clinical reports suggest that epi dural electric stimulation of motor cortex, originally used to reduce chronic pain after sub-cortical strokes, reduces hemiparetic impairments (Tsubokawa et al., 1993), motor weakness (K atayama et al., 2002), motor spasticity (Garcia-Larrea et al., 1999), action tremor (Nguyen et al., 1998) and dystonia (Franzini et al., 2003). The efficacy of CS/RT at enhanci ng motor recovery afte r stroke has been demonstrated in rats (Adkins-Muir and Jones 2003; Kleim et al., 2003; Teskey et al., 2003; Adkins et al., 2006) and monkeys (P lautz et al., 2003). Furthermore, the enhanced motor recovery is associated with increased cortical dendritic hypertrophy (Adkins-Muir and Jones, 2003) and synaptogenesis (Adkins et al., 2008) in comparison to animals in standard rehabilitation. The increased post-synaptic space is accompanied by an enlargement of the polysynaptic component of motor cortical evoked potentials (Teskey et al., 2003). Finally, CS/RT also induces a greater expansion of movement

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45 representations in rats (Kleim et al., 2003; Boychuk et al., 2009) and monkeys (Plautz et al., 2003). All of these data dem onstrate that CS/RT drives significantly greater motor recovery after stroke and that the functional gains are acco mpanied by an upregulation of the neuroplastic changes obser ved with standard rehabilitation. Translation of CS/RT into Clinical Practice Recently a phase III clinical trial of CS/RT failed to demonstrate a si gnificant effect of the treatment. The trial enrolled 146 patients (CS/RT= 91; RT=55) and the methods were the same as the phase II clinical trial where a significant CS /RT treatment effect was demonstrated (Huang et al., 2008; Levy et al., 2008). It is likely our lack of understanding of how to translate CS/RT fr om preclinical basic studies to human applications contributed to the lack of CS/RT effect observed in the phase III trial (Plow et al., 2009). Interestingly, some patients in the combined CS/RT group demonstrated robust motor improvements relative to the group receiving RT alone (Plow et al., 2009). One interpretation of this finding is t hat CS/RT procedure used was better suited for treating a subset of the stro ke patients. Identifying fact ors that can account for the differences between animal and human studies of CS/RT should support more effective CS/RT therapies in the future as well as contribute to our understanding of brain repair following stroke. In the present work rodent models of stroke we re used in an attempt to identify several of the import ant factors that impact the efficacy of CS/RT therapy. One important factor with CS/RT is the placement of stimulation on the cortex. This placement is dictated by the configuration of the CS/RT electrodes surface contacts. Animal studies of CS/RT have used stimulation that is distributed across the motor cortex. In contrast, hum an studies have used very foca l stimulation. Further, the focal stimulation in the clinical studies was localized to the largest fMRI activation zone

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46 during wrist/hand/finger movement (Plow et al ., 2009). In the present work a study was conducted that examined the effects of focal versus dist ributed cortical stimulation on both motor performance and motor map plastici ty. ICMS was used to identify movement representations at the begi nning and end of the study. In this study, the focal configuration applied st imulation to the largest set of wrist representations while the distributed configuration appl ied stimulation to non-wris t representations surrounding the large area of wrist repres entations located in the cent er of the motor map. In a second study, distributed forms of CS/RT were tested to examine the importance of the number of independent stimulati on sites, i.e., to compare the importance of the density of stimulation sites within a distributed configuration. Another important differenc e between animal and human studies of CS/RT is the amount of post injury motor training prior to the onset of CS/RT treatment. Human stroke patients have a minimum delay of four months between onset of stroke and enrollment into the clinical trial (Levy et al., 2008). While the amount of rehabilitative training given to these stroke patients is not reported it is expect ed that the majority received standard rehabilitative treatment in t he months following stro ke. In contrast, all of the documented animals studies of CS/RT have initiated the CS/RT-RT comparison without any preceding post injury motor traini ng. This discrepancy raises the issue of whether CS/RT is limited by prior rehabilitativ e experience. It is possible that early rehabilitative training results in persistent forms of neural plasticity that cannot be altered or hinder changes by subsequent CS/R T application. It is also possible early rehabilitative training produces a ceiling effect in the amount of motor improvements that can be induced by subseque nt CS/RT. Presently, a thir d study used a comparison

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47 between animals receiving CS/RT and RT alone was made after the animals had received post injury motor training to i dentify how the behavioral effects of CS/RT are affected by weeks of prior rehabilitative training. The subjects in animals and human studies of CS/RT also differ in the location of ischemic damage. Previous animal studies of CS/RT have exclusively modeled stroke by focal damage to motor cortex. In contrast human stroke patients with either cortical or capsular stroke damage were enrolled in CS/RT clinical trials (Brown et al., 2006; Huang et al., 2008; Levy et al., 2008). While it is clear that both cortical and capsular stroke often result in moto r impairments (Shelton and Reding 2001; Schiemanck et al., 2008) the types of neural reorganization that are relevant to improving motor function may be different (Plow et al., 2009). It is unclear whether CS/RT can enhance motor improvements after capsular ischemic damage by the same mechanisms as those suggested following ischemic damage to motor cort ex. In particular, it is not presently known whether CS/RTs enhanced reorganization of perilesional motor cortex can support greater motor improvements following i schemic damage to the internal capsule. Presently, a fourth study was performed to examine the behavioral effects of CS/RT following ischemic damage to either cortex or to the internal capsule in order to examine the efficacy of CS/RT after capsular stroke. Thesis Outline The goal of this dissertation is to further characterize the effectiveness of brain stimulation in enhancing motor improvements during motor rehabilitation after stroke injury. Specifically it examines how changes in the distribution of stimulation across the cortex, the effects of previous training ex perience and importance of locus of injury affect CS/RT. In the present experiments several rodent models are used to study the

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48 return of motor functions after stroke. Behav ioral changes in these models are assessed by performance on a skilled food-reaching task, and when possible, the physiology of the motor cortex is assessed using ICMS. Three primary questions will be addressed: First, does the configuration of the contacts on the CS/RT electrode affect its ability to drive motor improvements? Second, does prio r rehabilitative experi ence affect CS/RTs ability to drive motor improvements? Th ird, will CS/RT enhanc e motor improvements when the ischemic injury is located in the subcortical white matter? Collectively, the configuration of CS/RT cont acts, amount of prior rehabilit ative experience and location of the lesion represent import ant factors that need to be studied in order to facilitate the translation of CS/RT from animals studies to clinical practice with human stroke patients. The data from these studies will provide information regarding the situations that CS/RT will be most effective as well as contribute to t he understanding of brain repair following stroke.

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49 CHAPTER 2 DISTRIBUTED VERSUS FOCAL CORTICAL STIMULATION TO ENHANCE MOT OR FUNCTION AND MOTOR MAP PLASTICI TY AFTER EXPERIMENTAL ISCHEMIA Introduction Stroke remains the major source of adult disability and much of the reduction in quality of life can be attributed to motor im pairments. Of the 560 000 Americans that survive stroke each year, more than 80% will have acute motor impairments and most will receive some form of mo tor rehabilitation (Gresham et al., 1995; Rathore et al., 2002). However, the effectiveness of motor rehabilitation is highly variable (Duncan et al., 2000) and reflects our lack of understand ing of the neurobiological mechanisms underlying functional recovery. Improvements in motor function a fter stroke can be thought of as a relearning pr ocess whereby lost motor functions are reestablished through functional restoration and/or compen sation within spared brain regions (Cramer et al., 1997; Cramer and Bastings, 2000; Nudo, 2007). Animal models of stroke show that focal ischemic damage to motor cortex re sults in a loss of microstimulation-evoked motor representations (Nudo and Milliken, 1996) and a decrease in synapses (Hasbani et al., 2001; Zhang et al., 2005; Mu rphy et al., 2008) within resid ual cortical areas. In the absence of rehabilitative training, the majori ty of lost motor representations do not reappear and the topography of the remaining map is not significantly altered (Nudo and Milliken 1996; Friel et al., 2000). With moto r rehabilitation, however, improvements in motor performance are accompanied by motor map expansion and reorganization (Nudo et al., 1996b; Nudo, 2006) that is likely m ediated by synaptic plasticity (Kleim et a., 2002a; Kleim et al., 2004). Thus, adjuvant t herapies that promote cortical/synaptic plasticity may enhance improvements in motor performance after stroke. Indeed, several plasticity-promoting agents have been paired with motor rehabilitation to

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50 enhance motor function after stroke. Improved motor performance has been demonstrated with pharmacologica l manipulations such as amphetamine and nicotine that are associated with synaptic reorgani zation (Stroemer et al., 1998; Adkins and Jones, 2005; Ramic et al., 2006; Papadopoulo s et al., 2009). Additionally, enhancing intracellular signaling pathways known to pr omote synaptic plasticity augments skilled reaching ability in a rodent model of stroke that is associated with expansion of forelimb movement representations in motor cortex (Macdonald et al., 2007). In addition to pharmacological agents, electrical stimulation has been used to treat motor impairments associat ed with various neurological disorders. Initial clinical studies found that epidural motor cortex st imulation, intended to reduce chronic pain after sub-cortical strokes, reduced hemi paretic impairments (Tsubokawa et al., 1993), motor weakness (Katayama et al., 2002), motor s pasticity (Garca -La rrea et al., 1999), action tremor (Nguyen et al., 1998), and dy stonia (Franzini et al., 2003). Subsequent animal models of cortical stimulation combined with rehabilitation therapy after stroke demonstrate enhanced motor outcomes relative to rehabilitative training alone (AdkinsMuir and Jones, 2003; Adkins et al., 2006). T he improved motor performance is also associated with an expansion of microstimulation-evoked motor r epresentations within residual cortical areas of r odents (Kleim et al., 2003) and pr imates (Plautz et al., 2003) that is accompanied by increased synaps e density (Adkins et al., 2008) and enhanced synaptic potentials (Teskey et al., 2003) Together these results demonstrate the viability of CS/RT for enhancing motor improvem ent and concomitant cortical plasticity after stroke. However, results from a phase three clinical trial of CS/RT in stroke patients failed to show any significant effect of the treatment. This may in part be due to

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51 our lack of understanding as to how CS/RT should be translated from animal to human applications (Plow et al., 2009). One key param eter in CS/RT is the distribution of stimulation across the cortex. Clinically, CS/RT is delivered to a focal region of motor cortex whereas animal studies have used mu ch more distributed stimulation. This difference may account for the efficacy of the treatment in animal studies but lack thereof in human studies. Here we examine the effects of focal versus distributed cortical stimulation on both motor perform ance and motor map plasticity in an animal model of cerebral ischemia. Methods Subjects Forty-five adult male Long-Evans hooded rats (350-420g) were housed (1 animal/cage) in standard laboratory cages. Anim als were kept on a 12:12 hour light dark cycle throughout the experiment. All exper im entation was conduct ed during the light cycle. Rats were maintained on Lab Diet 5001 (PMI Feeds, St. Louis, MO) and water ad libitum, and were handled and cared for in ac cordance with the National Institutes Health Guide for the Care and Use of Labor atory Animals and with the approval of the University of Floridas Institutional Animal Care and Use Committee (IACUC). Reach Training Over the course of several days, all animals were placed on a restricted diet until they measured 90% of their original body we ight. A brief period of pretraining was then given to familiarize th e rats with the reachi ng task. Pretraining involved placing them into test cages (10 X 18 X 10 cm) with floor s constructed of 2 mm bars, 9 mm apart edge to edge. A 4 cm wide and 5 cm deep tray filled with food pellets (45 mg; Bioserv) was mounted on the front of t he cage. The rats were required to reach outside the cage

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52 and retrieve pellets from the tray. Rats were permitted to use either limb and the preferred limb was noted for each animal. All ra ts remained in pretraining until they had successfully retrieved 10 pellets (approximatel y 1 hour/day for 2 days). After pretraining, the rats were placed into a Plexiglas cage (11 cm X 40 cm X 40 cm) with a 1 cm slot located at the front of the cage. Animals were trained for 20 minutes each day to reach with their preferred limb through the slot and retrieve food pellets from a table outside the cage (Whishaw and Pellis, 1990). Each session was videotaped and later used to assess reaching performance. A successful r each was scored when the animal grasped the food pellet, brought it into the cage and to its mouth without dropping the pellet. The percentage of successful reaches [(# successfu l retrievals/the total # of reaches) x 100] was then calculated. All training sessions were video taped and used to measure reaching accuracy. Animals were trained fo r approximately two weeks on this task to establish a baseline measure of motor performance. Baseline was defined as the average accuracy across the 3 final days of trai ning. Animals failing to achieve a mean reaching accuracy of 40% across 3 consec utive days were not used in the study. Animals were sorted by their prelesion r eaching performance to create 5 groups with comparable baseline levels of reaching accuracy: nontrained (NT) (n=11), E1 configuration cortical stimul ation with rehabilitative trai ning (E1-CS/RT) (n=10), E2 configuration cortical stimul ation with rehabilitative trai ning (E2-CS/RT) (n=9), E3 configuration cortical stimulation with rehabilitative tr aining (E3-CS/RT) (n=7), and rehabilitative training alone (RT) (n=8). All conditions were subjecte d to the same motor mapping and infarction procedures described below.

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53 Electrophysiological Mapping Within two days of baseline training, standard ICMS techniques were used to generate detailed maps of forelimb regions of the motor cortex c ontralateral to the trained forelimb (Kleim et al., 1998; Remple et al., 2001). Prior to surgery animals were anesthetized with ketamine hydrochloride (70 mg/kg i.p.) and xylazine (5 mg/kg i.p.). Animals received low levels of isofluorane (0.15%) and supplemental doses of ketamine (20 mg/kg i.p.) as needed. Under sterile c onditions, a craniotomy was performed over the motor cortex contralateral to the trai ned paw of each animal. To prevent edema, a small puncture was made in the cisterna magna prior to removing the skull and dura. The exposed cortex was then covered in warm saline (37 degrees celsius). A digital image of the cortical surface was taken and a 375 mm grid was superimposed onto the image. A glass microelectrode (controlled by a hydraulic microdrive) was used to make systematic penetrations across the cortex using the cortical surface image and grid as a guide. At each penetration site, the electrode was lowered to approximately 1550 mm (corresponding to cortical layer V). Stimulat ion consisted of thirteen, 200 ms cathodal pulses delivered at 350 Hz from an electrically isolated stim ulation circuit. Animals were maintained in a prone position with the lim b consistently supported. Sites where no movement was detected at 60 mA were recorded as unresponsive. Forelimb movements were classified as either distal (w rist/digit) or proximal (elbow/shoulder) and representational maps were generated from the pattern of electrode penetrations. The caudal forelimb area (CFA) was defined by a medial boundary of vibrissa representations, a lateral and caudal boundary of non-responsive sites and a rostral boundary of head and neck representations (Kleim et al., 1998; Remple et al., 2001;

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54 Kleim et al., 2002a). An image analysis program (CANVAS v. 3.5) was used to calculate the areal extent of the CFA. Focal Infarction Following ICMS, focal ischemic infarcts were created within the CFA of motor cortex via bipolar electrocauterization of t he surface vasculature (Figure 2-1) (Nudo et al., 1996b; Kleim et al., 2003). The infarct targeted primarily the distal forelimb representations but in some cases included sm all regions of proximal representations. The coagulated vessels included fine arterial and venous capillaries as well as larger vessels but specifically avoided any bypassing arteries supplying other cortical areas. Coagulation was continued until all vessels within the targeted area were no longer visible and the tissue appeared white. Cortical Electrode Implantation Directly after the infarction, each animal was implanted with one of three types of electrodes that varied by the configuration of their surfac e contacts. Nine-pin electrode carriages (Plastics One Inc., Roanoke, VA) were implanted epidurally over top sensorimotor cortex in the hemisphere opposite each animals preferred paw. The surface electrode was placed directly over the entire exposed cortex between 1 mm posterior to 5 mm anterior to bregma and 0. 5 mm to 5.5 mm lateral to midline. The surface electrode was placed directly over the entire exposed co rtex including the devascularized region, and remaining forelimb representation area of motor cortex. A return lead was fixed to the skull in a pos ition posterior to Lambda and the craniotomy filled with gel foam. Both the electrode and gel foam were covered in non-exothermic PolyWave dental acrylic and cured with a brie f pulse (50 seconds) of ultraviolet light. The electrode was then fixed to skull scr ews with standard dental acrylic and the

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55 animals were given 4cc of warm ringers solu tion (s.c.) and metacam (0.10 mg/kg; s.c.). The electrode contacts were all 0.60 mm in diameter and placed in one of three configurations. E1 electrodes had four c ontacts positioned 2 mm equidistant from one another. E2 had the same four contacts but clustered in the cent er of the pedestal and E3 electrodes included a single co ntact in the center (Figure 2-2). Determining Motor Thresholds After surgery all animals were returned to their home cage for ten days where they were supervised for health concerns but othe rwise left to recover. Animals were given full access to food until the last day where food restricti on regimen (see above) was reimplemented. After the t en days, animals with implants had their individual motor thresholds (MTs) determined and then all anima ls (except those in the NT condition) were started in a motor rehabilitation paradigm. MTs were defined for each animal prior to training on each day as the minimum current to cause an involuntary motor response. Cortical stimulation was administered with a preclinical stimulation system (Northstar Neuroscience, Seattle WA). Briefly, each co rtical electrode was connected to a remote stimulator suspended above the training cage where information was sent wirelessly to a base-station connected to a Windows-based per sonal computer (Del l, Roundrock TX). Current was delivered through the remote stimulator and t he parameters were controlled by application software installe d on the computer. Stimulation for these thresholds consisted of 3 second trains of 1 millisecond 50 Hz monopolar cathodal pulses. Current was gradually increased by 5% increments until a movement of the contralateral forelimb could be clearly detected. MTs were tested in all animals in all three CS/RT conditions. Movements could only be evoked for animals in the E1 (n =10) and E2 (n=9) conditions. Animals in the E1 showed large-scale movement of the head,

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56 neck, forelimb and hindlimb in response to stimulation. Animals in the E2 condition showed very focal forelimb movements. This finding is consistent with the location of the electrode contacts in each of these c onditions because the E1 electrode contacts were spaced throughout the motor map while t he E2 contacts were localized to forelimb representations. No movements could be elicited in the E3 (n =7) animals at the maximum current (12 mA) output level. As a re sult, the animals from E3 remained in the study combined with the RT controls. Cortical Stimulation and Re habilitation Training (CS/RT) The motor rehabilitation paradigm cons isted of daily fifteen-minute training sessions on the skilled reaching task for 12 cons ecutive days. A probe trial was given at the end of training where all animals performed a final day of reaching but the stimulation condition received training wi thout stimulation. During these training sessions the CS/RT animals (E1 and E2) were treated with electric stimulation via the preclinical stimulation syst em. Monopolar cathodal stim ulation was administered continuous with a frequency of 50 Hz and a curr ent intensity of 50% of the subjects movement threshold. Each pulse was biphasic, charged balanced and asymmetric consisting of a square phase lasting 100 + 10 microseconds and a decaying exponential phase lasting ~19900+ 10 microseconds. Assessing Residual Motor Map Area and Topography Within three days of the final rehabilitation training sess ion, ICMS was again used to generate a second map of the CFA contralateral to the trained forelimb. Prior to surgery animals were anesthetized with ke tamine hydrochloride (70 mg/kg i.p.) and xylazine (5 mg/kg i.p.), receiving xylazine (0.02 mg/kg i.m.) and ketamine (20 mg/kg i.p.) as needed. Further, animals were plac ed on isofluorane (0.15%, 1.5% O2) when

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57 needed. The electrode was removed and the cortical surface covered in body temperature silicon oil. M apping procedures were identical to those used in the initial mapping (see above). Infarct area was calcul ated by outlining the cauterized area on the digital image of the cortical surface obtained from the first map. The area was clearly visible as a bleached or whitened area due to the loss of blood flow. Residual CFA prior to rehabilitation (Resid-PreRehab) was defined as the total area of CFA minus the area of infarct. Residual CFA post-rehabilitation (Resid-PostRehab) was the total area of CFA after rehabilitation. The percentage change in residual CFA was then calculated as [(Res-PostRehab) (Res-PreR ehab)/100]. In addition, the percentage of residual CFA that was occupied by distal movement representations was calculated both pre and post rehabilitation. Distal movement representations were analyzed because wrist/digit movement representatio ns expand during both normal skill learning and recovery of skill after cortical injury. Histology and L esion Verification Following the rehabilitation phas e the ani mals were given an overdose of pentobarbital and then transcardially perfused with 0.1 M sodium phosphate buffer followed by 4% paraformaldehyde solution in the same buffer. Brains were then extracted and post fixed in 4% paraformaldeh yde solution in 0.1 M sodium phosphate buffer. Serial 50 m coronal sections were then taken using a microtome. Ten sections spaced 600 m apart and spanning approximatel y 2.7 mm anterior and 3.3 mm posterior to bregma were sampled for lesion verification. The same number of sections was analyzed for each animal. The sampled sect ions were stained with Toluidine blue (a Nissl stain) and digitally scanned (E spon Perfection V500 Photo Scanner, Long Beach, CA) for lesion verification. The area of spared tissue, characterized by

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58 consistent Nissl staining, was traced using Image J software (Abramoff et al., 2004; Rasband, 2009) and cortical volumes were estimated with the Cavalieris unbiased estimator method using the formula: volume di 1 n yi ( t ) ymax Where yi is the cross sectional area of the ith section through the morphometric region, d is the distance between sections (600 m) and n is the total number of sections (12). ymax is maximum value for the area of one section and t is the section thickness (50 m) and their product is subtracted from the basic question as a correction for the overprojection (G undersen 1986; Gundersen and Jensen, 1987; Mayhew 1992). Estimated volumes for cortical and subcorti cal tissue were analyzed as the percent affected to unaffected side (volume lesioned hemisphere/volume non lesioned hemisphere*100) in order to account for individual differences in brain size. Results Reaching Accuracy A repeated measures ANO VA with CONDITION as a be tween subject factor and TIME as a within subj ect factor revealed a significant CONDITION x TIME interaction [F(26,403)= 15.712; p<0.01] on reaching accu racy (Figure 2-3). Subsequent multiple comparisons (Fishers PLSD; p<0.01) revealed that all conditions had significantly lower reaching accuracies on day 1 of rehabilitation in comparison with pre-stroke levels. Further comparisons (Fishers PLSD; p<0.05) showed that while all animals receiving rehabilitation showed significant increases in reaching accuracy during the 12 days of rehabilitation, the animals receiving cortic al stimulation (E1-CS/RT and E2-CS/RT) had significantly higher reaching accuracies that RT Controls on days 5 through 8. Animals

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59 in the E1-CS/RT condition performed significantly better than the E2 and RT Controls on days 3-4 and day 6 through the remainder of the study. The RT Control condition showed a progressive increase in reaching accuracy during the first 4 days of training that was followed by a significant decrease in accuracy on days 5-7 (Fishers PLSD; p<0.05). No significant improvements were seen in the stimulated conditions after 6 days of rehabilitation. NT control animals had a significantly lower final reaching accuracy than all groups with rehabilitati on even though a Paired T-Test revealed a small but significant increase in motor performance in the NT controls that was indicative of spontaneous recovery [T(10) =5 .006; p<0.01]. This demonstrates that while rehabilitation induced improvements in reaching accuracy the combination of rehabilitation and cortical stimulation wit h a distributed contact arrangement (E1) resulted in even greater increases in relative reaching accuracy following focal cortical ischemia. Movement Thresholds A repeated measures ANO VA with CONDITION and TIME showed a significant CONDITION x TIME interaction [F(2,34) = 4.115; p<0.05] on mean movement threshold (Figure 2-4). The mean MT required to elicit movement was signific antly higher in the E1 electrodes at all time points (FLPSD; p< 0.05). Animals in both E1 and E2 conditions showed a progressive decrease in MTs as training continued (Figure 3). There was a trend for the E1 condit ion to show a larger decrease in threshold than the E2 condition [F(1,17) = 3.736; p<0.07]. Residual Motor Maps CFA motor maps prior to infarction were 4.36 mm2 ( 0.40 mm2) in RT controls, 4.30 mm2 ( 0.74 mm2) in E1-CS/RT, 4.14 mm2 ( 0.38 mm2) in E2-CS/RT, and 4.25

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60 mm2 ( 0.45 mm2) in NT controls. The mean infarction area was 1.08 mm2 ( 0.18 mm2) in RT controls, 1.20 mm2 ( 0.22 mm2) in E1-CS/RT, 1.12 mm2 ( 0.20 mm2) in E2CS/RT, and 1.10 mm2 ( 0.19 mm2) in NT controls. A one-way ANOVA revealed a significant main effect of CONDITION on the area of residual CFA after rehabilitation [F(3,41) = 120.170; p<0.01]. Su bsequent multiple comparisons (Fishers PLSD; p<0.01) showed that the amount of residual CFA of all rehabilitation gr oups was greater than that of NT Controls (Figure 2-5A). The amount of residual CFA for both CS/RT groups was greater than that of eit her RT or NT controls (Fi gure 2-4A). A second one-way ANOVA revealed no significant main affect of CONDITION on the area of residual proximal representations afte r rehabilitation [F(3,41) = 1.402; p=0.256] (Figure 2-5B). A third one-way ANOVA revealed a significant main effect of CONDITION on the area of residual distal representations after rehabilitation [F(3,41) = 97.243; p<0.01]. Subsequent multiple comparisons (Fishers PLSD; p<0.01) showed that the amounts of residual distal representations for all rehabi litation groups was great er than that of NT Controls (Figure 2-5C). The amount of residual distal re presentations for both CS/RT groups was greater than that of the RT and NT contro ls. Furthermore, a one-way ANOVA revealed a significant main effect of CONDITION on the percent of distal representations after rehabilit ation [F(3,41) = 25. 732; p<0.01] (Figure 2-6). Subsequent multiple comparisons (Fishers PLSD; p< 0.01) showed that the percent distal representations in all rehabi litation groups was significantly greater than the NT Controls. The E1-CS/RT had a significant ly greater amount of residual distal representations than the RT controls (Fisher s PLSD; p<0.01). In addition, the RT and E2-CS/RT groups did not significantly diffe r in the percent of residual distal

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61 representations (Fishers PLSD; p<0.01). Fina lly, a significant positive correlation was found between the increase in the total CFA area and the increase in post rehabilitation reaching accuracy (Mean final 3 rehabilitation days Mean first 3 rehabilitation days) during rehabilitation [r=0.7838, p<0.0001] (Figur e 2-7). A significant positive correlation was found between the increase in the area of distal represent ations and the increase in post rehabilitation reaching accuracy (Mean final 3 rehabilitation days Mean first 3 rehabilitation days) during r ehabilitation [r=0.7619, p<0.0001]. Estimation of Remaining Tissue A one-way ANOVA revealed no s ignificant main effect of CONDITION on the estimates of remaining cortical tissue [F(4 ,40) = 0.3149; p>0.05]. Subsequent multiple comparisons (Fishers PLSD; p<0.05) showed t hat the amount of resi dual cortical tissue was similar for all conditions (Table 2-1). A one-way ANOVA revealed no significant main effect of CONDITION on the estimates of remaining subcortical tissue [F(4,40) = 1.3302; p>0.05]. Subsequent multiple compar isons (Fishers PLSD; p>0.05) showed that the amount of residual subcortical tissue was similar for all conditions. Discussion While the mechanisms underlying motor im provement after stro ke are not fully understood, there is a gr owing body of evidence to s upport the role of functional restoration/compensation withi n residual neural tissue. Further, the use of adjuvant therapies that promote such reorganizati on by augmenting endogenous neural plasticity may serve to increase functional outcome over and above that observed with standard rehabilitation. The present study provides addi tional evidence that cortical stimulation in combination with rehabilitation enhances motor function and motor cortex reorganization in an animal model of cortical ischemia. In addition, the results

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62 demonstrate the importance of the distribution of stimulation across the cortex for augmenting motor performance. In this study all groups that received rehabilitative motor training exhibited great er motor improvements than non-trained animals. Further, all animals receiving motor rehabilitation sh owed significant expansion/reorganization of movement representations in residual motor cortex relative to the NT group. These findings suggest that both behavioral improv ements and physiological remodeling after injury are experience dependent processes and rehabilitative motor training serves as a driving factor. CS/RT increased both the motor improvement and map reorganization observed with rehabilitation. Finally, the spat ial configuration of the contacts on the cortical electrode surface served as an import ant factor for CS/RT delivery as only the distributed configuration was associated with enhanced motor improvement over and above RT alone. The mechanisms of CS/RTs enhanced motor recovery are not clearly understood but likely involve enh ancing synaptic strength within residual cortical circuits (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Plautz et al., 2003; Teskey et al., 2003; Adkins et al., 2006; Adkins et al., 2008). The present study finds reorganization of cortical microstimulation-evoked motor representations withi n the affected hemisphere that occurs in parallel with behavioral recovery. Previous work using CS/RT substantiates this association between reco very and motor map plasticity in rodents (Kleim et al., 2003) and primates (Plaut z et al., 2003). Learning-dependent motor map plasticity is accompanied with increases in synapse number (Kleim et al., 2002a; Kleim et al., 2004) and these same changes likely support CS/RT motor improvements as increases in synapse density (Adkins et al ., 2008) and synaptic responses (Teskey et

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63 al., 2003) have been observed in CS/RT animals rela tive to a RT condition. Studies with CS/RT have observed changes in the MTs used to calibrate the amount of current administered for CS/RT. MTs typically decrease across days of rehabilitation, and CS/RT conditions that display enhanced motor recovery demonstrate the greatest reductions in MTs (Kleim et al., 2003; Te skey et al., 2003). These reductions in MT suggest changes in cortical excitability t hat could be mediated by increased synaptic input onto corticospinal neurons (Monfils et al., 2005). In the present study, MTs decreased across days of rehabilitation and t here was trend for larger threshold reductions for the E1-CS/RT condition rela tive to the E2-CS/RT in a manner that paralleled E1-CS/RTs enhanced motor impr ovements relative to E2-CS/RT. This suggests that CS/RT is associated with an incr ease in cortical excitability that may be mediated by increased in synapse number or strength (Adkins et al., 2008). Although animals in both CS/RT conditi ons showed larger residual motor maps than RT or NT controls after training, the E2-CS/RT animals exhibited motor improvements that were markedly less than the E1-CS/RT group and similar to those observed with the RT control group. This dissociation between map restoration and behavioral motor recovery may suggest that map restoration may be necessary but not sufficient to support motor rehabilitation, at l east in the context of CS/RT therapy. This finding has been reported previously in rodent studies where el ectric (Kleim et al., 2003) or pharmacological stimulation (Macdonald et al., 2007) induces motor map changes without robustly impacting behavior. These findings indicate that there is not a linear relationship between injured brain plasticity and final motor recovery. Map plasticity

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64 appears necessary as all an imals that demonstrated enhanced motor recovery also exhibited significantly greater mo tor maps within residual tissue. The results also demonstrate that distribut ed cortical stimulation is more effective at enhancing motor performance than focal stimulation even when contact area was held constant. There are several possible expl anations for this finding. First, the focal stimulation contacts may result in overlapping areas of cortical stimulation. That is, two electrodes may be stimulating the same cortic al area resulting in a net decrease in the amount of cortex that is being stimulated as compared to the distributed contact configuration. Second, the lo cation of cortical stimulatio n due to contact configuration also differed between the two electrode configurations. The focal arrangement tended to stimulate within the forelimb motor maps wh ile the distributed stimulated both forelimb and nonforelimb (whisker, neck etc) areas. This was evident when determining MTs, focal stimulation only evoked forelimb mo vements whereas distributed evoked a wide range of forelimb, whisker and neck movements. Stimulation of areas outside of the forelimb representations during rehabilitation may hav e promoted an expansion of forelimb representations into non-forelimb ar eas. The distributed stimulation may have further facilitated timing-depende nt recruitment of resid ual cortex into forelimb representations (Jackson et al., 2006). That is, having stimulation within head and neck or vibrissa areas in conjunction with forelimb movements produced during forelimb training may have recruited those areas in to the forelimb motor map. The focal electrode configuration would ha ve limited this possibility. The present findings further support t he viability of CS/RT for enhancing motor function after stroke. The results also dem onstrate the importance of stimulation

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65 distribution in CS/RT. Future clinical trials of CS/RT must consider the importance of stimulating wide areas of cortex to promote functi onal reorganization and motor recovery.

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66 Figure 2-1. Representative Ni ssl stained coronal section fr om the MCAo lesion. The medial-lateral extent of brain damage at 24 days following ischemic insult by electrocoagulation of the surface vasculature withi n sensorimotor cortex is shown. Figure 2-2. The three different electrode confi gurations examined. A ll contacts were 0.6 mm in diameter. E1 consisted of 4 contacts each in the corner of a 2mm x 2mm square. E2 clustered all four contac ts into the center of the pedestal and E3 had a single contact in the center.

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67 Figure 2-3. Reaching performance prestr oke and during rehabilitation. Mean (SD) percent reach accuracy both prestroke and across 12 days of rehabilitative training on the skilled reaching task. One probe session (P) was given at the end of training without stim ulation. All groups receiv ing rehabilitation (E1, E2 and RT) demonstrated greater motor im provement than the NT animals. The RT Control condition showed a transient decrease in accuracy on days 6-8. Both CS/RT conditions (E1 and E2) showed greater reaching accuracies than RT Controls on days 5-8. Animals in t he E1-CS/RT condition had significantly higher reaching accuracies than all ot her conditions on days 3-4 and day 6 through the remainder of t he study (Fishers PLSD; p<0.05; indicated by the symbol ).

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68 Figure 2-4. Mean (SD) movement thresholds for animals in the E1 and E2 conditions. Both E1 and E2 conditions showed a progressive decrease in threshold as training continued. The mean threshold required to elicit movement was significantly higher in the E1 animals at all time points (Fishers PLSD; p<0.05; indicated by the symbol ). There was a trend for the E1 condition to show a larger decrease in threshold than the E2 condition [F(1,17) = 3.736; p<0.07]

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69 Figure 2-5. Changes in the am ount of movement representat ions during rehabilitation. A) Mean (SD) size of the residual CF A after rehabilitative training. CS/RT animals (E1 and E2) had a larger area of residual CFA than both the RT and NT controls (Fishers PLSD; p<0.05; indicated by the symbol ). B) Mean (SD) size of the residual proximal r epresentations after rehabilitative training. C) Mean (SD) size of the residual dist al representations after rehabilitative training. All rehabilitation groups (RT, E1 and E2) possessed significantly greater areas of distal motor repres entations than NT controls (Fishers PLSD; p<0.05; indicated by the symbol ).

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70 Figure 2-6. Changes in the proportion of movement representations during rehabilitation. Area of dist al and proximal representations given as a percent of total residual CFA before and after rehabilitation. The percent distal representations in all rehabilitation groups (RT, E1 and E2) were significantly greater than in NT Controls. The E1-C S/RT group had a significantly larger percent of residual distal representati ons than the RT controls. (Fishers PLSD; p<0.01). The RT and E2-CS/RT gr oups did not significantly differ in the percent of residual distal representation.

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71 Figure 2-7. Relationship between behavior and motor map changes. A) A significant positive correlation was found between the increase in the total CFA area and the increase in post rehabilitation reaching accuracy (Mean final 3 rehabilitation days Mean first 3 rehabi litation days) duri ng rehabilitation [r=0.7838, p<0.0001]. B) A significant positive correlation was found between the increase in the area of distal r epresentations and the increase in post rehabilitation reaching accuracy (Mean fi nal 3 rehabilitation days Mean first 3 rehabilitation days) during reha bilitation [r=0.7619, p<0.0001].

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72 Table 2-1. Estimate of spared cortical and subcortical tissue from experiment 1. Estimated volumes for cortical and s ubcortical tissue were analyzed as the percent affected to unaffected side (vol ume lesioned hemisphere/volume non lesioned hemisphere*100) in order to a ccount for individual differences in brain size. Data is represented as mean percent SEM. Condition Sample Size Estimates of Spared Tissue Volume Cortical (% Contralesional ) Subcortical (% Contralesional) E1-CS/RT 10 96 99 RT 8 98 100 E2-CS/RT 9 97 97 E3-CS/RT 7 96 99 NT 11 97 98

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73 CHAPTER 3 CORTICAL STIMULATION PLUS REHABI LITATIVE TRAINING ENHANCES MOTOR FUNCTION INDEP ENDENT OF THE NUMBER OF STIMULATING CONTACTS AFTER EXPERIMENTAL ISCHEMIA Introduction Stroke is a major contributor to adult di sabilit y and limits the qua lity of life of its survivors. It is estimated that more t han 500 000 Americans survive stroke each year with at least 70% of these survivors exhibi ting motor deficits in the upper extremities (Roth et al., 1998; Luke et al., 2004; Urton, et al., 2007). While stroke survivors often receive intensive rehabilitative practice or training, complaints of residual motor deficits are found in over half of this population (Urton, et al., 2007). In addition to being limited, motor outcomes following rehabilitation are highly variable (Duncan et al., 2000; Pedersen et al., 1998) thus reflecting our lack of understanding of the neurobiological mechanisms underlying functional recovery. Behavioral improvements following stroke are believed to be supported by the restorat ion and/or recruitment of spared brain regions to perform lost functions. Animal models have demonstrated that stroke is associated alterations in neural connectivity that is demonstrated by reduced dendritic arbor (Zhang et a., 2005) and synaptic atrophy (Hasbani et al., 2001; Zhang et al., 2005; Murphy et al., 2008). The disruption in neural connectivity is further demonstrated by a persistent loss of microstimulation-evoked movement representations within motor cortex (Nudo and Milliken, 1996; Friel et al ., 2000). Although some motor function may be restored spontaneously after stroke (Nakay ama et al., 1994; Cr amer, 2008), there is considerable evidence that mo tor training can drive improv ements in motor performance and changes in neural circuitry within moto r brain regions. In non-human primates, appropriate motor rehabilitative training induc es motor improvements that are paralleled

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74 by an expansion/reemergence of microstimulation evoked movement representations that were previously lost by the ischemic damage (Nudo et al., 1996a; Kleim et al., 2003). Animals not given motor training do not s how the same gains in motor function or map reorganization (Nudo et al ., 1996b). Similarly, map re storation in rodents is dependent upon skilled rehabilitation and does not occur in animals that experience unskilled rehabilitation involving extensive repetition of simple limb movement (Kleim et al., 2003; Kleim et al., 2004). The func tional reorganization of movement representations following stroke within motor cortex is likely mediated by a reinstitution and/reorganization of cortical circuitry in volving synaptogenesis (Stroemer et al., 1995; Buonomano and Merzenich, 1998; Brown and Murphy, 2007). Therefore, adjuvant therapies that promote synaptic plasti city within motor cortex may enhance rehabilitation-dependent improvements in motor performance after stroke. Indeed, greater behavioral gains have been observed wit h rehabilitative training in animals receiving pharmacological interventions known to stimulate synaptic plasticity such as the administration of amphet amine (Stroemer et al., 1998; Adkins and Jones, 2005; Ramic et al., 2006; Papadopoulos et al., 2009) or nicotine (Gonzalez et al., 2005; Gonzalez et al., 2006). Pharmacological st imulation of the intracellular signaling pathways known to promote synaptic plasti city increases animals skilled reaching ability and the size of forelimb movement re presentations following cortical ischemia (MacDonald et al., 2007). Electrical stimulation can increase or decrease synaptic efficacy at a variety of sites within the nervous system (Blu ndon and Zakharenko 2008) including sensorimotor cortex (Trepel and Racine, 1998; Froc et al ., 2000; Teskey et al., 2002;

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75 Monfils et al., 2004). These changes in synap tic efficacy may support the encoding of behavioral experience (Martin and Morris, 2002). For example, synapses in sensorimotor cortex are potentiated during skilled motor learning (Rioult-Pedotti et al., 1998; Monfils and Teskey, 2004). Skilled motor tr aining also results in a reorganization of motor representations as representations for movements involved in the motor task expand their territories at the expense of others. The reorganizat ion of motor cortex occurs later in skilled training after initial improvements in skill behavior (Kleim et al., 2004) and is temporally paralleled by in creased synaptogenesis (Kleim et al., 1996; Kleim et al., 2002a; Kleim et al., 2004). Electric al stimulation protocols that can induce a form of long-term potentiation in awake behavi ng animals expands their motor maps in the absence of skilled motor learning (Monfils et al., 2004). Similarly, kindling-type electrical stimulation used to study of epileps y results in large expansions of movement representations within motor cortex (van Ro oyen et al., 2006). The output of the motor cortex can also be altered by facilitat ing the connection of previously unconnected motor regions by conditioning with an stimul ating neural implant (Jackson et al., 2006). The similarities between synaptic changes d ue to electric stimulation and skilled motor learning have raised the possibility that el ectric stimulation can facilitate behavioral encoding in the nervous system. There is now evidence from animal models of stroke that CS/RT enhances restoration of motor fu nction relative to the training alone and induces many neural plastic changes with brain tissue. These studies have assessed post injury motor performance using tasks such as single pellet retrieval (Kleim et al., 2003; Adkins et al., 2006; Adkins et al., 2008), pasta matrix (Te skey et al., 2003), Montoya staircase (Adkins-Muir and Jones 2003), and pellet retrieval from a 5 well

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76 apparatus in primates (Plautz, et al., 2003). Combined CS/RT therapy has resulted in a greater return of motor performance in a ll of these tasks following focal ischemic damage to motor cortex. CS/RT has also been associated with an expansion and/or reorganization of movement representations within motor cort ices of rodents (Kleim et al., 2003) and primates (Plautz et al., 2003). In addition, CS/RT results in an increased number of synapses (Adkins et al., 2008) as well as enhanced synaptic responses (Teskey et al., 2003). Recently, a phase three clinical trial of CS/RT in stroke patients failed to show any significant effect of the treatment. This may in part be due to clear methodological differences between the preclinical animal studies and the human c linical applications (Plow et al., 2009). Specifically, CS/RT is delivered to a much more focal region of motor cortex within human stroke patients th an in animal models. A recent animal study demonstrated that CS/RT with contacts dist ributed across motor cortex significantly enhanced behavioral improvements relative to CS/RT that had stimulation focal to one region (Boychuk et al., 2009). While this sugges ts that CS/RT is more efficacious when stimulation is delivered across motor co rtex the significance of the number of independent contact sites ac ross the cortex is unknown. Here, the importance of contact arrangement was assessed by testing several types of CS/RT that differed in their number of independent stimulation sites in a rodent model of middle cerebral artery (MCA) stroke. Methods Subjects Fifty-six adult male Long-Evans hooded rats (350-420g) were housed (1 animal/cage) in standard laboratory cages on a 12: 12 hour light dark cycle throughout the

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77 experiment. Rats were ma intained on Lab Diet 5001 (PMI Feeds, St. Louis, MO) and water ad libitum, and were handled and cared for in accordance with the National Institutes Health Guide for the Care and Use of Laboratory Animals and with the approval of the University of Floridas In stitutional Animal Care and Use Committee (IACUC). Reach Training Over the course of several days, all an imals were placed on a restricted diet until they measured 90% of their original body weight. A brief period of pretraining was then given to familiarize th e rats with the reachi ng task. Pretraining involved placing them into test cages (10 X 18 X 10 cm) with floor s constructed of 2 mm bars, 9 mm apart edge to edge. A 4 cm wide and 5 cm deep tray filled with food pellets (45 mg; Bioserv) was mounted on the front of t he cage. The rats were required to reach outside the cage and retrieve pellets from the tray. Rats were permitted to use either limb and the preferred limb was noted for each animal. All ra ts remained in pretraining until they had successfully retrieved 10 pellets (approximatel y 1 hour/day for 2 days). After pretraining, the rats were placed into a Plexiglas cage (11 cm X 40 cm X 40 cm) with a 1 cm slot located at the front of the cage. Animals were trained for 15 minutes each day to reach with their preferred limb through the slot and retrieve food pellets from a table outside the cage (Whishaw and Pellis, 1990). Each session was videotaped and later used to assess reaching performance. A successful r each was scored when the animal grasped the food pellet, brought it into the cage and to its mouth without dropping the pellet. The percentage of successful reaches [(# successfu l retrievals/the total # of reaches) x 100] was then calculated. All training sessions were video taped and used to measure reaching accuracy. Animals were trained fo r approximately two weeks on this task to

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78 establish a baseline measure of motor performance. Baseline was defined as the average accuracy across the 3 final days of tr aining. Animals failing to achieve a mean reaching accuracy of 40% across 3 consecutiv e days were not used in the study. Prior to surgery, all animals were sorted by their prelesion reaching performance to create groups with comparable baseline levels of r eaching accuracy. This was done to ensure that reaching performance prior to infarction was similar across conditions. Infarction Following the 2 weeks of mo tor training, focal ischemic damage was given to the lateral aspect of sensorimotor cortex by temporary occl usion of the middle cerebral artery (MCAo). Briefly, animals were anes thetized with ketamine hydrochloride (70 mg/kg i.p.) and xylazine (5 mg/kg i.p.). Animals receiv ed low levels of isofluorane (0.15%) and supplemental doses of ketamine (20 mg/kg i.p.) as needed. Under sterile conditions, an incision was made midline and the skull exposed. Small portions of the skull were then removed over the hemisphere c ontralateral to each animals trained to allow injection of the vasoconstr icting peptide endothelial-1 ( ET-1: 0.2 g/ L; American Peptide, Sunnyvale, CA) via the Nanolit re injection system (World Precision Instruments, Sarasota, Fl) controlled by the SYS-Micro 4 Controlle r (World Precision Instruments, Sarasota, Fl). Stereotaxic coordi nates of the injection site with respect to bregma were as follows: anteroposterior +0.9 mm; mediol ateral, -5.2mm; and dorsoventral, -8.7 mm (Biernaskie and Corbe tt 2001). ET-1 (320 pMol dissolved in 0.9% sterile saline) was injected at a rate of 7nL/sec through a glass pipette and pipette was left in the injection site for 5 minutes to avoid backflow. A group of animals that never received an infarction were included as Healthy Controls (HC; n=10) and were given training on the same skilled reaching task on all days of rehabilitation.

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79 Cortical Electrode Implantation Directly following the infarction, nine-pin elec trode carriages (Plastics One Inc., Roanoke, VA) were implanted epidurally over top sens orimotor cortex in the hemisphere opposite each animals preferred paw. The skull was removed between 1 mm posterior to 5.5 mm anterior to bregma and 0.5 mm to 5.5 mm lateral to midline to allow the electrode to be lowered onto the cortical surfac e. A return lead was fixed to the skull in a position posterior to Lambda and the craniotom y filled with gel foam. Both the electrode and gel foam were covered in non-exothermi c PolyWave dental acrylic and cured with a brief pulse (50 seconds) of ultraviolet ligh t. The electrode was then fixed to skull screws with standard dental acrylic and then dental cement was applied on top of the dental acrylic. The skin was sutured and given topical antibiotics and the animals were given 4cc of warm ringers solution (s.c.) and meta cam (0.10 mg/kg; s.c.). The Dual Rail configuration (n=10) consisted of two parallel 0.4 mm by 3 mm stainless steel strips separated by 2 mm. This configuration was incl uded in this study to allow comparison to previous rodent studies of CS/RT that have used similar Dual Rail-type configurations (Adkins-Muir and Jones, 2003; Teskey et al., 2003; Adkins et al., 2006; Adkins et al., 2008). The 2x2 configuration (n=10) consis ted of four contacts positioned 2mm equidistant from one another each with a diam eter of 0.60 mm. The 3x3 configuration (n=10) consisted of 9 contacts positi oned 1.1 mm equidistant from one another each with a diameter of 0.2 mm (figure 1). Movement Thresholds After surgery all animals were returned to their home cage for three days where they were supervised for health concerns bu t otherwise left to recover. Animals were given full access to food until the last day where food restriction regimen (see above)

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80 was reimplemented. After t he three days, animals with implants had their individual motor thresholds (MTs) det ermined and then animals we re started in a motor rehabilitation paradigm (except those in the NT condition). MTs were assessed on post lesion training days 1, 11 and 20. MTs were defined for each animal as the minimum current to cause an involuntary motor respons e and were tested in all animals that had received a cortical electrode. The animals were placed into a transparent cylinder and observed while 3 second trains of 1 millis econd 100 Hz monoploar cathodal pulses were given. Current was gradually increased by 5% increments until a movement of the contralateral forelimb could be clearly detec ted. Cortical stimulation during the post injury motor training phase was then deliv ered at 50% of each animals MT during rehabilitation for t he CS/RT condition. Cortical Stimulation and Re habilitation Training (CS/RT) Following motor threshold testin g the animals received twenty daily bouts of motor training intended to model motor rehabilitation. Each bout consisted of 20 minutes of the skilled reach training used dur ing the training procedur e described earlier or the combination of skilled reach training and cort ical stimulation (C S/RT). One group of animals were included that received the stroke model and rehabilitative training alone (RT; n=10). Another group of animals were in cluded that received the stroke model but did not receive rehabilitative training (NT; n= 6). The post injury reaching behavior of the NT group was assessed by probe trials on da ys 1 and 20 of motor training. All sessions were video taped for analysis of reaching accuracy and number of reach attempts. Animals receiving the combinat ion of cortical stimulation and rehabilitative training were stimulated via the Vertis St imulation System during t hese sessions. The cortical electrode was connected to a remote stim ulator suspended abov e the training cage

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81 where information was then sent wirelessly to the rest of th e system. CS/RT was delivered as monopolar cathodal stimulation and was adminis tered continuous with a frequency of 100 Hz with a current intensity dictated by the subjects movement threshold. Each pulse was biphasic, c harged balanced and asymmetric consisting of a square phase lasting 100 + 10 microseconds and a decaying exponential phase lasting ~9900+ 10 microseconds. Histology and L esion Verification Following the rehabilitation phas e the ani mals were given an overdose of pentobarbital and then transcardially perfused with 0.1 M sodium phosphate buffer followed by 4% paraformaldehyde solution in the same buffer. Brains were then extracted and post fixed in 4% paraformaldeh yde solution in 0.1 M sodium phosphate buffer. Serial 50 m coronal sections were then taken using a microtome. Ten sections spaced 600 m apart and spanning approximately 2.7 mm anterior and 3.3 mm posterior to bregma were sampled for lesion verification. The same number of sections was analyzed for each animal. The sampled sect ions were stained with Toluidine blue (a Nissl stain) and digitally scanned (Espon Perfection V500 Photo Scanner, Long Beach, CA) for lesion verification. The area of spared tissue, characterized by consistent Nissl staining, was traced using Image J software (Abramoff et al., 2004; Rasband, 2009) and cortical volumes were estimated with the Cavalieris unbiased estimator method using the formula: volume di 1 n yi ( t ) ymax Where yi is the cross sectional area of the ith section through the morphometric region, d is the distance between sections (600 m) and n is the total number of

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82 sections (12). ymax is maximum value for the area of one section and t is the section thickness (50 m) and their product is subtracted from the basic question as a correction for the overprojection (G undersen 1986; Gundersen and Jensen, 1987; Mayhew 1992). Estimated volumes for cortical and subcorti cal tissue were analyzed as the percent affected to unaffected side (volume lesioned hemisphere/volume non lesioned hemisphere*100) in order to account for individual differences in brain size. Results Reaching Accuracy A repeated measures ANOVA with CONDITION as a between subject factor and TIME as a within subj ect factor that incl uded all conditions re vealed a significant CONDITION x TIME interaction [F(10, 100)= 12.0559; p<0.0001] on reaching accuracy (Figure 3-1A). A repeated measures ANO VA with CONDITION as a between subject factor and TIME as a within subject factor that excluded the NT condition revealed a significant CONDITION x TIME interact ion [F(80,900)= 2.8347; p<0.0001] on reaching accuracy. Subsequent multiple comparisons (Fishers PLSD; p<0.01) revealed that all groups that received lesions had significantly lower reaching accuracies on day 1 of rehabilitation in comparison with pre-stroke levels and with the performance of the HC group. On the final day of motor rehabilitat ion, all groups receiving rehabilitation performed significantly higher reaching accuracies than the NT group (Fishers PLSD; p<0.05). Further comparisons (Fishers PL SD; p<0.05) found that the Dual-CS/RT exhibited greater reaching im provements than the RT condition on days 8, 11-13, 16 and 18-20. The 2x2-CS/RT exhibited greater reaching improvements than the RT condition on days 8, 10, 16 and 18-20. The 3x3-CS/RT exhibited greater reaching improvements than the RT condition on days 10, 12-13, 16 and 18-20. Thus, all three

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83 CS/RT conditions had significantly higher reaching accuracies than RT Controls on several of the final days of rehabilitation (Fishers PLSD; p<0.05). All CS/RT and RT groups exhibited similar number of days w here significant motor improvements were observed: RT=15 days, 2x2-CS/RT=16 days, 3x3-CS/RT=10 days, Dual-CS/RT=13 days. The NT condition did not exhibit si gnificant motor impr ovements between day 1 and 20 (Fishers PLSD; p<0.05). These result s demonstrate that while rehabilitation following focal cortical ischemia induced improvements in reaching accuracy, the combined therapy of CS/RT resulted in en hanced behavioral gains following injury. Reaching Attempts A repeated measures ANOVA with CONDITION as a between subject factor and TIME as a within subj ect factor that incl uded all conditions re vealed a significant CONDITION x TIME interaction [F(10,100) = 5.7307; p<0.0001] on the number of reach attempts (Figure 3-1A). A repeated meas ures ANOVA with COND ITION as a between subject factor and TIME as a within subjec t factor that excluded the NT condition revealed a significant CONDITION x TIME interaction [F(80,900)= 2.0599; p<0.0001] on the number of reach attempts. Subsequent multiple comparisons (Fishers PLSD; p<0.01) revealed that all groups that received lesions had significantly lower numbers of reach attempts on day 1 of r ehabilitation in comparison wit h pre-stroke levels and with the performance of the HC group. Further co mparisons (Fishers PLSD; p<0.05) found that none of the groups with lesions were signi ficantly different from one another on any day except that all groups given rehabilitation performed significantly more reach attempts than the NT group on day 20. The RT condition demonstrated a significantly smaller number of reach a ttempts than the HC conditi on on days 1-14 and 16-20. The Dual-CS/RT condition demonstrated a significantly smaller number of reach attempts

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84 than the HC condition on days 1-14 and 16-20. T he 2x2-CS/RT condition demonstrated a significantly smaller number of reach a ttempts than the HC condition on days 1-12, 14-16, 18 and 20. The 3x3-CS/RT condition de monstrated a significantly smaller number of reach attempts than the HC condition on days 1-10, 12-14 and 17-20. Thus, all conditions with lesions per formed fewer reach attempts t han the healthy controls on almost all days of rehabilitation (Fishers PL SD; p<0.05). No significant improvements in the number of reach attempts were seen in the RT condition after 8 days of rehabilitation, in the Dual-CS/RT condition after 9 days, in the 2x2-CS/RT condition after 10 days or in the 3x3-CS/RT condition afte r 9 days. The NT condition did not perform a significantly higher number of reach atte mpts between the assessments made on days 1 and 20. These results demonstrate that a ll groups receiving rehabilitation performed similar numbers of reach attempts and that all reached less than healthy controls. Movement Thresholds A repeated measures ANOVA with CONDITION and TIME showe d a significant CONDITION x TIME interaction [F(6,72) = 5.5221; p<0.001] on mean movement threshold (Figure 3-2A). Subsequent multip le comparisons (Fishers PLSD; p<0.01) revealed that all conditions had significantly lower reaching accuracies on the third assessment of MTs relative to the first. Further comparisons (Fishers PLSD; p<0.05) found that the Dual-CS/RT, 2x2-CS/RT and 3x3-CS/RT groups exhibited significantly larger decreases in the perc ent MT between the first and fi nal assessment relative to the RT group. Finally, a significant negative correlation was found between the percent reduction in movement threshold (MT2-MT1/MT1*100) and the increase in post rehabilitation reaching accuracy (Mean fi nal 3 rehabilitation days Mean first 3 rehabilitation days) [r=-0.3861, p<0.01] (Fi gure 3-2B). This demonstrates that the

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85 combination of CS/RT resulted in signific antly greater reducti ons than RT alone and reductions in MTs have a significant correlation with increased motor improvements following cortical ischemia. Estimation of Remaining Tissue A one-way ANOVA revealed a significant main effect of CONDITION on the estimates of remaining cortical tissue [F(5 ,48) = 16.1341; p<0.05] Subsequent multiple comparisons (Fishers PLSD; p<0.05) showed t hat the amount of resi dual cortical tissue was similar for all conditions except that the HC condition exhibi ted a significantly greater amount of remaining cortical tissue than all other conditions (Table 3-1). A oneway ANOVA revealed no significant main ef fect of CONDITION on the estimates of remaining subcortical tissue [F(5,48) = 10.372; p<0.05]. Subsequent multiple comparisons (Fishers PLSD; p<0.05) showed that the amount of residual subcortical tissue was similar for all condit ions except that the HC conditi on exhibited a significantly greater amount of remaining subcorti cal tissue than all other conditions. Discussion Although the neural mechanisms of stro ke recovery are not fully understood, there is evidence that motor improvements can be elicited by t he recruitment and/or retraining of residual tissue within motor co rtex. Further, motor experience is an important mediator of the neuroplastic changes that lead to functional reorganization within motor cortex. The pres ent experiments further show the capacity of motor rehabilitation to improve mo tor function post-stroke as all animals that received rehabilitative training demonstrated greater motor improvements than animals given no post injury training. This work also serv es as corroborating ev idence that combining rehabilitative training with adjuvant therapi es can augment motor improvements. Here,

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86 the combination of rehabilitative training and electrical stimulation of motor cortex achieved greater motor improvements than r ehabilitative training al one. The benefit of CS/RT was observed regardless of the number of independent stimulating contact sites within each cortical electrode. The ability of distributed configurations consisting of either four or nine contac t sites to enhance behavioral improvements was unexpected as it was thought a larger number of stim ulation sites would facilitate greater neuroplastic change in motor cortex. While a body of evidence suggests that el ectric stimulation of motor cortex can enhance behavioral gains during rehabilitation, the appropriate paramet ers to translate to human therapy are not fully understood and may contribute to the lack of efficacy observed in a recent clinical trial (Plow et al., 2009). It has previously been reported in a rodent model of stroke that CS/RT stim ulation needs to be distributed across motor cortex and encompasses several ICMS-evoked movement representations in order to be efficacious (Boychuk et al ., 2009). In that study, dist ributed CS/RT was associated with a reemergence of distal representations that had been compromised by the experimental lesion and this reemergence occurred at the expense of surrounding representations (Boychuk et al., 2009). St imulation distributed across motor cortex targets more non-forelimb (whisker, neck etc) areas and therefore may promote a greater amount of timing-depend ent recruitment of residua l cortex into forelimb representations (Jackson et al., 2006; Boychuk et al., 2009). It has previously been noted that CS/RT using one stimulation contact was insufficient to drive neurons within motor cortex in order to evoke movement whereas four contact sites were sufficient (Boychuk et al., 2009).

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87 While prior work demonstrated that a dist ributed configuration of CS/RT using a small number of contacts is capable of induc ing enhanced motor improvements, it is not clear what number of stimulat ing sites in a distributed arrangement will result in the greater rehabilitative benefi t. Here, two configurations of CS/RT (2x2 and 3x3) that differed in the number and proxim ity of stimulating contact sites were compared by their ability to enhance motor improvements in a rodent model of stroke. The 2x2-CS/RT configuration has previously been associ ated with enhanced behavioral gains (Boychuk et al., 2009). These two configurations were also compared to a dual rail configuration that was associated with a behavioral benefit in many previous studies of CS/RT with rodent models of stroke (Adkins-Muir and Jones 2003; Kleim et al., 2003, Teskey et al., 2003; Adkins et al., 2006). In the current study, the electrodes for the 2x2-CS/RT condition contained very few contacts distri buted across the motor cortex while the 3x3CS/RTs electrodes contained a large number of contacts distributed across the cortex but in closer proximity to one another. It was hypothesized that 3x3-CS/RT would induce greater motor improvements than 2x2-CS /RT because of its relatively greater number of stimulation sites making contact with different areas of motor cortex. The 3x3-CS/RT configuration induc ed greater motor improvement s than rehabilitative alone but unexpectedly conferred no additional benefit relative to 2x2-CS/RT or Dual-CS/RT. Additionally, the three types of CS/RT di d not significantly differ in changes in movement threshold amongst each other but di d exhibit significantly greater movement threshold reductions compared to the RT group. These reductions in movement thresholds are indicative of changes in co rtical excitability and may reflect increased synaptic input onto corticospinal neurons (Monfils et al., 2005).

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88 It is not clear why the 3x3-CS/RT confe rred no additional benefit relative to 2x2CS/RT except to say that 2x2-CS/RT provided a sim ilar amount of necessary stimulation to drive enhanced mo tor improvements relative to 3x3-CS/RT. An important similarity between 2x2-CS/RT and 3x3-CS/RT is that both configurations are widely distributed across motor cortex rather than focally distribut ed within it. The majority of forelimb representations are located within the interior of motor map whereas nonforelimb representations are located in areas surrounding them. Boychuk et al. (2009) found that CS/RT stimulation di rected at the outer regions of motor cortex (areas with predominantly non forelimb representations) induced a greater expansion of forelimb areas into these surrounding representations that was accompanied by enhanced motor improvements. In contrast, CS/RT directed at t he interior of the forelimb map resulted in less expansion of forelimb areas into surrounding parts of the motor map and no behavioral benefit relative to r ehabilitative traini ng alone (Boychuk et al., 2009). It is possible the four contacts from the 2x2 co nfiguration provided adequate stimulation of the distributed (more peripheral) areas of motor cortex such that the nine sites in the 3x3 configuration offered no additional benef it by having more independent sites of stimulation within this outer portion of the motor map. It is also possible that the additional 3x3-CS/RT contacts in the interi or of the motor map are ineffective at recruiting non-forelimb areas during rehabilitati on in same manner that Boychuk et al. (2009) observed with focal CS/RT. Another important consideration is that there may be an upper limit to the extent that stimulation of moto r cortex can enhance motor improvements during rehabi litative training. It is possible that 3x3-CS/RT is driving

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89 neuroplastic changes in a larger proportion of motor cortex than 2x2-CS/RT but resulting in similar behavioral ben efits because of a ceiling effect. CS/RT may be enhancing motor improvement by one or a combination of a number of processes including restoration of function within motor areas, reorganization of motor cortex, recruitment of areas outside of motor cortex, angiogenesis, glialgenesis, neuroprotection, or growth factor production. Rehabilitative training alone often induces a reorganization of motor cortex involving an expans ion of movement representations when the training is specif ic to these representations (Nudo et al., 1996a). For example, a food-reaching task in duces reemergence of distal forelimb representations (Nudo et al., 1996a). Seve ral studies have found that CS/RT is associated with an enhanced ex pansion/reorganization of microstimulation evoked movement representations in ipsilesional motor cortex relative to training alone (Kleim et al., 2003, Plautz et al., 2003; Boychuk et al., 2009). CS/RT is also associated with increased synaptic density (Adkins et al., 2008) and enhanced synaptic responses (Teskey et al., 2003) within ip silesional motor cortex that may support its functional reorganization. Further, animal studies have shown that electrical stimulation can induce the expansion/reorgani zation of movement representations in healthy motor cortex as well as increase synaptic dens ities and dendritic hypertrophy all in the absence of motor training (Teskey et al., 2002; Monfils et al., 2004; van Rooyen et al., 2006). Detailed electrophysiological measures in human stroke patients given CS/RT are limited due their invasiveness, however, Brown et al. (2006) noted that during intraoperative cortical mapping after CS/RT therapy individual fi nger representations were observed in some patients who were unable to perform the movements with

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90 volitional effort. These data s upport that CS/RT is enhancing behavioral gains by driving functional reorganization of motor cortex above and beyond the remodeling that occurs with rehabilitative training alone. The MTs used to assign CS/RT current amplitudes also provide evidence that CS/RT induces functional reorganization. The minimum threshold to evoke movement is reduced across days of rehabilitation in animals receiving RT or CS/RT, however, CS/RT gro ups often exhibit the greatest reductions (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Adkins et al., 2006; Boychuk et al., 2009). In the present study significantly greater reductions in MT for the CS/RT condition relative to the RT condition were observed. These reductions in MTs suggest increased excitability in moto r cortex and parallel the motor map changes observed with CS/RT (Monfils et al., 2005). In the present study, CS/RT was associated with enhanced behavioral improvements in a rodent model of stroke While it has previ ously been shown that distributed CS/RT is efficacious and focal CS/RT is not, the present work finds that increasing the number of stimulation sites within a distributed configuration imparts no additional benefit to a distribut ed configuration with few sites. This speaks to the robust effect of electrical stimulation of the co rtex for enhancing functi onal improvement after stroke. These findings suggest motor cortex can respond to a small number of independent stimulation sites prov ided the sites are located in areas of motor cortex that can be recruited to facilitate disrupted mo tor patterns and behavior. This work lends further support for the use of cortical st imulation for magnifying the behavioral benefits of rehabilitation in human stroke patients.

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91 Figure 3-1. Representative Ni ssl stained coronal section fr om the MCAo lesion. The medial-lateral extent of brain damage at 24 days following ischemic insult by 4 l injection of the vasoc onstricting peptide ET-1 (0.2 g/ l) is shown. Figure 3-2. The three different electrode confi gurations examined. A ll contacts were 0.6 mm in diameter. The Dual Rail configurat ion consisted of tw o parallel 0.4 mm by 3 mm stainless steel strips separ ated by 2 mm. The 2x2 configuration consisted of four contacts positioned 2 mm equidistant from one another each with a diameter of 0.60 mm. The 3x3 c onfiguration consisted of 9 contacts positioned 1.1 mm equidistant from one another each with a di ameter of 0.40 mm.

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92 Figure 3-3. Reaching performance prestrok e and during 20 days of rehabilitation. A) Mean (SD) percent reach accuracy. All groups receiving rehabilitation performed significantly higher reaching accuracies than the NT group. All three CS/RT conditions had significantly higher reaching accuracies that RT Controls on rehabilitation days 16, 18-20 (Fishers PLSD; p<0.05; indicated by the symbol ). B) Mean (SD) number of reach attempts. Multiple comparisons (Fishers PLSD; p<0.05) found that none of the groups with lesions were significantly different from one another on any day except that all groups given rehabilitation performed signifi cantly more reach attempts than the NT group on day 20.

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93 Figure 3-4. Mean (SD) movement threshol ds for animals in the RT and all CS/RT conditions during rehabilitation. The RT and all CS/RT conditions exhibited a significant decrease in threshold on the third assessment of movement threshold relative to the first (Fisher s PLSD; p<0.05). Further comparisons (Fishers PLSD; p<0.05) found that the all CS/RT groups exhibited significantly larger decreases in the percent movement threshold between the first and third assessment relative to the RT group (Fishers PLSD; p<0.05; indicated by the symbol ). B) A significant negative correlation was found between the percent reduction in move ment threshold (MT3-MT1/MT1*100) and the increase in post rehabilitati on reaching accuracy (Mean final 3 rehabilitation days Mean first 3 rehabi litation days) [r=-0.3861, p<0.01].

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94 Table 3-1. Estimate of spared cortical and subcortical tissue from experiment 2. Estimated volumes for cortical and s ubcortical tissue were analyzed as the percent affected to unaffected side (vol ume lesioned hemisphere/volume non lesioned hemisphere*100) in order to a ccount for individual differences in brain size. Data is represented as mean percent SEM. Condition Sample Size Estimates of Spared Tissue Volume Cortical (% Contralesional ) Subcortical (% Contralesional) HC 10 98 101 RT 8 85 91 Dual-CS/RT 10 84 91 2x2-CS/RT 10 83 89 3x3-CS/RT 10 84 92 NT 6 86 93

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95 CHAPTER 4 CORTICAL STIMULATION WITH REH ABI LITATIVE TRAINING CAN ENHANCE MOTOR IMPROVEMENTS AFTER EARLY APPLICATION OF REHABILITATION ALONE IN A RODENT MODEL OF ISCHEMIA Introduction More than half of stroke pati ents exhibit motor deficits afte r stroke (Roth et al., 1998; Luke et al., 2004; Urton, et al., 2007). While some spontaneous recovery is observed in stroke survi vors (Nakayama et al., 1994; Cramer 2008) it is estimated that a majority of this population suffers from residual moto r impairments (Urton, et al., 2007). Current rehabilitative strategies demonstrate some benefit on a population level, however, individual patterns of motor im provements after stroke are highly variable and typically incomplete (Gresham et al., 1995; Duncan et al., 2000). The inability to amerliorate persistent motor impairments in chronic stroke survivors reflects our lack of understanding on how to facilitat e brain repair after injury. It is expected that more effective stroke treatments will develop as the mechanisms of stroke recovery are characterized. Human patients with a high degree of moto r improvements after cortical stroke typically exhibit a reorganization of spared pr imary motor cortex wit hin the ipsilesional hemisphere (Rossini et al., 1998; Cramer et al., 2000; Wittenberg et al., 2003). In animal models, ischemic damage is associat ed with a loss of dendritic structure (Zhang et a., 2005) as well as a loss of synapses (Hasbani et al., 2001; Zhang et al., 2005; Murphy et al., 2008). Cortical ischemia is also associated with a loss of microstimulation-evoked movement represent ations within motor cortex (Nudo and Milliken, 1996; Friel et al., 2000). The loss of movement representations following cortical ischemia persists in the absence of rehabilitative training (Nudo et al., 1996b).

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96 Appropriate rehabilitative training followi ng cortical ischemia results in motor improvements and an expansion and reemergence of movement representations that were lost by the infarct (Nudo et al., 1996a; Kleim et al., 2003). The areas of motor cortex that exhibit the expansion and reorgani zation of movement r epresentations also demonstrate increased synaptogenesis (Kleim et al., 2002a; Kleim et al., 2004). Synaptogenesis following cortical ischemia is associated wit h behavioral improvements that are though to arise from a reinstitution of cortical circ uitry (Stroemer et al., 1995; Buonomano and Merzenich, 1998; Brown and Murphy, 2007). Indeed, adjuvant therapies that promote synaptic plasticity with in motor cortex such as the administration of amphetamine (Stroemer et al., 1998; Ad kins and Jones, 2005; Ramic et al., 2006; Papadopoulos et al., 2009) or nicotine (Gonz alez et al., 2005; Gonzalez et al., 2006) enhance rehabilitation-dependent im provements in motor performance after stroke. Administration of a type IV-specific phosphodies terase inhibitor (PDE4) that stimulates synaptic plasticity in a cAMP/CREB depe ndent manner increases animals skilled reaching ability and the size of forelimb movement representations following cortical ischemia (MacDonald et al., 2007). Excitability in the central nervous system can also be altered by electrical stimulation (Blundon and Zakharenko, 2008). For example, several daily bouts of stimulation in sensorimotor cortex results in potentiated synaptic responses in this tissue in awake behaving animals (Trepel and Racine, 1998; Froc et al., 2000; Teskey et al., 2002; Monfils et al., 2004). These cha nges in synaptic efficacy may support the encoding of behavioral experience (Martin and Morris, 2002). Interestingly, synaptic potentiation in sensorimotor cortex has also been observed following skilled motor

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97 learning (Rioult-Pedotti et al., 1998; M onfils and Teskey, 2004). The similarity in sensorimotor cortices respons e to both electrical stimul ation and behavioral experience suggest electrical stimulation can be used to facilitate the encoding of information within this tissue. Jackson et al. (2006) used a neur al implant to demons trate that repeated conditioning with a closed loop electronic device can alter the output of motor cortex by creating stable connections between previously unconnected areas within this cortical region. Electrical stimulation of motor cortex combined with rehabilitative training (CS/RT) also enhances the motor improvements normally associated with rehabilitative training in rodent (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Adkins et al., 2006; Adkins et al., 2008) and primate (Plautz, et al., 2003) models of stroke. The enhanced behavioral gains with CS/RT are paralleled by increased reorganization of movement representations within motor cort ices of rodents (Kleim et al., 2003) and primates (Plautz et al., 2003). In addition to motor map changes, CS/RT is associated with an increased number of sy napses (Adkins et al., 2008) as well as enhanced synaptic responses (Teskey et al., 2003). CS/RT was associated with enhanced motor improvements in a case report of a single human stroke patient (Brown et al., 2003). CS/RT also induced significant improvements relative to RT alone in a human phase I clinical trial with eight participants (Brown et al., 2006) and a phase II clinical trial with twenty four participant s (Huang et al., 2008; Levy et al., 2008). A recent phase III clinical trial with one-hundred and forty-six participants failed to show enhanced behavioral gains in patients rece iving CS/RT relative to RT (Plow et al., 2009). An important difference bet ween the preclinical animal st udies and clinical trials

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98 of CS/RT is the amount of rehabilitative expe rience prior to the onset of CS/RT. In the clinical trials humans stroke patients were recruited a minimum of four months post stroke after conventional rehabilitation ther apies had been administered (Brown et al., 2003; Brown et al., 2006; Huang et al., 2008; Le vy et al., 2008). In the preclinical animal studies, CS/RT has always been initiated within two weeks of experimental ischemia in the absence of any prior rehabilitative traini ng (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Plautz, et al., 2003; Adkins et al., 2006; Adkins et al., 2008). Prior rehabilitative trai ning may limit subsequent behavioral gains by CS/RT by reducing the brains capacity to reorganize in response to CS/RT. Motor training before CS/RT may also interfere with CS/RTs effi cacy by reinforcing connections in motor cortex that do not support behavioral im provements induced CS/RT. While both rehabilitative training and CS/RT appear to induce motor improvements through similar mechanisms, eg., reorganization of movement representations in motor cortex, it is possible that differences in their neuroplastic responses may result in interference when RT is applied prior to CS/RT. Here, rodents given experimental ischemia were given sixteen days of rehabilitative training bef ore a comparison was made between RT and CS/RT over an additional sixteen days. Methods Subjects Thirty-one adult male Long-Evans hooded rats (350-420g) were housed (1 animal/cage) in standard laboratory cages. Animals were kept on a 12:12 hour light dark cycle throughout the experiment. All experimentati on was conducted during the light cycle. Rats were maintained on Lab Diet 5001 (PMI Feeds, St. Louis, MO) and water ad libitum, and were handled and car ed for in ac cordance with the National Institutes

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99 Health Guide for the Care and Use of Labor atory Animals and with the approval of the University of Floridas Institutional Animal Care and Use Committee (IACUC). Reach Training Over the course of several days, all an imals were placed on a restricted diet until they measured 90% of their original body weight. A brief period of pretraining was then given to familiarize th e rats with the reachi ng task. Pretraining involved placing them into test cages (10 X 18 X 10 cm) with floor s constructed of 2 mm bars, 9 mm apart edge to edge. A 4 cm wide and 5 cm deep tray filled with food pellets (45 mg; Bioserv) was mounted on the front of the cage. The rats were required to reach outside the cage and retrieve pellets from the tray. Rats were permitted to use either limb and the preferred limb was noted for each animal. All ra ts remained in pretraining until they had successfully retrieved 10 pellets (approximatel y 1 hour/day for 2 days). After pretraining, the rats were placed into a Plexiglas cage (11 cm X 40 cm X 40 cm) with a 1 cm slot located at the front of the cage. Animals were trained for 15 minutes each day to reach with their preferred limb through the slot and retrieve food pellets from a table outside the cage (Whishaw and Pellis, 1990). Each session was videotaped and later used to assess reaching performance. A successful r each was scored when the animal grasped the food pellet, brought it into the cage and to it s mouth without dropping the pellet. The percentage of successful reaches [(# successfu l retrievals/the total # of reaches) x 100] was then calculated. All training sessions were video taped and used to measure reaching accuracy. Animals were trained fo r approximately two weeks on the reaching task to establish a baseline measure of motor performance. Baseline was defined as the average accuracy across the 3 final days of training. Animals failing to achieve a mean reaching accuracy of 40% across 3 cons ecutive days were not used in the study.

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100 Prior to surgery, all animals were sorted by their prelesion reaching performance to create groups with comparable baseline levels of reaching accuracy. This was done to ensure that reaching performance prior to infarction was similar across conditions. The thirty-one animals were divide d into four groups. A group of animals that never received the experimental lesion was in cluded as Healthy Controls (HC; n=8) and was given training on the same skilled reaching task on all days of rehabilitation (32 days). A second group that received the stroke model but did not receive rehabilitative training was included as non-trained controls (NT; n=7). The post injury reaching behavior of the NT group was assessed by probe trials on days 1 and 32 of motor training. The remaining animals (n=16) received the stroke model and subs equent rehabilitative training in two phases (16 days plus 16 days). Infarction Following the 2 weeks of motor training, focal ischemic damage was given to the lateral aspect of sensorimotor cortex by temporary occl usion of the middle cerebral artery (MCAo). Briefly, animals were anes thetized with ketamine hydrochloride (70 mg/kg i.p.) and xylazine (5 mg/kg i.p.). Animals receiv ed low levels of isofluorane (0.15%) and supplemental doses of ketamine (20 mg/kg i.p.) as needed. Under sterile conditions, an incision was made midline and the skull exposed. A small burr hole was made in the hemisphere contralateral to each animals trained to allow a 3 L injection of sterile saline and the vasoconstricting peptide endothelin-1 (ET-1: 0.2 g/ L; American Peptide, Sunnyvale, CA) via the N anolitre injection system (World Precision Instruments, Sarasota, Fl) controlled by the SYS-Micro 4 Controlle r (World Precision Instruments, Sarasota, Fl). Stereotaxic coordi nates of the injection site with respect to bregma were as follows: anteroposterior +0.9 mm; mediol ateral, -5.2mm; and

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101 dorsoventral, -8.7 mm (Biernaskie and Corbe tt 2001). ET-1 (240 pMol dissolved in 0.9% sterile saline) was injected at a rate of 7nL/sec through a glass pipette and the pipette was left in the injection site for 5 minutes to avoid backflow. After the MCAo surgery all animals were returned to their home cage for three days where they were supervised for health concerns but otherwise left to recove r. Animals were given full access to food until the last day where the food restricti on regimen (see above) was reimplemented. Rehabilitative Training To examin e the effect of prior rehabili tative experience on CS/RTs ability to enhance motor improvements, the animals from the remaining group, i.e., animals not placed into the HC or NT conditions, were given two phases of post injury motor training. In the first phase these animals re ceived rehabilitative training with their affected limb on the skilled reaching task for twenty minutes/day for sixteen days. The behavior of this group was considered stable in the last five days of the sixteen-day period because no significant improvements in reaching performance were observed (days 12-16) (Fishers PLSD; p<0.05). On t he seventeenth day, all animals in this group were given a second surgery where surface cortical electrodes were implanted over motor cortex. The implanted animals were t hen divided so that half continued to receive rehabilitative training (RT; n=8), while the other half received cortical stimulation and rehabilitative training (CS/RT; n=8), each fo r an additional sixteen days. Animals from the third group were divided into the RT and CS/RT conditions by their reaching performance on the last three da ys of the first sixteen days of rehabilitation. This sorting was performed in order to create two groups with comparable levels of reaching accuracy. All sessions were video taped for analysis of reaching accuracy and number of reach attempts.

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102 Cortical Electrode Implantation In the second surgery a craniotomy was ma de over the motor cortex in the same hemispher e as the ischemic injury. The craniotomy was made between 1 mm posterior to 5.5 mm anterior to bregma and 0.5 mm to 5.5 mm lateral to midline. For each RT and CS/RT animal, a nine-pin electrode carriage (Plastics One Inc., Roanoke, VA) with a dual rail contact configuration was implanted directly on motor cortex. The Dual Rail electrode configuration consisting of two para llel 0.4 mm by 3 mm stainless steel strips separated by 2 mm was used to allow comparison to previous rodent studies of CS/RT (Adkins-Muir and Jones, 2003; Teskey et al., 2003; Adkins et al., 2006; Adkins et al., 2008). A return lead was fixed to the skull in a position posterio r to Lambda and the craniotomy filled with gel foam. Both the electrode and gel foam were covered in nonexothermic PolyWave dental acrylic and cured with a brief pulse (50 seconds) of ultraviolet light. The electrode was then fixe d to skull screws with standard dental acrylic and then dental cement was applied on top of the dental acrylic. The skin was sutured and given topical antibiotics and the animals were given 4cc of warm ringers solution (s.c.) and metacam (0.10 mg/kg; s.c.). Af ter the implant surgery all animals were returned to their home cage for three days where they were supervised for health concerns but otherwise left to recover. Anim als were given full access to food until the last day where food restriction re gimen (see above) was reimplemented. Movement Thresholds After three days, the anim als had their individual motor thresholds (MTs) determined. MTs were assessed for RT and CS/RT animals on r ehabilitation days 17, 25 and 32 (days 1, 9 and 16 of the second phas e). MTs were defined for each animal as the minimum current to cause an invol unt ary motor response. The animals were

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103 placed into a transparent cylinder and observ ed while 3-second trains of 1 millisecond, 100 Hz, monoploar cathodal pulses were given. Current was gradually increased by 5% increments until a movement of the contralateral forelimb could be clearly detected. Cortical stimulation during the post injury motor training phase was then delivered at 50% of each animals MT during rehabilitation for the CS/RT condition. Cortical Stimulation and Re habilitation Training (CS/RT) Animals receiving the combi nation of cortical stimulat ion and rehabilitative training were stimulated via the Vertis Stimulation System duri ng the twenty-minute training sessions. The cortical electrode was connected to a remote stimulator suspend ed above the training cage where information was then sent wirelessly to the rest of the system. CS/RT was delivered as monopolar ca thodal stimulation and was administered continuous with a frequency of 100 Hz with a curr ent intensity dictated by the subjects movement threshold (see above). Each pulse was biphasic, charged balanced and asymmetric consisting of a square phase lasting 100 + 10 microseconds and a decaying exponential phase lasting ~9900+ 10 microseconds. Histology and L esion Verification Following the rehabilitation phas e, the animals were given an overdose of pentobarbital and then transcardially perfused with 0.1 M sodium phosphate buffer followed by 4% paraformaldehyde solution in the same buffer. Brains were then extracted and post fixed in 4% paraformaldeh yde solution in 0.1 M sodium phosphate buffer. Serial 50 m coronal sections were then taken using a microtome. Ten sections spaced 600 m apart and spanning approximately 2.7 mm anterior and 3.3 mm posterior to bregma were sampled for lesion verification. The same number of sections was analyzed for each animal. The sampled sect ions were stained with Toluidine blue

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104 (a Nissl stain) and digitally scanned (Espon Perfection V500 Photo Scanner, Long Beach, CA) for lesion verification. The area of spared tissue, characterized by consistent Nissl staining, was traced using Image J software (Abramoff et al., 2004; Rasband, 2009) and cortical volumes were estimated with the Cavalieris unbiased estimator method using the formula: volume di 1 n yi ( t ) ymax Where yi is the cross sectional area of the ith section through the morphometric region, d is the distance between sections (600 m) and n is the total number of sections (12). ymax is maximum value for the area of one section and t is the section thickness (50 m) and their product is subtracted from the basic question as a correction for the overprojection (G undersen 1986; Gundersen and Jensen, 1987; Mayhew 1992). Estimated volumes for cortical and subcorti cal tissue were analyzed as the percent affected to unaffected side (volume lesioned hemisphere/volume non lesioned hemisphere*100) in order to account for individual differences in brain size. Results Reaching Accuracy A repeated measures ANOVA with CONDITION as a betw een subject factor and TIME as a within subj ect factor that incl uded all conditions re vealed a significant CONDITION x TIME interaction [F(6,54) = 20.9405; p<0.0001] on reaching accuracy (Figure 3-3A). A repeated measures ANO VA with CONDITION as a between subject factor and TIME as a within subject factor that excluded the NT condition revealed a significant CONDITION x TIME interact ion [F(64,672)= 4.3392; p<0.0001] on reaching accuracy. Subsequent multiple comparisons (Fishers PLSD; p<0.01) revealed that the

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105 NT, RT and CS/RT groups had significantly lower reaching accuracies on day 1 of rehabilitation in comparison with pre-stroke levels and with the performance of the HC group. The NT condition did not significant ly change its reaching accuracy between post injury training days 1 and 32. Further compar isons (Fishers PLSD; p<0.05) showed that while all animals receiving rehabilitation dem onstrated significant increases in reaching accuracy during the 32 days of rehabilitation, the animals receiving CS/RT had significantly higher reaching accuracies than RT Controls on days 26 and 28-32. The CS/RT conditions reaching accuracy was not significantly diffe rent from healthy controls on days 21, 24, 26 and 28-32 whereas the RT condition was not significantly different from healthy controls only on day 21 (Fishers PLSD; p<0.05). No significant improvements were seen in the RT condition after 10 days of rehabilitation. In contrast, significant improvements were observed in the CS/RT condition through 25 days of rehabilitation (Fishers PLSD; p<0.05). These results demonstrate that while rehabilitation following ischem ic injury induced improvements in reaching accuracy, CS/RT can enhance motor improvements after ear ly application of r ehabilitation alone. Reaching Attempts A repeated measures ANOVA with CONDITION as a between subject factor and TIME as a within subj ect factor that incl uded all conditions re vealed a significant CONDITION x TIME interaction [F(6,54) = 3.3090; p<0.01] on t he number of reach attempts (Figure 3-3B). A repeated meas ures ANOVA with COND ITION as a between subject factor and TIME as a within subjec t factor that excluded the NT condition revealed a significant CONDITION x TIME interaction [F(64,672)= 1.3591; p<0.05] on the number of reach attempts. Subsequent multiple comparisons (Fishers PLSD; p<0.01) revealed that the NT, RT and CS/R T groups had significantly lower numbers of

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106 reach attempts on day 1 of r ehabilitation in comparison wit h pre-stroke levels and with the performance of the HC group. Further co mparisons (Fishers PLSD; p<0.05) found that the RT and CS/RT groups were not signi ficantly different from one another on any day. On day 32 of training the NT, RT and CS/RT performed similar numbers of reach attempts (Fishers PLSD; p<0.05). The RT condition demonstrated a significantly smaller number of reach a ttempts than the HC conditi on on days 1-13, 16-18, and 2324 (Fishers PLSD; p<0.05). The CS/RT cond ition demonstrated a significantly smaller number of reach attempts than the HC condition on days 1-13, 16-18, 20 and 26-27 (Fishers PLSD; p<0.05). No significant impr ovements in the number of reach attempts were seen in the RT condition after 20 da ys of rehabilitation whereas no significant improvement were observed in the CS/RT condition after 24 days (Fishers PLSD; p<0.05). The NT condition also performed a significantly higher number of reach attempts between the assessments made on days 1 and 32 (Fishers PLSD; p<0.05). These results demonstrate that the RT and CS /RT groups performed similar numbers of reach attempts and that the NT, RT and CS/RT groups demonstrated significant increases in the number of reach atte mpts in the final days of the study. Movement Thresholds A repeated measures ANOVA with CONDITION and TIME showed a significant CONDITION x TIME interaction [F(1,15) = 43.7268; p<0.001] on mean movement threshold (Figure 3-4A). Animals in the RT and CS/RT conditions showed a significant decrease in MTs as training continued: RT: [T(7) = -5.26512; p<0.001] and CS/RT [T(7) = -5.80717; p<0.001]. While the MTs for th e two conditions were not significantly different on the first day of a ssessment, the thresholds were significantly smaller in the CS/RT relative to the RT condition on the second assessment (Fishers PLSD; p<0.05).

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107 Furthermore, the CS/RT condition exhibited a significantly larger percent reduction in MT compared to the RT condition [F(1,14) = 8.8931; p<0.01]. Finally, a significant negative correlation was found between the percent reduction in MT (MT2MT1/MT1*100) and the increase in post rehabi litation reaching accuracy (Mean final 3 rehabilitation days Mean first 3 rehabilitatio n days) [r=-0.5433, p<0.05] (Figure 3-4B). This demonstrates that the combination of CS/RT was associated with the greatest reductions in MT relative to RT and reduction s in MTs are significantly correlated with increased motor improvements fo llowing cortical ischemia. Estimation of Remaining Tissue A one-way ANOVA revealed a significant main effect of CONDITION on the estimates of remaining cortical tissue [F(3 ,27) = 5.7673; p<0.05]. Subsequent multiple comparisons (Fishers PLSD; p<0.05) showed t hat the amount of resi dual cortical tissue was similar for all conditions except that the HC condition exhibi ted a significantly greater amount of remaining cortical tissue than all other conditions (Table 4-1). A oneway ANOVA revealed no significant main ef fect of CONDITION on the estimates of remaining subcortical tissue [F(3,27) = 2.6208; p>0.05]. Subsequent multiple comparisons (Fishers PLSD; p<0.05) s howed that the HC cond ition exhibited a significantly greater amount of remaining subcortical ti ssue than all other conditions except the RT condition. All of the groups that received experimental lesions (RT, CS/RT and NT) exhibited similar amounts of remaining subcortical tissue (Fishers PLSD; p<0.05). Discussion The motor cortex is a lo cus for neuroplastic changes that are associated with motor improvements after cortical ischemia. Mo tor training after cortical ischemia results

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108 in motor improvements along with an exp ansion and reemergence of movement representations that were lo st by the infarction (Nudo et al., 1996a; Kleim et al., 2003). The current experiment also observed motor improvements in animals that received rehabilitative training that were not observ ed in animals that received the same lesion without any post injury motor training. Several studies have observed magnified behavioral gains with CS/RT relative to RT (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Plautz, et al., 2003; Adkins et al., 2006; Adkins et al., 2008). In the present experiment, CS/RT magnifi ed motor improvements after experimental ischemia in animals that had already rece ived prior rehabilitative training. These findings indicate the nervous system can be continually remodeled following stroke in ways that support further behavioral gains. The data also suggest that CS/RT can drive behavioral gains in human stroke patients who have already received standard forms of rehabilitation following stroke. CS/RT is likely inducing enhanced motor improvements by driving functional reorganization of ipsilesional motor cortex. Electrical stimulation in the absence of motor training can induce the expansio n of movement representations in healthy motor cortex as well as increase synaptic densities and dendr itic hypertrophy (Teskey et al., 2002; Monfils et al., 2004; van Rooyen et al., 2006). CS/RT is associated with increased synaptic density (Adkins et al., 2008) and enhanced synaptic responses (Teskey et al., 2003) within ipsilesional motor cortex. Relative to RT alone, CS/RT also results in a greater expansion of microstimulation evoked movement representations in ipsilesional motor cortex (Kleim et al ., 2003, Plautz et al., 2003; Boyc huk et al., 2009). In the present study motor maps were not anal yzed, however, MTs that measure the

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109 minimum current necessary to evoke forelim b movement by CS/RT stimulation were recorded. The CS/RT condition was associated with a significantly smaller MT on the final day of assessment relative to the RT condition. Similarly, t he CS/RT condition was associated with significantly greater percent reduction in MT across days of rehabilitation. While these MTs lack the spatial resolution of ICMS, they do reflect changes in cortical excitabi lity (Monfils et al., 2005). T hese data support that motor improvements can be mediated by reorganization of motor cortex and that CS/RT can magnify these processes. Other neuroplastic changes that may support CS/RTs enhanced behavioral gains include angiogenesis, gliogenesis or glial activation, neuroprotection, or secreti on of growth factors. Here, 16 days of the combined therapy of CS/RT enhanced motor improvements above and beyond 16 prior days of RT alone in a rodent model of stroke. Previous studies have found magnified behavioral gain s with CS/RT relative to RT, however, CS/RT has always been initiated without any prior rehabilitative experience (AdkinsMuir and Jones, 2003; Kleim et al., 2003; Teske y et al., 2003; Adkins et al., 2006; Adkins et al., 2008). In the present study t he RT condition exhibi ted no increase in reaching accuracy after 10 days of rehabilitat ion while the CS/RT condition increased reaching accuracy for 25 days of rehabilitation. These data indicate cortical stimulation can induce functionally relevant change in the nervous system after it has already responded to rehabilitative experience and argue that rehabilitative training does not exhaust or disrupt neuroplastic changes associated with CS/RT. The data also suggest that CS/RT will not be limited in human stroke patients if they have previously received rehabilitative training.

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110 In the experiment CS/RT was shown to enhance motor improvements in a rodent model of MCAo. Previous CS/RT using r odent models of stroke have used focal ischemic damage within motor cortex (Adk ins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Plautz, et al., 2003; Adkins et al., 2006; Adkins et al., 2008). These results are some of the first evidence that CS/RT can be applied to motor cortex to induce motor improvements when ischemic injury is located in remote, lateral cortex (see Chapters 3 and 5). It is not clear how CS/RT enhances motor improvements following MCAo. CS/RT may be inducing greater behavioral gains by the same mechanisms that have been observed with CS/R T after focal ischemic damage to motor cortex. It is known that MC Ao disrupts forelimb movement representations within motor cortex (Gharbawie et al., 2005a; Gharbawie et al., 2008) as well as skilled reaching behavior (Gharbawie et al., 2005b; Gharbawie et al., 2008). Given that CS/RT is known to promote the reemergence of movement representations (Kle im et al., 2003; Plautz et al., 2003), in particular forelimb representatio ns (Boychuk et al., 2009) within spared motor cortex, it is possible that CS/RT can cause enhanced motor improvements by driving reorganization of motor cortex a fter MCAo as well. The present studys association with CS/RT and enhanced reductions in movement threshold demonstrate a striking similarity to the same phenomenon ob served with CS/RT after focal damage to motor cortex. In both cases, there appears to be changes in cortical excitability that indicate reorganization of motor cortex. CS/RT induced motor improvements in animals that had already received rehabilitative training alone indicating that the nervous system can be continually remodeled by appropriate interventions and experience. The present findings lend

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111 further support for the use of CS/RT in enhancing behavioral gains in human stroke patients.

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112 Figure 4-1. Representative Ni ssl stained coronal section fr om the MCAo lesion. The medial-lateral extent of brain damage at 40 days following ischemic insult by 3 l injection of the vasoc onstricting peptide ET-1 (0.2 g/ l) is shown.

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113 Figure 4-2. Reaching performance prestroke and during rehabilitati on. A) Mean (SD) percent reach accuracy both prestroke and across 20 days of rehabilitative training on the skilled reaching task. Multiple comparisons (Fishers PLSD; p<0.05) showed that while all animals receiving rehabilitation demonstrated significant increases in reaching accura cy during the 32 days of rehabilitation, the animals receiving CS/RT had signific antly higher reaching accuracies that RT Controls on days 26 and 28-32 (Fishers PLSD; p<0.05; indicated by the symbol ). B) Mean (SD) number of reac h attempts both prestroke and across 20 days of rehabilitative training on the skilled reaching task. Further comparisons (Fishers PLSD; p<0.05) found that none of the rehabilitation groups were significantly different from one another on any day.

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114 Figure 4-3. Movement thresholds during r ehabilitation. A) Both the RT and CS/RT conditions showed a progressive decrease in mean (SD) movement threshold as training continued. The CS/R T condition exhibited a significantly larger percent reduction in movement th reshold compared to the RT condition ([F(1,14) = 8.8931; p<0.01] indicated by the symbol ). B) A significant negative correlation was found between t he percent reduction in movement threshold (MT2-MT1/MT1*100) and the increase in post rehabilitation reaching accuracy (Mean final 3 r ehabilitation days Mean first 3 rehabilitation days) [r=-0.5433, p<0.05].

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115 Table 4-1. Estimate of spared cortical and subcortical tissue from experiment 3. Estimated volumes for cortical and s ubcortical tissue were analyzed as the percent affected to unaffected side (v olume lesioned hemisphere/volume non lesioned hemisphere*100) in order to a ccount for individual differences in brain size. Data is represented as mean percent SEM. Condition Sample Size Estimates of Spared Tissue Volume Cortical (% Contralesional ) Subcortical (% Contralesional) HC 8 99 100 CS/RT 8 90 93 RT 8 89 94 NT 7 92 93

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116 CHAPTER 5 CORTICAL STIMULATION DOES NOT ENHANCE MOTOR FUNCTION AFTER SUBCORT ICAL STROKE Introduction In the United States approximately 795, 000 new or recurrent strokes occur each year (American Heart Association, 2009). Th e nature and severity of the functional deficits observed after stroke are widely varied and reflect the fact that infarctions occur virtually anywhere in the brain (Tekin et al., 2002; Saczynski et al., 2009). Because of the cerebral cortex represents the largest brain area, most strokes involve loss of cortical tissue. However, more than 20% of all ischemic stroke cases involve damage to subcortical structures (Bryan et al., 1999; Saczynski et al., 2009). Many of the motor impairments present after subcortical stroke o ccur as a result of damage to descending white matter connections (Norrving, 2003; Arakawa et al., 2006). Indeed, stroke damage in the posterior region of the inte rnal capsule has been associated with relatively severe impairments and little restor ation of motor function (Morecraft et al., 2002; Lie et al., 2004; Wenzelburger et al ., 2005). In some ca ses, upper extremity impairments show less functional improvement after damage within the posterior limb of the internal capsule than after damaged to motor cortex (Shelton and Reding, 2001; Schiemanck et al., 2008). Recent advances in MR imaging have allowed measurement of the degree of damage in white matter in human stroke patients based on impairments of water diffusion within these tracts (Werring et al., 2000). Reduced fractional anisotropy, one measure of axonal integrity, within the first weeks after stroke correlates with further loss of fractional anisotropy and motor function in pat ients with poor motor outcome (Mller et al., 2007). More symmetric al bilateral fractional anisotropy after stroke damage with the coronal r adiata or internal capsule is correlated with improved

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117 motor function at a three-month assessment (Jang et al., 2005). MR spectroscopic measurements of the neuronal marker compound N-acetyl aspartate in the posterior limb of the internal capsule found that the extent the stroke damage intersected with motor pathways exhibited a stronger relationsh ip with the resulting motor deficit than the total size of the lesion (Pineiro et al ., 2000). Functionally, the absence of TMS responses within the first forty-eight hours is associated with complete hand palsy (Pennisi et al., 1999). In patients with some motor recovery, there is also evidence for cortical reorganization after subcortical damage withi n the corticospinal tract. For instance, there is a positive correlation between the extent of subcortical CST damage and the recruitment of the contralesi onal motor and sensory cortices (Schaechter et al., 2008). The recruitment of secondary motor areas in the contralesional hemisphere is also associated with motor improvements in cases of subcortical stroke involving white matter damage (Ward et al., 2003; Ward et al., 2006) including capsular stroke (Gerloff et al., 2006; Lotze et al., 2006). Motor im provements after ischemic damage to subcortical white matter have also been asso ciated with the recruitment of several ipsilesional primary and secondary moto r areas (Loubinoux et al., 2003). Further, Weiller et al. (1993) found a ventral shift in PET activation of motor cortex during a hand movement of stroke patients with damage to the posterior limb of the interior capsule. While studies of human patients with subcortical white matter damage suggest ipsilesional reorganization of motor areas may have a func tionally significance, the similarity to functional reorganization of motor cortex following cortical ischemia is not known. Similarly, it is not clear whether st roke injuries to subcortical white matter will

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118 respond to the same treatments as stroke injuries to cortex. The unique injury profiles of cortical and subcortical stroke sug gest that the underly ing mechanisms of recovery/compensation may not be the same. A greater of the understanding of the importance of lesion location will help guide the appr opriate selection of treatments. Stroke patients with good recovery c an demonstrate fMRI activations patterns with reorganized activity around the central sulcus (Cramer, 2000). Cortical stroke followed by a high degree of motor recovery exhibits an enlargement and posterior shift of the sensorimotor areas (Rossini et al., 1998). Patients given constraint-induced movement therapy after cortical stroke exhibit decreased PET activation during motor tasks and increased TMS evoked motor maps in the ipsilesional hemisphere (Wittenberg et al., 2003). In animal studies, isc hemic damage to motor cortex results in a loss of synapses (Hasbani et al., 2001; Zhang et al., 2005; Murphy et al., 2008) and a loss of ICMS evoked movement representat ions (Nudo and Milliken, 1996; Friel et al., 2000). In the absence of rehabilitative training the majority of lost motor representations do not reappear and the topography of the remaining map is not significantly altered (Nudo and Milliken, 1996; Friel et al., 2000). Appropriate moto r training after cortical ischemia results in motor improvem ents and an expansion and reemergence of movement representations (N udo et al., 1996a; Kleim et al ., 2003). The reemergence of perilesional motor maps is also associ ated with synaptogenesis (Kleim, 2009). Thus, cortical ischemia results in a loss of move ment representations t hat are likely restored by rehabilitative training through synaptic pl asticity including the formation of new synapses (Stroemer et al., 1995; Buonoman o and Merzenich, 1998; Kleim et al., 2002a; Kleim et al., 2004; Brown and Murphy, 2007). It is unclear whether these same forms of

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119 cortical plasticity also occur in response to motor rehabilitation afte r subcortical stroke Animal studies comparing neuroplastic changes within motor cortex a fter cortical versus subcortical stroke are lacking because most me thods for experimentally inducing stroke involve damage to the cortex. In addition to the potentially different neur al substrates for both motor impairment and functional improvement after cortical vers us subcortical stroke, these two different lesions may respond differentially to plastici ty promoting, adjuvant therapies. Several adjuvant therapies that prom ote synaptic plasticity within motor cortex such as amphetamine (Stroemer et al., 1998; Adkins and Jones, 2005; Ramic et al., 2006; Papadopoulos et al., 2009) and nicotine (G onzalez et al., 2005) have magnified behavior improvements in models of cortical ischemia. Further, enhanced motor improvements and expansions in forelimb movement representations have been demonstrated after cortical ischemia by pha rmacological stimulation of intracellular pathways that participate in synaptic plasticity (MacDonald et al., 2007). Electrical stimulation of sensorimotor cortex increases synaptic efficacy (Trepel and Racine, 1998) and expands movement repr esentations (Teskey et al., 2002; Monfils et al., 2004). In animal models of cortical ischemia electric stimulation of motor cortex during rehabilitative training (CS/RT) enhances be havioral improvements (Adkins-Muir and Jones, 2003; Teskey et al., 2003; Adkins et al ., 2006; Adkins et al., 2008). CS/RT also results in further expansion and reorganization of movement represent ations (Kleim et al., 2003; Plautz, et al., 2003; Boychuk et al., 2009). In addition, CS/RT results in a greater increase in the number of synapses (Adkins et al., 2008) as well as enhanced synaptic responses (Teskey et al., 2003). However, none of these treatments have

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120 been used to enhance motor performance after subcor tical stroke. Specifically, all of the animal studies have examined the efficacy of CS/RT after cortical stroke where stimulation is being applied to both damaged and healthy tissue. This is an important clinical question given the re cent finding that CS/RT faile d to confer enhanced motor improvements in a large populati on of stroke patients (Plow et al., 2009). An important difference between the basic and clinical studies is the location of ischemic damage. Clinical studies of CS/RT have enrolled pati ents with cortical and capsular ischemic damage (Brown et al., 2006; Levy et al., 2008). In contrast, preclinical animal studies have used focal cortical models of ischemia (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Adkins et al., 2006; Boychuk et al., 2009). Without animal studies of subcortical white matter stroke to compare the effects of CS/RT or even more generally the functional reorganization patterns to cortical stroke damage the efficacy of CS/RT after capsular infarct is not known. Here, a model of predominantly cortical stroke and a model of subcortical capsular stroke were used to assess how lesion location impacts the benefits of combined CS/RT therapy. Methods Subjects Forty adult male Long-Evans hooded rats (350-420g) were pair housed (2 animals/cage) in standard laboratory cages on a 12:12 hour light-d ark cycle within the University of Floridas Communicore Res earch Building vivarium All experimentation was conducted during the light cycle. Rats were maintained on Lab Diet 5001 (PMI Feeds, St. Louis, MO) and water ad libitum, and were handled and cared for in accordance with the National In stitutes Health Guide for the Care and Use of Laboratory

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121 Animals and with the approval of the University of Florida s Institutional Animal Care and Use Committee (IACUC). Reach Training Over the course of several days, animals we re placed on a restricted diet until they measured 90% of their original body weight. A brief period of pretraining was then given to familiarize the rats with the reaching task Pretraining involved placing them into test cages (10 X 18 X 10 cm) with floors constr ucted of 2 mm bars, 9 mm apart edge to edge. A 4 cm wide and 5 cm deep tray fill ed with food pellets (45 mg; Bioserv) was mounted on the front of the cage. The rats were required to reach outside the cage and retrieve pellets from the tray. Rats were permitted to use either limb and the preferred limb was noted for each animal. All rats remained in pretraining until they had successfully retrieved 10 pellets (approximatel y 1 hour/day for 2 days). After pretraining, the rats were placed into a Plexiglas cage (11 cm X 40 cm X 40 cm) with a 1 cm slot located at the front of the cage. Animals were trained for 15 minutes each day to reach with their preferred limb through the slot and retrieve food pellets from a table outside the cage (Whishaw and Pellis, 1990). Each session was videotaped and later used to assess reaching performance. A successful r each was scored when the animal grasped the food pellet, brought it into the cage and to its mouth without dropping the pellet. The percentage of successful reaches [(# successfu l retrievals/the total # of reaches) x 100] was then calculated. All training sessions were video taped and used to measure reaching accuracy. Animals were trained fo r approximately two weeks on this task to establish a baseline measure of motor performance. Baseline was defined as the average accuracy across the 3 final days of trai ning. Animals failing to achieve a mean reaching accuracy of 40% across 3 consec utive days were not used in the study.

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122 Animals were sorted by their prelesion reaching performance to create groups with comparable baseline levels of reaching accuracy. This was done to ensure that reaching performance prior to infarction was similar across conditions. Infarction Following the 2 weeks of mo tor training, focal ischemic damage was induced in either in the area of the proximal branches of the middle cerebral ar tery (MCA) or in the subcortical territory associated with the inte rnal capsule (IC). Briefly, animals were anesthetized with ketamine hydrochloride (70 mg/kg i.p.) and xylazine (5 mg/kg i.p.). Animals received low levels of isofluorane (0.15%) and supplemental doses of ketamine (20 mg/kg i.p.) as needed. Under sterile c onditions, an incision was made midline and the skull was removed overtop the appropriate injection site. The vasoconstricting peptide endothelin-1 (ET-1: 0.2 g/ L; American Peptide, Sunnyvale) was then injected either adjacent to the middle ce rebral artery to induce cortic al ischemia or adjacent to the internal capsule to induce subcortical white matter ischemia. ET-1 was delivered through a glass pipette via the Nanolitre inje ction system (World Precision Instruments, Sarasota, Fl) controlled by the SYS-Micro 4 Controller (World Precision Instruments, Sarasota, Fl). Stereotaxic coordinates of the injection site for the middle cerebral artery occlusion (MCAo) were: anteroposterior, +0.9 mm; mediolat eral, -5.2mm; and dorsoventral, -8.6 mm with respect to bregma (Biernaskie and Corbet 2001). Stereotaxic coordinates of the injection site near the internal capsule were: anteroposterior, +0.9 mm; medi olateral, -5.2mm; and dorsoventral, -6.5 mm with respect to bregma (Frost et al., 2006). For the MCAo 3 l of ET-1 (240 pMol dissolved in 0.9% sterile saline) were used while for the IC stroke model 1 l of ET-1 (80 pMol dissolved in 0.9% sterile saline). For all injections, t he ET-1 was delivered at a rate of 7nL/sec

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123 through a glass pipette and the pipette was left in the injection site for 5 minutes to avoid backflow. After the surgery all animals were returned to thei r home cage for three days where they were supervised for health concerns but otherwise left to recover. Animals were given full access to food until t he last day where food restriction regimen (see above) was reimplemented. Cortical Electrode Implantation Following the infarction procedure nine-pin electrode carriages (Plastics One Inc., Roanok e, VA) were implanted epidurally over sens orimotor cortex in the hemisphere contralateral to each animals preferred paw The surface electrode was placed directly over the entire exposed cortex between 1 mm posterior to 5 mm anterior to bregma and 0.5 mm to 5.5 mm lateral to midline. A re turn lead was fixed to the skull in a position posterior to Lambda and the craniotomy filled with gel foam. Both the electrode and gel foam were covered in non-ex othermic PolyWave dental acrylic and cured with a brief pulse (50 seconds) of ultraviolet light. T he electrode was then fixed to skull screws with standard dental acrylic and the animals were gi ven 4cc of warm ringers solution (s.c.) and metacam (0.10 mg/kg; s.c.). Movement Thresholds After surgery all animals were returned to their home cage for three days where they were supervised for health concerns bu t otherwise left to recover. Animals were given full access to food until the last day where food restriction regimen (see above) was reimplemented. After t he three days, animals with implants had their indiv idual motor thresholds (MTs) det ermined and then animals we re started in a motor rehabilitation paradigm. MTs were assessed on post lesion training days 1, 10 and 19. MTs were defined for each animal as the minimum current to cause an involuntary

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124 motor response and were tested in all animals that had received a cortical electrode. The animals were placed into a transparent cylinder and observed while 3 second trains of 1 millisecond 100 Hz monopolar cathodal pul ses were given. Current was gradually increased by 5% increments until a movement of the contralateral forelimb could be clearly detected. Cortical st imulation during the post inju ry motor training phase was then delivered at 50% of each animals MT during rehabilitation for the CS/RT condition. CS/RT and RT Three days after surgery began receiving daily twenty-mi nute sessions of motor rehabilitation. Each session consisted of sk illed reach training using the same training parameters described earlier. All sessions were video taped for analysis of reaching accuracy and number of reach attempts. Animal s receiving the combination of cortical stimulation and rehabilitative training were st imulated via the Vertis Stimulation System during these sessions. The cortical electrod e was connected to a remote stimulator suspended above the training cage where info rmation was then sent wirelessly to the rest of the system. CS/RT was delivered as monopolar cathodal st imulation and was administered continuous with a frequency of 100 Hz with a current intensity dictated by the subjects movement threshold. Each pulse was biphasic, charged balanced and asymmetric consisting of a square phase lasting 100 + 10 microseconds and a decaying exponential phase lasting ~9900+ 10 microsec onds. Half the animals that received MCAo were given rehabilitative training alone (MCA-RT; n=8) wh ile the other half received the combination of cortical stimulation and rehabilitative training (MCA-CS/RT; n=8). Similarly, in the two groups that received subcortica l ischemic damage near the internal capsule, half of the animals received rehabilitative training alone (Sub-RT; n=8) while the other half received the combination of cortical stimulation and rehabilitative

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125 training (Sub-CS/RT; n=8). A group of anima ls that never received an infarction were included as Healthy Controls (HC; n=8) and were given training on the same skilled reaching task on all days of rehabilitation. Histology and L esion Verification Following the rehabilitation phas e the ani mals were given an overdose of pentobarbital and then transcardially perfused with 0.1 M sodium phosphate buffer followed by 4% paraformaldehyde solution in the same buffer. Brains were then extracted and post fixed in 4% paraformaldeh yde solution in 0.1 M sodium phosphate buffer. Serial 50 m coronal sections were then taken using a microtome. Ten sections spaced 600 m apart and spanning approximately 2.7 mm anterior and 3.3 mm posterior to bregma were sampled for lesion verification. The same number of sections was analyzed for each animal. The sampled sect ions were stained with Toluidine blue (a Nissl stain) and digitally scanned (Espon Perfection V500 Photo Scanner, Long Beach, CA) for lesion verification. The area of spared tissue, characterized by consistent Nissl staining, was traced using Image J software (Abramoff et al., 2004; Rasband, 2009) and cortical volumes were estimated with the Cavalieris unbiased estimator method using the formula: volume di 1 n yi ( t ) ymax Where yi is the cross sectional area of the ith section through the morphometric region, d is the distance between sections (600 m) and n is the total number of sections (12). ymax is maximum value for the area of one section and t is the section thickness (50 m) and their product is subtracted from the basic question as a correction for the overprojection (G undersen 1986; Gundersen and Jensen, 1987;

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126 Mayhew 1992). Estimated volumes for cortical and subcorti cal tissue were analyzed as the percent affected to unaffected side (volume lesioned hemisphere/volume non lesioned hemisphere*100) in order to account fo r individual differences in brain size. Results Reaching Accuracy A repeated measures ANOVA with CONDITION as a between subject factor and TIME as a within subj ect factor revealed a significant CONDITION x TIME interaction [F(80,700)= 2.1027; p<0.0001] on reaching accuracy (Figure 4-1A). Subsequent multiple comparisons (Fishers PLSD; p<0. 01) revealed that groups given either experimental stroke had significantly lo wer reaching accuracies on day 1 of rehabilitation in comparison with pre-stroke levels and with performance levels of the HC group. No significant impr ovements were seen in the Sub-RT condition after 13 days of rehabilitation and in the Sub-CS/RT condition after 12 days of rehabilitation. No significant improvements were seen in the MCA-RT condition after 5 days of rehabilitation and in the MCA-CS/RT condition after 8 days of rehabilitation. The SubRT and Sub-CS/RT groups were not significant ly different from one another on any day (Fishers PLSD; p<0.05). In contrast, mult iple comparisons of the MCA-RT and MCACS/RT groups found that the MCA-CS/RT pe rformed significantly greater reaching accuracy than MCA-RT on days 8-9 and 18-20. The Sub-RT condition demonstrated significantly smaller percent reach accuraci es than the HC conditi on on days 1-20. The Sub-CS/RT animals demonstrated significant ly smaller percent reach accuracies than the HC condition on days 1-14 and 16-20. The MCA-RT group demonstrated significantly smaller percent reach accura cies than the HC condit ion on days 1-20. The MCA-CS/RT condition demonstrated significant ly smaller percent reach accuracy than

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127 the HC condition on days 1-15 and 17-20. Thes e results demonstrate that while the combined therapy of CS/RT following MC Ao magnified motor improvements, the combined therapy of CS/RT conferred no a dditional benefit following subcortical ischemia. Reaching Attempts A repeated measures ANOVA with CONDITION as a between subject factor and TIME as a within subj ect factor revealed a significant CONDITION x TIME interaction [F(80,700)= 1.6819; p<0.001] on the number of reach at tempts (Figure 4-1B). Subsequent multiple comparisons (Fishers PLSD; p<0.01) revealed that all groups that received lesions had significantly lower reachi ng accuracies on day 1 of rehabilitation in comparison with pre-stroke levels and with per formance levels of the HC group. Further comparisons (Fishers PLSD; p<0.05) found t hat the Sub-CS/RT a nd Sub-RT conditions performed significantly similar numbers of reach attempts on a ll days. Similarly, multiple comparisons found that the MCA-CS/RT and MCA-RT performed significantly similar numbers of reach attempts on all days. The Sub-RT condition demonstrated a significantly smaller number of reach attemp ts than the HC conditi on on days 1-20 while the Sub-CS/RT condition demonstra ted a significantly smaller number of reach attempts than the HC condition on days 1-16 and 18-20. The MCA-RT condition demonstrated a significantly smaller number of reach atte mpts than the HC condit ion on days 1-13 and 16-20 while the MCA-CS/RT condition demonstr ated a significantly smaller number of reach attempts than the HC condition on days 1-11, 13 and 18-20. No significant increases in reach attempts were seen in t he Sub-RT condition after 11 days or in the Sub-CS/RT condition after 10 days of rehabilita tion. No significant increases in reach attempts were seen in the MCA-RT condi tion after 8 days or in the MCA-CS/RT

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128 condition after 9 days of rehabilitation. Thes e results demonstrate that while all groups increased the number of number of reach attempts during rehabilitation, no differences in the number of reach attempts were obs erved between the RT and CS/RT conditions following subcortical ischemic damage near IC or MCAo. Movement Thresholds A repeated measures ANOVA with TIME revealed a significant effect of TIME [F(6,56) =17.5017; p<0.0001] on mean movem ent threshold (Figure 4-2). Subsequent multiple comparisons (Fishers PLSD; p< 0.01) revealed that all conditions had signific antly lower MTs on the third assessm ent relative to the first. No significant difference in MT was found between the SubCS/RT and Sub-RT conditions during any of the three assessments. In contrast, t he MCA-CS/RT group exhibi ted a significantly lower MT relative to the MCA-RT group on the third threshold assessment. Further comparisons (Fishers PLSD; p<0.05) found t hat all four groups ex hibited significant decreases in the percent MT between t he first and final assessment. Again, no significant difference was found between the Sub-CS/RT and Sub-RT conditions while the MCA-CS/RT condition exhibited a signifi cantly greater percent decrease in MT relative to the MCA-RT condition (Fisher s PLSD; p<0.05). Multiple comparisons (Fishers PLSD; p<0.05) also found the Sub-CS/RT and Sub-RT groups to have significantly higher thresholds relative to both the MCA-CS/RT and MCA-RT groups at all time points. Estimation of Remaining Tissue A one-way ANOVA revealed a significant main effect of CONDITION on the estimates of remaining cortical tissue [F(4 ,35) = 10.3268; p<0.05] Subsequent multiple comparisons (Fishers PLSD; p<0.05) showed t hat the amount of resi dual cortical tissue

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129 was similar for the HC, SubRT and Sub-CS/RT conditions (Table 5-1). The amount of residual cortical tissue was also similar between the MCA-RT and MCA-CS/RT conditions (Fishers PLSD; p<0.05). In addition, the HC, SubRT and Sub-CS/RT conditions exhibited a significantly greater am ount of residual cortical tissue relative to the MCA-RT and MCA-CS/RT conditions (Fis hers PLSD; p<0.05). A one-way ANOVA revealed a significant main effect of CONDITION on the estimates of remaining subcortical tissue [F(4,35) = 3.0039; p< 0.05]. Subsequent multiple comparisons (Fishers PLSD; p<0.05) showed that the amount of residual subcortical tissue was similar for the HC, Sub-RT and Sub-CS/RT conditions. The amount of residual cortical tissue was also similar between the MCA-RT and MCA-CS/RT conditions (Fishers PLSD; p<0.05). Finally, the HC, Sub-RT and Sub-CS/RT conditions exhibited a significantly greater amount of residual cortical tissue rela tive to the MCA-RT and MCACS/RT conditions (Fishers PLSD; p<0.05). Discussion Animal studies have provided clear evi denc e that cortical stimulation in combination with motor rehabilitation can si gnificantly enhance both motor performance and cortical plasticity after cortical ischem ia (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Plautz, et al., 2003; Adkins et al., 2006; Adkins et al., 2008). The present study examined the efficacy of this treatment for augmenting motor performance after subcortical ischemia. T he animals received either a predominantly cortical infarct by MCAo or subcortical infarc t involving damage to the internal capsule. Although both injury models resulted in forelimb motor impairments, motor improvements during twenty days of rehabilitative training were greater in the MCACS/RT condition than the Sub-CS/RT group. Indeed, the Sub-CS/RT animals showed

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130 no significant benefit of CS/RT over rehabilitat ion training alone. This suggests that the efficacy of adjuvant therapies may be dependent upon lesion location. Prior animal studies of CS/RT have model ed stroke by inducing focal ischemic damage within motor cortex (Adkins-Muir and Jones, 2003, Kleim et al., 2003; Teskey et al., 2003). The present finding that CS/RT can also enhance behavioral gains following MCAo has not been previously p ublished but is supported by other experiments in this dissertation (see C hapters 3 and 4). The MCAo model in rodents results in a disruption of mo vement representations (Ghar bawie et al., 2008) that is likely due to a loss of synapses and dendrites within motor cortex (Zhang et al., 2005). After ischemic damage within motor cortex the reemergence of perilesional motor maps following rehabilitative traini ng is associated with synaptogenes is (Kleim, 2009). CS/RT after focal ischemic damage within motor co rtex results in m agnified motor map reorganization (Kleim et al., 2003; Plaut z et al., 2003; Boychuk et al., 2009) and synaptogenesis (Adkins et al., 2008). Rehabilita tive training after MCAo may result in similar motor map reorganization and synapt ogenesis that can be facilitated by CS/RT in a similar fashion to what has been observ ed in the focal cortical models. Indeed, Stroemer et al. (1995) found an association between behavior improvements and synaptogenesis within sensorimot or cortex following MCAo that indicates a functional importance for restoring synapt ic input in motor cortex. Sparing of the CST is an important predictor of motor outcome following subcortical stroke (Pennisi et al., 1999; Pineiro et al., 2000; Werring et al., 2000). In the present study animals receiving subcortica l lesions were given ischemic damage near the internal capsule. The integrity of the CST follo wing ischemic insult near the internal

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131 capsule was not directly measured but can be inferred by the animals responses during MT assessments. Movement could be elicited in animals given the subcortical lesion, however, the amounts of current needed was ma rkedly higher than in animals given the cortical lesion. These responses suggest the CST was intact but damaged in the subcortical lesion animals. Behaviorally, animals that received the subcortical lesion exhibited smaller initial impai rments than animals that received the cortical lesions by MCAo. These behavioral results also suggest that the subcortical lesions did not eliminate all descending mo tor output through the internal capsule with the consideration that other motor tracts or uncrossed CST fiber s from the unaffected hemisphere may have also contributed to the post injury reaching behavior. There is no immediate explanation for the lack of enhanced behavioral gains with CS/RT after ischemic damage to the internal capsule. The motor impairments observed in the present study are suppor ted by previous reports where focal ischemic damage to the internal capsule resulted in impairments in forelimb placing (Frost et al., 2006) and forelimb exploration within a cylin der (Lecrux et al., 2008). Injuri es to the CST result in a loss of cells in motor cortex (Pernet and Hepp-Reymond, 1975; Hains et al., 2003) and a retraction of damaged pyramidal cell axons (Galea and Darian-Smith, 1997). Injury to the CST is also associated alterations in motor cortex pyramidal cells including cell shrinkage and a decrease in Nissl staining (W annier et al., 2005). While ICMS has not been performed on a model of capsular infarc t, motor mapping has been performed on a more downstream portion of the CST within the pyramid tracts. Complete unilateral lesions of the pyramid tract (pyramidotomy) results in impaired forelimb function and a loss of forelimb movement representations within motor cortex (Pie charka et al, 2005).

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132 In contrast to cortical isc hemic insult, rehabilitation after pyramidotomy does not result in reorganization of movement representations (Piecharka et al, 2005). Similarly, large ischemic lesions in primate motor cortex result in an expansion of movement representations within the se condary motor area PMv rather than within primary motor cortex (M1) (Nudo, 2007). In a primate model of stroke a linear relationship between increasing lesion size within M1 and incr easing reorganization of PMv was observed (Dancause et al., 2005). Collectively, thes e studies suggest that post infarct reorganization of movement representations within motor cortex requires sufficient sparing of this motor area and its axonal projections. In the absence of sufficient remaining motor cortex, postinfarct reor ganization of the motor system may depend on the recruitment of secondary motor areas outside of motor cortex. For example, human stroke patients with damage to the CST oft en demonstrate a recrui tment of secondary motor areas (Ward et al., 2003; Ward et al., 2006). It is possible then that CS/RT was ineffective in enhancing behavioral improvements following experimental damage within internal capsule because its promotion of reorganization of movement maps within motor cortex was not as functionally relevant as recruitment of secondary motor areas. Even though the model of capsular infarct used here likely spared many pyramidal cells it is possible they were in insufficient quantities to support behavioral gains through reorganization of motor cortex. Enhanced motor improvements with CS/RT have been observed in several animal studies but the mechanisms underlying the enhancement are not clear. Cortical stimulation may have neuroprotective, angiogenic, anti-inflammatory or growth factorreleasing properties (Baba et al., 2009). CS/RT is also associated with increased peri-

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133 infarct reorganization of movement representations. Appropriate reha bilitative training following cortical ischemia results in motor improvements that are accompanied by an expansion and reemergence of mo vement representations t hat are specific to the training (Nudo et al., 1996b; Nudo, 2006) that is likely mediated by synaptic plasticity (Kleim et al., 2002; Kleim et al., 2004). Cort ical stimulation is associated with greater reorganization of the motor map than RT alone (Kleim et al ., 2003; Plautz et al., 2003; Boychuk et al., 2009). In particular, CS/R T results in increased distal forelimb representations that have a strong posit ive correlation with skilled reaching ability (Boychuk et al., 2009). CS/RT after cortical ischemia is also associated with a greater number of synapses (Adkins et al., 2008) and increased synaptic responses (Teskey et al., 2003) that likely support the increased reorganization of perinfarct motor maps. The present findings further support the viability of CS/RT for enhancing motor function after cortical stroke. The result s also demonstrate the importance of lesion location in CS/RT as cortical stimulation was ineffective in magnifying behavioral improvements in a model of subcortical ischemia involving white matter damage. Future clinical trials of CS/RT must consider t he location of stroke to promote functional reorganization and motor recovery.

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134 Figure 5-1. Representative Ni ssl stained coronal section fr om the MCAo lesion. The medial-lateral extent of brain damage at 24 days following ischemic insult by 3 l injection of the vasoc onstricting peptide ET-1 (0.2 g/ l) is shown. Figure 5-2. Representative Nissl stained coronal section from the capsular lesion. The subcortical white matter damage at 24 da ys following ischemic insult by 1 l injection of the vasoconstricting peptide ET-1 (0.2 g/ l) is shown.

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135 Figure 5-3. Reaching performance prestrok e and during 20 days of rehabilitation. A) Mean (SD) percent reach accuracy. The Sub-RT and Sub-CS/RT groups were not significantly different from one another on any day (Fishers PLSD; p<0.05) while the MCA-CS/RT exhibit ed significantly greater reaching accuracies than MCA-RT on days 89 and 18-20 (Fishers PLSD; p<0.05; indicated by the symbol ). B) Mean (SD) number of reach attempts. The Sub-CS/RT and Sub-RT conditions did not exhibit significantly different number of reach attempts on any day (F ishers PLSD; p<0.05). Similarly, the MCA-CS/RT and MCA-RT groups not exhibit significantly different number of reach attempts on any day (Fishers PLSD; p<0.05).

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136 Figure 5-4. Mean (SD) movement thresholds a fter cortical or subcortical ischemia. All conditions had significantly lower mo vement thresholds on the third assessment relative to the first (Fis hers PLSD; p<0.01). The Sub-CS/RT and Sub-RT groups exhibited significantly hi gher thresholds relative to both the MCA-CS/RT and MCA-RT groups at all ti me points (Fishers PLSD; p<0.01; indicated by the symbol #). No signifi cant difference in movement threshold was found between the Sub-CS/RT and SubRT conditions. In contrast, the MCA-CS/RT group exhibited si gnificantly lower movement thresholds relative to the MCA-RT group on the third th reshold assessment (Fishers PLSD; p<0.05; indicated by the symbol ).

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137 Table 5-1. Estimate of spared cortical and subcortical tissue from experiment 4. Estimated volumes for cortical and s ubcortical tissue were analyzed as the percent affected to unaffected side (vol ume lesioned hemisphere/volume non lesioned hemisphere*100) in order to a ccount for individual differences in brain size. Data is represented as mean percent SEM. Condition Sample Size Estimates of Spared Tissue Volume Cortical (% Contralesional ) Subcortical (% Contralesional) HC 8 99 98 MCA-RT 8 91 94 MCA-CS/RT 8 89 94 Sub-RT 8 100 98 Sub-CS/RT 8 99 99

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138 CHAPTER 6 GENERAL DISCUSSION Summary The present set of experiments explored the use of electrical stimulation of motor cortex as a treatment adjuvant to magnify the behavior al benefit of rehabilitative training after ischemic brain damage. The experiments have produced several important findings. First, the distribution of stimulat ing contacts was ident ified as an important factor for cortical stimulat ion and rehabilitative training (C S/RT). CS/RT with stimulating contacts distributed across motor cortex resulted in enhanced motor improvements while CS/RT with focal stimulation confe rred no benefit. Second, within distributed configurations, the number of independent contact points did not alter the efficacy of the treatment. Third, prior rehabilitative training did not prevent enhanced motor improvements with CS/RT. Finally, the location of injury influenced the effectiveness of CS/RT. Animals with subcortical stroke did not respond differently to the combined therapy relative to animals that recieved RT alone. The goal of these studies is to have biologically relevant basic science inform pot ential clinical applications. To that end, these results are highly relevant. While t hese and other studies show that cortical stimulation paired with rehabilitative training has demonstrat ed a behavioral benefit in preclinical animal stud ies, a recent phase III of the t herapy in stroke patients failed (Plow et al., 2009). This demonstrates the co mplexity of translating such animal data into clinical application. The results of the present set of studies demonstr ate that there are critical parameter s of both delivery of CS/RT (electrode distribution) and the nature of the injury (cortical versus subcortical) that influence the effica cy of the therapy.

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139 Study #1: Distributed Versus Focal Co rtical Stimulation To Enhance Motor Function And Motor Map Plasticity After Experimental Ischemia. The first two studies were performed to ident ify the most effective configuration of stimulating contacts for CS/R T. The first experiment determined how the distribution of cortical stimulation affects motor improvem ent, and motor map plasticity, by comparing focal versus distributed electrode configurati ons. The total contact surface area between the distributed and focal configurations was kept constant and focal ischemic damage within motor cortex was used as the experi mental lesion. Distri buted CS/RT stimulated a greater proportion of non-distal forelimb representations relative to focal CS/RT based on the location of contacts within the residual motor map as well as by the responses to motor threshold testing. CS/R T with a distributed configurati on of stimulating contacts induced greater motor improvements than RT alone or CS/RT with a focal distribution of contacts. Distributed CS/RT was also associ ated with a greater expansion of movement representations compared to RT alone and the size of the post rehabi litation motor map positively correlated with motor improvements. CS/RT has previously been associated with magnified expansion of the motor map (Kleim et al., 2003; Plautz et al., 2003). CS/RT in this first experiment was specific ally associated with an expansion of distal representations. Changes in di stal representations in the intact motor cortex reflect changes in the ability to perform skilled wrist and forelimb movements during skilled reach training (Monfils et al., 2005). The si ze of post rehabilitati on distal movement representations positively correlated with motor improvements. Similarly, the increase in distal representations during rehabilitation also positively correlated with motor improvements. There was al so a trend for distributed CS/RT to exhibit a greater decrease in the current needed to evoke a movement with CS/RT stimulation (MT)

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140 relative to focal CS/RT. While lacking the spatial resolution of intracortical microstimulation (ICMS), these changes in movement threshold reflect changes in cortical excitability and reorganization of motor cortex (Monfils et al., 2005). It is possible that distributed CS/RTs association with greater motor improvements was due to its stimulation in movement representations adjacent to the repr esentations that were lost by the ischemic damage. Appropriate rehabi litative training alone results in some reemergence of movement repres entations that were lost by the injury (Nudo et al., 1996a; Kleim et al., 2003). Stimulation may be facilitating t he reemergence of movement representations by reinforci ng coincident activity during appropriate rehabilitative training (Jackson et al., 2006). Distributed CS/RT s stimulation of neighboring representations may facilitate t he invasion of representations that are emphasized by motor training into these areas. Although CS/RT with focal and distribut ed stimulation resulted in greater expansions of movement r epresentations only distributed CS/RT resulted in enhanced motor improvements. The dissociati on between motor map reorganization and behavioral improvements suggests that these measures share a non-linear relationship. This finding has previously been reported in rodent studies where electric (Kleim et al., 2003) or pharmacological stimulation (Ma cdonald et al., 2007) induces motor map changes without robustly impacting behavior. It is possible that motor map restoration is necessary but not sufficient to support moto r improvements and that other neuroplastic changes are needed to induce behavioral gains. Map plasticity appears necessary as all animals that demonstrated enhanced motor recove ry also exhibited si gnificantly greater motor maps within residual tissue. It is also possible that certain patterns of motor map

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141 reorganization are more relevant than others. In a primate model of focal ischemia within motor cortex, Plautz et al. (2003) observed a 410% expansion of hand representations after CS/RT using a pelle t retrieval task that required fine hand movement. These hand expans ions occurred in areas of motor cortex that had previously been occupied by proximal fore limb representations and the greatest expansions of hand representations occurr ed near the stimulating contacts of the CS/RT electrodes (Plautz et al., 2003). In the present study comparing focal versus distributed types of CS/RT, both types of CS/RT resulted in map expansions, however, only distributed CS/RT was associated with a greater proportion of distal representations compared to RT controls whil e the focal was not. Further, the total area and expansion of distal representations obs erved by ICMS at the end of the study positively correlated with post injury reaching improvements. Electrical stimulation of motor cortex in the absence of behavioral trai ning expands motor maps, however, it is does not alter the proportion of different type s of movement representations (Monfils et al., 2004). The close relationship between the expansion of distal forelimb representations and motor im provements with distributed CS /RT suggests that changes in specific movement repr esentations are an important measure of functional reorganization after injury. Study #2: Cortical Stimulation Plus Rehabilitative Training Enhances Motor Function Independent of The Number of Stimula ting Contacts After Experimental Ischemia. The results of the first study showed that distributed stimulation was more effective than focused stimulation at enhancing motor improvement and inducing motor map expansion. To further exami ne the importance of how the topography of stimulation influences motor improvement, the second study used a distributed contact

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142 configuration but varied the number of independent contacts. The total electrode contact area was held constant at (1.2 mm2) but distributed through either a 2x2 array (four contacts) or a 3x3 array (nine contacts). The 2x2 array had previously demonstrated enhanced behavioral gains (see chapter 2). The st imulation from CS/RT is likely driving motor improvements by inducing reorganization of motor cortex throu gh the facilitation of coincident activity during rehabilitative tr aining (Jackson et al., 2006). It was expected that the 3x3-CS/RT configur ation would drive greater behavioral gains than 2x2-CS/RT because its additional contact sites placed with in independent areas of motor cortex should promote greater reorganization of motor cortex. The result s how that 3x3-CS/RT induced greater behavior gains compared to RT alone but not relati ve to 2x2-CS/RT. Instead, the 2x2 and 3x3 arrays were associated with highly similar patterns of motor improvements in rodents giv en middle cerebral artery occlusion (MCAo). The 2x2CS/RT and 3x3-CS/RT also exhi bited similar patterns of motor improvement as a Dual Rail-CS/RT condition that was included in the study to allow comparison to previous CS/RT work in rodent models of stroke (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Adkins et al., 2006; Adkins et al., 2008). The close similarity in patterns of behavioral improvements betw een 2x2-CS/RT, 3x3-CS/RT and Dual RailCS/RT suggests the number of contact site s does not alter the effectiveness of CS/RT at least within the range of contact sites examined here. There is likely a limit to the smallest number of contacts that can support CS/RT. For instance, CS/RT through one contact is unable to evoke involuntary move ment during motor threshold testing (see chapter 2). The similarity bet ween 2x2-CS/RT and 3x3-CS/RT i ndicate there is also an upper limit to the number of CS/RT stimulating sites needed to enhance motor

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143 improvements. There is also the caveat with the model used in these studies, in that because of the small size of t he motor cortex relative to the electrodes, there may not be sufficient space in which to test for subtle differences in the number of contacts. For example, in an animal with a larger cortex mo re dramatic differences in contact number could be compared. Regardless, the efficacy of both the 2x2 and 3x3 electrodes speaks to the robustness of the therapy. Study #1 demonstrated that distributed CS/RT magnifies behavioral gains while focal CS/RT does not, i.e., focal CS/RT direct ed at the center of the motor map does not enhance motor improvements (see chapter 2). This suggests that stimulation of the borders of the motor cortex may be critical. This is consistent with the finding that no additional behavioral benefit is observed with the Dual Rail and 3x3 configurations that both stimulate a larger pr oportion of the center of motor cortex than the 2x2 configuration. In other words, the 3x3 and Dual Rail configurat ions may confer no additional benefit relative to the 2x2 conf iguration because they are not stimulating additional sites on the edges of the forelim b map where forelimb areas could expand into non-forelimb areas such as whisker, jaw or neck. Indeed these border areas have been shown to be malleable in response manipu lations that increase neural excitability including the application of GABA antagonists (Sanes and Donoghue, 2000) and intracortical stimulation (Nudo et al., 1990). The similarity of behavioral outcomes with these three configurations is also likely the re sult of all of them su fficiently stimulating motor cortex in a distributed manner that targets the more peripheral movement representations. While the exact similariti es in the neuroplastic changes induced by these three configurations are not known, all three of these CS /RT configurations

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144 resulted in similar reductions in motor thre sholds relative to RT alone. The motor thresholds represent a general measure of exci tability as well as the ability of CS/RT stimulation to activate the corticofugal proj ections of motor cortex Reductions in motor thresholds also positively correlated with mo tor improvements during the rehabilitation phase of this experiment. Study #3: Cortical Stimulation With Re habilitative Training Can Enhance Motor Improvements After Early Application of Rehabilitat ion Alone in a Rodent Model of Ischemia Currently most stroke interventions focu s on the acute phase and involve trying to reduce the amount of tissue lo ss by promoting reperfusion wit h mechanical or chemical clot busters. While these treatments can succe ssfully reduce infarction size and reduce functional impairment, most stroke victims fail to receive the treatment in time and suffer motor deficits of varying degrees. In these pat ients the only current treatment is motor rehabilitation and most patients receive severa l weeks of rehabilitation after the insult. Thus there is a large population of individu als with chronic stroke that have undergone motor rehabilitation that, regar dless of the efficacy, has likely induced changes within residual neural tissue. These changes may influence the efficacy of CS/RT. In all previous CS/RT studies, the animals receiv e treatment after stroke but without prior rehabilitation experience. The third study was performed to investigate whether prior rehabilitation interacted with CS/RT. Anim als were given an experimental model of MCAo stroke followed by a phase of RT and then a phase of RT or CS/RT. The results showed that even after extensive motor rehabi litation and significant gains in motor performance, CS/RT could induce further motor improvements. It is important to note that the sa me neurophysiological and neuroanatomical changes that occur in residual motor cortex after RT alone also occur with CS/RT.

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145 Specifically, rehabilitative training results in an expansion of movement representations within motor cortex (Nudo et al., 1996a; Kleim et al., 20 03) and CS/RT results in an even greater expansion (Kleim et al., 2003; Plautz et al., 2003). While the areas of motor cortex that exhibit expansions of movement repr esentations also exhibit synaptogenesis in response to motor training (Kleim et al., 2002a; Kleim et al., 2004), CS/RT is associated with even greater incr eases in synaptogenesis (Adkins et al., 2008). Finally, CS/RT is also associated with enhanced synaptic responses following therapy (Teskey, et al., 2003). By thes e measures CS/RT appears to be enhancing behavioral improvements by magnifying the i nnate neuroplastic changes that occur in response to standard RT. These data sugge st that CS/RT and RT are generally complimentary to one another and that the nervous system can be continually remodeled after injury by experience and ad juvant therapies. Motor mapping was not performed in this third experiment but MTs were assessed. RT was associated with a reduction in motor thresholds while CS/R T was associated with enhanced reductions in MTs. Reduced MTs suggest increased synaptic strength consistent with prior observations of enhanced synaptic responses (Teskey et al., 2003) and synaptogenesis (Adkins et al., 2008). Study #4: Cortical Stimulation Does Not Enhance Motor Function After Subcortical Stroke The fourth experiment examined the si gnific ance of lesion location for CS/RTs ability to enhance motor improvements after brai n ischemia. In the clinical trials of CS/RT, patients are enrolled who have suffered either cortical or capsular ischemic damage (Brown et al., 2006; Levy et al., 2008). In contrast, preclinical animal studies have only used cortical models of ischemia to study CS/RT (Adkins-Muir and Jones,

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146 2003; Kleim et al., 2003; Teskey et al., 2003; Adkins et al., 2006; Boychuk et al., 2009). For the fourth experiment a r odent model of capsular isc hemia was developed in order to test CS/RTs ability to drive motor impr ovements in a rehabilitation phase. The effects of CS/RT following damage to th e internal capsule (IC) were compared to the effects of CS/RT following a model of middle cerebral ar tery (MCA) stroke. RT following ischemic damage in the internal capsule or MCAo resu lted in motor improvements. Compared to RT alone, CS/RT enhanced motor improvement s in animals that had received MCAo but conferred no benefit in animals that had received capsular ischemia. MTs were significantly higher following capsular isc hemia compared to thre sholds after MCAo. After MCAo, animals that received RT exhibi ted a reduction in their MTs. Animals given CS/RT after MCAo exhibited an even greater decrease in MTs. Animals given CS/RT also exhibited a reduction in MTs following capsular ischemia, however, the reduction was statistically similar to the reduction observed in animals given the same type of lesion and RT alone. Potential reasons for CS /RTs lack of effect in this model of capsular stroke are described below. Possible Neural Bases for CS/RTs Enhanced Motor Outcomes The effect of CS/RT appears to be mediated through an enduring c hange in the synaptic connectivity of the motor cortex as evidenced by motor map reorganization (Kleim et al., 2003), enhanced synaptic responses (Teskey et al., 2003) and synaptogenesis (Adkins et al., 2008). Persistent enhancements in motor performance have been observed four to fourteen weeks post CS/RT therapy in pr imates (Plautz et al, 2003) and as long as nine to ten months post CS/RT therapy in rodents (A. OBryant, A. A. Sitko, and T. A. Jones, unpublish ed observations). Studies with human stroke patients have observed enhanced motor performance in individuals given three weeks

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147 of CS/RT as far as twelve w eeks later (Brown et al., 2006) There are numerous sites of plasticity in the nervous system that suppor t motor improvements a fter injury. Following ischemic injury, CS/RT may be inducing brain plasticity such as peri-infarct reorganization (Nudo and Milliken 1996), recruitm ent of ipsilesional (Loubinoux et al., 2003; Ward et al., 2003) or contralesional areas (Johansen-Berg et al., 2002), shifts in interhemispheric interactions (Murase et al., 2004; Duque et al., 2005;) or shifts in bihemispheric connectivity (Seitz et al., 2004). These brain changes during CS/RT may be the result of processes in cluding expansion of motor m aps, angiogenesis, alterations in synaptic strength, neuroprotection, sy naptogenesis, dendritic hypertrophy, release of growth factors or suppression of inflammatory responses. Direct Effects of CS/RT Stimulation Little is known of the direct actions of CS/RT stimulation in human motor cortex. The electrical stimulation used in CS/RT is administered at a frequency in the range of fifty to one-hundred pulses per second. Each pulse is biphasic, charged balanced and asymmetric consisting of a square phase la sting 100-250 microseconds followed by a decaying exponential phase. Models of CS in human M1 have given some details of the location of current density. CS electrodes placed directly above a model of the precentral gyrus result in half of the current flow entering the gyrus while the remainder passes do wn the banks of the sulci (Wongsarnpigoon and Grill, 2008). Neuronal activation with this electrode placement is thought to be isolated to the crown of the gyrus whereas cells further down the banks of sulcus would have increased membrane potentials that are not sufficient for ac tivation (Wongsarnpigoon and Grill, 2008). The magnitude of the activation is inversel y related to the thickness of CSF (Wongsarnpigoon and Grill, 2008). Changes in the electrode placement on the modeled

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148 precentral gyrus or changes in the thickness of the gyrus alters the activation pattern largely by changes in the orientation of neurons beneath the electrode (Wongsarnpigoon and Grill, 2008). Neuronal axons have the highest probability of becoming activated by electrical stimulat ion because they have the highest density of ion channels (Huerta and Volpe, 2009). Thus the predominant effect of electrical stimulation in the cortex is the presynaptic release of neur otransmitter onto postsynaptic cells by axonal stimulation. Most cortical neurons release glutamate while a smaller portion release GABA. However, electric stimul ation of axons in motor cortex may lead to the presynaptic release of glutamate, GABA, acetychol ine, dopamine, norepinephrine or serotonin (Huerta and Volpe, 2009). The re sponse to even a single pulse of electrical stimulation is likely mixture of activation in excitatory, inhibitory and neuromodulatory pathways from intracortical afferents. Dir ect responses by activation of axons of corticofugal fibers are also po ssible by stronger electrical stimuli, however, the primary responses observed by electric stimulation of the cortex demonstrate latencies more indicative of indirect responses by the activa tion of intracortical afferents (Jankowska et al., 1975). Serial trains of elec trical stimuli in motor cortex have the capacity to activate entire networks over wide areas of cortex bas ed on the projections of postsynaptic cells receiving neurotransmitter release withi n motor cortex (Fetz and Cheney, 1980). However, weak electrical stimuli in moto r cortex are thought to activate organized groups of inputs to pyramidal tract neur ons (Porter and Lemon, 1995). Finally, the temporal and spatial facilitation of electric al stimulation within the cortex may support coincident activity of incoming afferent signals that does not originate from the electrical stimulation resulting in t he strengthening of neuronal connections (Jackson et al., 2006).

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149 Potential Neuroplastic Responses to CS/RT Electric stimulation of s ensorimotor cortex immediately after MCAo results in neuroprotection, angiogenesis, the release of growth factors, and suppression of inflammatory responses (Baba et al., 2009). CS /RT is typically initiated beyond the acute phase of ischemic injury, however, each of these processes merit attention because of their potential to al ter the brains response to stroke damage. Of this list, only the role of neuroprotection in CS/RT has been investigated. Early applic ation of electrical stimulation after ischemic insult decreases cell death and infarct volume (Glickstein et al., 2003; Maesawa et al., 2004) Some animals CS/RT studies have not reported spared tissue volumes (Kleim et al ., 2003; Teskey et al., 2003). Of those that do report spared tissue volume, most observe enhanced motor improvements with no differences relative to RT groups (Adkin s-Muir and Jones, 2003; Adkins et al., 2008; Boychuk et al., 2009). In the present ex periment no differences in spared tissue volumes were observed. Adkins et al. (2006) found that relative to an RT group, one group of CS/RT initiated 10-14 days after ischemia was associated with enhanced motor improvements and a greater perilesional neuronal density that was not due to an increase in newly born BrdU -labeled cells. In this same study, a second group with CS/RT initiated 10-14 days after ischem ia was associated with enhanced motor improvements and no elevated pe rilesional neuronal density re lative to the RT group (Adkins et al., 2006). These authors also obs erved a CS/RT group that demonstrated enhanced motor improvements and relatively le ss spared tissue (a greater difference in contralesional versus ipsilesional cortical volume) but the decreased spared tisse was attributed to the CS/RT group tending to have the greatest contralesional and lowest ipsilesional volumes (Adkins et al., 2006). Further, studies have reported enhanced

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150 motor improvements when CS/RT is initiated 3 months after experimental ischemia in primates (Plautz et al., 2003) or in human patients at leas t 4 months post stroke with average delays of 28 months (Brown et al ., 2006) and 32.8 months (Levy et al., 2008). In these studies CS/RT ability to enhance moto r improvements in a more chronic phase of stroke suggests that the underlying mechanism of CS/RT is not neurprotection. The relationship between CS/RT and the release of growth factors deserves attention. Many growth factors have demonstrated neuroprotective effects when administered acutely in animal models of stro ke. For instance, administration of BDNF early after ischemic injury reduces the loss of cells and infarct volume (Wu and Pardridge, 1999; Zhang and Pardridge, 2006). Ad ministration of vector expressing glialderived neurotrophic fact or (GDNF) during MCAo is associated with smaller infarcts and less apoptotic markers such as TUNEL staini ng (Tsai et al., 2000; Yagi et al., 2000). Intravenous administration of vascular-endothelia l growth factor ( VEGF) within two days after ischemic damage induces angiogenesis and restores neuronal function (Zhang et al., 2000). The chronic effect s of increasing neur otrophic factor levels are less understood making it difficult to characterize t heir relationship with CS/RT. For example, administration of HSV-amplicon-based vector encoding GDNF promoted behavioral improvements after MCAo when delivered four days prior but not three days after the lesion (Harvey et al., 2003). Chronic VEGF administration after stroke is cautioned because of its secondary effects of incr easing vessel permeability, edema and the activation of inflammatory responses (S hibuya, 2009). Understan ding the relationship between BDNF and CS/RT is becoming increasingly important because delayed administration of BDNF following ischemic injury enhances motor recovery most likely

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151 by increasing synaptic connections within the br ain (Schabitz et al., 2004; Schabitz et al., 2007; Muller et al., 2008). It is known that motor learning increases BDNF levels in the motor cortex (Klintsova et al., 2004). Furt her, blocking BDNF signaling by infusions of antisense BDNF oligonucleotide attenuates motor improvements during motor training after MCAo (Ploughman et al., 2009). BDNF may facilitate reorganization of motor cortex by influencing neuroplastic changes such as synaptogenesis or dendritic hypertrophy (Biernaskie and Corbett, 2001; Klei m et al., 2003; Monfils et al., 2005; Mller et al., 2008). Given that no differences infarct volume were observed in the present studies, if growth fact ors are involved they are likely promoting plasticity rather than neuroprotection. The relationship between CS/RT and angiogenesis, glial responses and the suppression of inflammatory responses has not been examined. Glial responses are of interest as interventions such as skill ed reach training or exposure to enriched environments result in behavioral improvem ents that are associated with suppressed proliferation of microglia/m acrophages and increased prolifer ation of astrocytes in perilesional cortex (Keiner et al., 2008). Angiogenesis and the suppression of inflammatory responses are particularly important in the acute phase. Lowering inflammatory responses may increase the number of cells that survive brain ischemia by blocking the production of cytokines and NO and preventi ng neutrophil and macrophage infiltration into brain parenchyma (Vexler and Yenari, 2009). Angiogenesis may be supporting neurons that are endangered by low blood su pply in the ischemic penumbra (Noshita et al., 2001). It is likely CS /RT is initiated outside of the window to fully influence acute mechanisms such as neuroprotection, angiogenesis, and

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152 suppression of inflammatory responses. However, because CS/RT is administered relatively earlier in preclinical animal studi es than in clinical trials using human stoke patients it is important to study these responses as important translational issues. Reorganization of Motor Cortex With CS/RT Many animal studies of CS/RT have r eported reorganization at the site of stimulation: the ipsilesional motor cortex. The pa tterns of reorganization in motor cortex after CS/RT share a high degree of similari ty with patterns in the same area during healthy motor training. Skille d motor traini ng results in a reorganization of motor representations as representations for mo vements involved in the motor task expand their territories at the expens e of others. The reorganization of motor cortex occurs later in skilled training after initial improvements in skill behavior (Kleim et al., 2004) and is temporally paralleled by in creased synaptogenesis (Kleim et al., 1996; Kleim et al., 2002a; Kleim et al., 2004). Following the disco very that LTP can be induced in the neocortex of awake behaving rodents (Racine et al. 1995, Trepel and Racine, 1998). Rioult-Pedotti et al. (1998) demonstrated th at the acquisition of skilled motor behavior results in LTP-like potentiation in the horiz ontal afferents within motor cortex. This potentiation of motor co rtex occurs late in training after initial improvements in skill behavior (Monfils and Teskey, 2004). Interesti ngly, motor cortex plasticity is task specific with neither strength training (Rem ple et al., 2001) nor endurance training (Kleim et al., 2002b) resulting in reorganization of motor cortex. Furt her, reaching on an unskilled task does not result in motor map reorganization (Kleim et al., 2004). In animal studies, ischemic damage to moto r cortex results in a loss of synapses (Hasbani et al., 2001; Zhang et al., 2005; Mur phy et al., 2008) and a loss of movement representations (Nudo and Mill iken, 1996; Friel et al., 2000). In the absence of

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153 rehabilitative training the majo rity of lost motor represent ations do not reappear and the topography of the remaining m ap is not significantly altered (Nudo and Milliken, 1996; Friel et al., 2000). Appropriate motor training after cortical ischemia results in motor improvements and an expans ion and reemergence of mi crostimulation-evoked movement representations (N udo et al., 1996a; Kleim et al ., 2003). The reemergence of perilesional motor maps is also associat ed with synaptogenesis (Kleim et al., 2002a; Kleim et al, 2004). CS/RT studies of experimental ischemia have observed a heightened increase in reorganization of movement repres entations within motor cortex during rehabilitation (Kleim et al., 2003; Pl autz et al., 2003; Boychuk et al., 2009). Increases in the size of movement r epresentations following CS/RT on a skilled reaching task positively correlated with increases in motor improvements (Boychuk et al., 2009). CS/RT appears to expand motor maps by emphasizing the reemergence of representations that were lost by the ischemic injury such as hand representations in a primate model of stroke (Plautz et al., 2003) or distal forelimb representations in a rodent model of stroke (Boychuk et al., 2009). Electrical stimulation of motor cortex in the absence of behavioral training expands motor maps, however, it is does not alter the proportion of different types of movement representations (Monfils et al., 2004). In chapter 2, increases in the proportion of distal forelimb representations following CS/RT on a skilled reaching task positively correlat ed with increases in motor improvements (Boychuk et al., 2009). While the invasive ness of ICMS prevents its use in human stroke patients, the possibility of cortical reorganization was raised in a study that noted individual finger movements in response to intraoperative cortical mapping that patients

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154 were not capable of voluntarily making before cortical stimulation and rehabilitative training (Brown et al., 2006). CS/RT is also associated with synaptic changes that may support the functional reorganization of movement representations. CS/RT results in an increased density of axodendritic synapses including increases in (presumed efficacious) perforated synapses and multiple synaptic boutons within layer 5 of mo tor cortex (Adkins et al., 2008). In addition, CS/RT is associated wit h enhanced polysynaptic responses in motor cortex (Teskey et al., 2003). In CS/RT studi es, MTs are typically assessed on the first day of rehabilitation following experimental stroke as well as every seven to ten days subsequent. It is common to fi nd that all conditions rece iving RT or CS/RT exhibit decreases in MTs through days of rehabilitat ion (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Adkins et al., 2006; Boychuk et al., 2009). In chapters 2-5 of this dissertation the CS/RT groups t hat demonstrate enhanced motor improvements also exhibited the greatest reductions in MT In several of the present experiments, decreases in MT positively correlated with motor improvements after brain ischemia. While these MTs lack spatial resolution compared to ICMS they do reflect general levels of excitability in motor cortex (Monfils et al., 2005). Excitabi lity can be altered by the number of cells or the intr insic excitability of individual cells and the strength of their synaptic connections (Redecker et al., 2000) Given that CS/RT is not typically associated with neuroprotection or the proliferation of new cells it is likely that affects the intrinsic excitability of cells within motor cortex (Adkins et al., 2006). Shifts in Excitability With CS/RT CS/RT may be mediated through a resolution of cortical hyperexcit ability or hyperinhibition. Ischemic insult results in a di sruption of excitability at many levels such

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155 as through a loss of cells, their connections and the expression of their receptors (Redecker et al., 2002). Early after ischem ia, many regions of cortex become hyperexcitable as NMDA receptors are upregulated and GABA-subt ype A receptors are downregulated (Redecker et al., 2000). For example, the PM v motor area demonstrates hyperexcitability and reduced motor maps suggesting that increases in excitability may disrupt the function of co rtical areas (Nudo, 2007). After the acute phase of stroke there is typically a reduced output or activation of the affected hemisphere (Desrosiers et al., 2006) whereas the opposite case is found in the contralesional hemisphere (Cauraugh et al., 2000). The contralesional hemisphere may also be exerting excess inhibition on the affected hemisphere (Platz et al., 2005) Many stroke treatments using noninvasive stimulation employ a general strategy of tryi ng to encourage activity in the ipsilesional hemisphere or discourage it in the contra lesional hemisphere (Talelli and Rothwell, 2006). In the ipsilesional hemisphere, stim ulation protocols that facilitate neuronal activity such as high-frequency repetitive transcranial magnetic stimulation (rTMS) (Khedr et al., 2005, Kim et al., 2006) anodal transcranial direct current stimulation (Hummel and Cohen, 2005) or intermittent theta-burst stim ulation delivered ipsilesionally (Talelli et al ., 2007, Di Lazzaro et al., 2008) result in motor improvements. Similarly, low-frequency rTMS (Fregni et al., 2006, Mansur et al., 2005) cathodal transcranial direct current stimulation (Fr egni et al., 2005) or continuous theta-burst stimulation (Talelli et al., 2007, Di Lazzaro et al., 2008) deliv ered contralesionally also result in behavioral improvements. The enhanced synaptic responses, synaptogenesis and motor map expansions with CS/RT suggest that it is facilitating or activating the ipsilesional hemisphere. It is possible that CS/RT is enhancing motor improvements

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156 after brain ischemia by encouraging the activi ty and output of the motor cortex in the injured hemisphere. The Effect of CS/RT in New Lesion Models CS/RT After MCAo In chapters 3-5, CS/RT enhanced motor im provements following a middle cerebral artery occl usion type stroke model. E nhanced behavioral improvements following MCAo have not been reported in the literature. The original animals studies that found enhanced behavioral gains with CS/RT indu ced ischemic damage focally to motor cortex by electrocoagulation of surface vasculature (Kleim et al., 2003; Plautz et al., 2003), pial stripping (Teskey et al., 2003) or topical application of the vasoconstricting peptide ET-1 (Adkins-Muir and Jones, 2003; Ad kins et al., 2006; Adkins et al., 2008). The present results suggest that CS/RT ca n induce functional benefits when ischemic damage is outside of motor cortex. It is unclear how CS/RT affects brain repair following MCAo. The MCAo model in rodents results in forelimb motor impairments (Gharbawie et al., 2005a; Gharbawie et al., 2005b; Ghar bawie and Whishaw, 2006) and a disruption of movement representations within motor cortex (Gharbawie et al., 2008). Depending the extent of MCAo lesion, motor map dysfun ction may be the result of disruption of the cortico-basal ganglia-thalamo-cortical loops (Karl et al., 2008), however, neurotoxic striatal damage affecting cells but sparing axon s in the striatum di d not alter motor map topography (Karl et al., 2008). Further, smalle r MCAo lesions that spare basal ganglia, motor cortex and the CST still resulted in a di sruption of the motor maps (Gharbawie et al., 2005b). The loss of motor m aps following MCAo may be due to a loss of cells or synaptic connections within motor cortex (Garci a et al., 1997). MCAo is associated with decreased excitability within mo tor cortex indicated by a reduction in field potential

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157 amplitudes (Neumann-Haefelin an d Witte, 2000). MCAo also results in a loss of large neurons in layer five of moto r cortex (Moisse et al., 2008) as well as a loss of synapses and dendrites within sensorimotor cortex (Z hang et al., 2005). Stro emer et al. (1995) found an association between behavior improvements and sy naptogenesis within sensorimotor cortex following MCAo that indicates a functi onal importance for restoring synaptic input in motor cortex. Further, the ICMS techniques that produce motor maps rely on both direct stimulation of pyramidal ce ll axons as well as indi rect stimulation of pyramidal cells by transynaptic recruitment (Jankowska et al., 1975; Fetz and Cheney, 1980). Using ICMS, Bolay and Dalkara (1998) found a recovery of the direct wave whereas a persistent deterioration of the indirect (transynaptic) wave in the pyramidal tract following MCAo. The persistent dysfunc tion of the indirect wave suggests the associated map dysfunction is due to a loss of intrahemispheric connections within motor cortex rather than a loss of pyramidal cells. Given the damage within motor cortex following MCAo, CS/RT may be enhancing motor improvements by restoring connections within motor cortex. This may be mediated by strengthening residual connections within motor cortex by facilitating coincident activity (J ackson et al., 2006). In support of this notion, in chapters 35 where MCAo was used to cause ischemic damage, CS/RT was associated with greater reductions in motor threshold than RT alone suggesting that cortical stimulation did increase ex citability within the motor cortex. CS/RT After Capsular Infarct The motor impairments observed in the pr esent study are supported by previous reports where focal ischemic damage to the in ternal capsule res ulted in impairments in forelimb placing (Frost et al., 2006) and forelim b exploration within a cylinder (Lecrux et

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158 al., 2008). Capsular infarct is typically a ssociated with increased motor thresholds to electric stimulation that are likely due to a loss of or dysfunction within pyramidal tract cells (Liepert et al., 2005). In juries that damage the corticospinal tract at the level of either the pyramids (Wohlfarth, 1932; Per net and Hepp-Reymond, 1975) cervical spinal cord (Holmes and May, 1909; Levin and Bradford 1938) or thoracic spinal cord (Hains et al., 2003) result in a loss of large pyrami dal neurons in the motor cortex. The loss of pyramidal cells following CST injury is a graded response. For example, unilateral pyramidotomy affecting 60%, 90% or 100% resu lts in a decrease in the proportion of pyramidal cells in motor cortex of 27%, 35% or 53% respectively (Pernet and HeppReymond, 1975). Injury to the CST is also associated with alterations in surviving pyramidal cells including cell shrinkage and a dec rease in Nissl staining (Wannier et al., 2005). Retrograde tracing experiments have dem onstrated a reduction in CST axons to the hemicord caudal to a C3 hemisecti on (Galea and Darian-Smith, 1997). While detailed histological assessment of motor cortex following internal capsule has not been performed to confirm similar findings as the spin al cord injury models, it is expected that internal capsule damage results in a loss and di sruption of pyramidal tract neurons from layer five of motor cortex. Ischemic damage in motor cortex is associated with a loss of neural connections that is demonstrated by a loss of dendritic arbor (Zhang et a., 2005) as well as by a loss of synapses (Hasbani et al., 2001; Zhang et al., 2005; Murphy et al., 2008). The disruption in neural connectivity is furt her demonstrated by a persistent loss of microstimulation-evoked movement represent ations within motor cortex (Nudo and Milliken, 1996; Friel et al., 2000) Stroke damage to the lateral aspect of the brain by

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159 occlusion of the middle cerebral artery also causes a disr uption of movement representations within motor cortex (G harbawie et al., 2008). The disruption of movement representations within motor co rtex was due to a loss of intracortical synapses indicated by the persistent impairment of the indirect (transynaptic) response to ICMS in the pyramidal tract (Bolay and Dalkara, 1998). In support, histological examination of sensorimotor cortex following MCAo shows a loss of synapses and dendrites (Zhang et al., 2005) and the restorati on of these synapses is associated with behavioral improvements (Stroemer et al., 1995). Rehabilita tion after focal ischemic injury to motor cortex or MCAo results in motor improvements t hat are likely mediated by a restoration of local cortical circuitr y in motor cortex in volving synaptogenesis (Stroemer et al., 1995; Buonomano and Merzenich, 1998; Kleim et al., 2002a; Kleim et al., 2004; Brown and Murphy, 2008). Giv en CS/RTs enhancement of motor map expansions (Kleim et al., 2003; Plaut z et al., 2003; Boychuk et al., 2009), synaptogenesis (Adkins et al., 2008) and reduc tions in MT (Adkins-Muir and Jones, 2003; Kleim et al., 2003; Teskey et al., 2003; Adkins et al., 2006; Boychuk et al., 2009) it is likely CS/RT is enhancing motor impr ovements by magnifying the brains response to rehabilitation. CS/RTs inability to enhance motor improv ements after capsular infarct in the present rodent model suggests it is not able to facilitate the restoration of local circuitry in motor cortex. One possibility is that the damage with this model does not spare enough pyramidal cell fibers to permit a restoration of loca l circuitry because there is insufficient corticofugal output to a llow appropriate feedf orward and feedback mechanisms. However, the animals motor improvements in response to rehabilitative

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160 training and CS/RTs ability to evoke movement during MT testing indicate that there was substantial sparing of CST fibers. Another possibility is that focal ischemic motor cortex damage and MCAo result in a predominant loss of intracortical afferents that can be restored with RT and CS/RT whereas capsular infarct predominantly results in a loss of pyramidal tract cells and thei r arbor. In this case it is not that pyramidal tract cells have been completely eliminated, but rather, that CS/RT cannot reinforce connections to the remaining pyramidal cells as it ma y be doing with the other two injury types. CS/RT may be unable to reinforce spared connections when the loss of synapses is due to a reduced number of postsynaptic pyra midal cells rather than due to a loss of presynaptic intracor tical afferents. There are many other possi bilities for the lack of CS/R T effect after capsular infarct. Ischemic damage within the internal capsule may be damaging an additional motor tract besides the CST that is impor tant for CS/RT-enhanced motor improvements. Capsular infarct may also be damaging s ubcortico-cortical connections such as thalamic inputs into motor cortex that are necessary for CS/RT-enhanced motor improvements (Axer and Keyserlingk, 2000). If CS/RT is magnifying behavioral improvements by increasing the excitability of the ipsilesional motor cortex, perhaps its inability to do so after capsular infarct is because of a unique pattern of aberrant excitability following this injury. Cortical stroke typically results in acute patterns of hyperexcitability (Redecker et al., 2000) followed by a loss of excitability involving decreases in output and neural activation (D esrosiers et al., 2006). The presumed loss of pyramidal cells following capsular infarct would result in a net loss of excitability. Relative to the cortical lesion models used here, the net loss of pyramidal cells coupled

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161 with the preservation of inhibitory interneurons may result in a state of hyperinhibition. If CS/RT is facilitating motor improvements after co rtical stroke by relieving inhibition it is possible a state of hyperinhibition followi ng capsular stroke prevents CS/RT from increasing cortical excitability to sufficient levels. Contrary to this possibility, however, stroke patients with motor cortex lesions demonstrate higher rate s of intracortical inhibition than patients with internal capsul e lesions (Liepert et al., 2005). Motor improvements after partial stroke damage within the coronal radiata have been associated with a reorganization of the residual fibers (Jang et al., 2005). If motor improvements are mediated thr ough the reconfiguration of CST fibers, CS/RT may lack efficacy in treating these injuries because it cannot facilitate this type of plasticity. It is also possible that brain repair after capsul ar infarct is best accomplished by the recruitment of secondary motor areas that project to spinal co rd motor neurons. In contrast to cortical ischemic insult, rehabilitation after py ramidotomy does not result in reorganization of movement representations (Piecharka et al, 2005). Similarly, large ischemic lesions in primate motor cortex result in an expansion of movement representations within the secondary motor area PMv rather than within M1 (Nudo, 2007). In this primate model of stroke, a li near relationship between increasing lesion size within M1 and increasing reorganization of PMv was observed (Dancause et al., 2005). Finally, some reports from non-invasi ve imaging of human stroke patients have observed an association between stroke damage to the CST and the recruitment of secondary motor areas (Ward et al., 2003; Ward, 2006). Translation of CS/RT From An imal to C linical Studies The lack of a CS/RT treatment effect in a recent phase III clinical trial was unexpected given the many pr evious preclinical and Phase II demonstrations of CS/RT-

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162 enhanced behavioral gains after brain ischemia Preclinical animal studies of CS/RT have also demonstrated a CS/RT-induced enhancement of motor improvements on tasks such as single pellet retrieval (Kleim et al., 2003; Adkins et al., 2006; Adkins et al., 2008), pasta matrix (Teskey et al., 2003), M ontoya staircase (Adkins-Muir and Jones, 2003), and pellet retrieval from a 5 well apparatus in primat es (Plautz, et al., 2003). Clinical reports have indicated that epidural motor cortex st imulation, used to reduce chronic pain after sub-cortical strokes, r educes hemiparetic impairments (Tsubokawa et al., 1993), motor weakness (Katayama et al., 2002), motor spasticity (Garcia-Larrea et al., 1999), action tremor (Nguyen et al., 1998) and dystonia (Franzini et al., 2003). CS/RT resulted in substantial behavioral gain s in a single case report of human stroke patient (Brown et al., 2003). CS/RT has be en associated with greater behavioral gains in both phase I (Brown et al., 2006) and phase II clinical trials (Huang et al., 2008; Levy et al., 2008) using the same procedures as the phase III trial. Interestingly, some patients in the combined CS/R T group of the phase III clinical trial demonstrated robust motor improvements relative to the group re ceiving RT alone (Plow et al., 2009). These studies serve as evidence that electrically stimulating the brain during rehabilitative training can magnify the trainings benefit. Di fficulty in translating CS/RT from the preclinical basic studies to it s application in hum an patients likely cont ributed to the lack of CS/RT effect observed in the phase III trial (Plow et al., 2009). One of the present experiments identified the placem ent of CS/RT stimulation as an important predictor of CS /RT efficacy. CS/RT distri buted across motor cortex enhanced motor improvements while CS/RT adm inistered focally within motor cortex conferred no benefit in a rodent model of st roke (see chapter 2). Distributed CS/RT was

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163 also associated with a significantly gr eater reemergence of distal forelimb representations relative to RT while focal CS/RT was not. Furthermore, the reemergence of distal forelimb represent ations positively correlated with motor improvements during r ehabilitation. Distribut ed CS/RT stimulated a greater proportion of non-distal forelimb representations relative to focal CS/RT based on the location of contacts within the residual motor map as we ll as by the responses to motor threshold testing. These data suggest that stimulation for CS/RT should be targeted to movement representations surrounding an d adjacent to the representations that are have compromised by brain ischemia. CS/RT elec trodes in animal studies are sufficiently large enough to cover the entire motor cortex whereas the CS/RT electrodes in clinical studies only cover a portion of human motor cortex. The volume of human motor cortex is approximately 10.87 cm3 based on histological estimates (Rademacher et al., 2001). The total contact area of the CS/RT electrodes in human clinical trials is constrained by the capacity of the power s upplies and has been between 28.26 mm2 and 42.39 mm2 (Brown et al., 2006; Huang et al., 2008; Levy et al., 2008). Given that CS/RT in human stroke patients will be administered to only a portion of motor cortex, an understanding of the most effective location within motor cortex to administer the stimulation will likely improve CS/RTs effectiveness in human stroke patients. The clinical trials of CS/RT have localized the placement of the stimulating contac ts based on fMRI activation during hand/wrist/finger movement (Brown et al., 2006; Huang et al., 2008; Levy et al., 2008). More detailed localization with fM RI using craniomet er landmarks and phase reversal to localize the central sulcus will likely provide better localization of the appropriate target for CS/RT stimulation movements (Plow et al., 2009). Further, TMS

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164 mapping of ipsilesional motor cortex may prov ide more optimal plac ement of stimulating contacts by providing details of the size and location of each motor map (Plow et al., 2009). If the present data are correct and stim ulation for CS/RT should be directed to movement representations surrounding the representations compromised by brain ischemia, then it will be necessary to perfo rm TMS motor mapping in order to identify each individuals movem ent representations. Additionally, proper localization of CS/RT stimulation will require a greater understanding of the current distributions within human mo tor cortex after stroke. Current distribution modeli ng in rodents is informative but limited due to species differences that affect current such as the size of cortex and dura as well as lissencephalic nature of rodent cortex (Wongsarnpigoon and Grill, 2008). Using MRIderived finite head modeling to examine the effects of TMS stimulat ion, Wagner et al. (2006) demonstrated that cortical stroke in the human brain displaces the location of maximal current density, alters the magnitude of maximal current density and causes the current density to be disj ointed and multifocal around the infarct. More current modeling using noninvasive techniques in human as well as studies with non-human primate motor cortices should permit better lo calization of stimulation for CS/RT. The use of fMRI localization of hand/wrist/finger movement to identify the site of CS/RT stimulation in the clinical studies have ignored the type of rehabilitative training used for CS/RT as many of the rehabilitative tasks tr ained proximal limb movements (Plow et al., 2009). The animal studies have observed littl e transference of CS/RT-induced motor improvements from the trained task to other motor tasks sugges ting that the localization of stimulation and rehabilitative training should be directed at the same type of

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165 movements (Adkins et al., 2006). The activa tion site of hand/wrist/finger movement was used as the site for CS/RT stimulation regar dless of the location of each individuals stroke or its clinical manifestations. Great er clinical benefits of CS/RT will likely be observed if the location of stimulation and rehabilitative training are directed at each patients motor impairments. In one of the present experiments the locati on of stroke injury was shown to be an important predictor of CS/RT efficacy. Here, CS/RT co nferred no additional behavioral benefit relative to RT alone in a rodent model of capsular infarct. These data indicate that CS/RT may not be a viable therapy for human stroke pati ents with capsular damage yet the clinical studies of CS/RT have enrolled patients with cortical and capsular ischemic damage (Brown et al., 2006; Levy et al., 2008). While it is not known if CS/RT can enhance motor improvements in human stroke patients with capsular infarct, CS/RTs lack of effect in the present animal model suggests that the inclusion of these patients in the phase III clinical st udy may have contributed to the failure to observe enhanced motor improv ements with CS/RT. The data also offer the possibility that there may be other loca tions of stroke that do not respond to CS/RT therapy. Additional animal studies with other stroke models should contribute a better understanding to how lesion location affects CS/RT therapies. Clinical studies of CS/RT should also include lesion location as a co variate in analysis. In order to better characterize each patients stroke, the int egrity of the descending motor pathways could be assessed using TMS prior to enrollment for CS/RT therapy because viability of these pathways is an important factor for motor prognos is (Stinear et al., 2007). The integrity of the descending motor pathways after stroke is also an important predictor of brain

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166 activation patterns that may i ndicate different mechanisms of brain repair (Ward, 2006). Interestingly, only 16% of the participants in the CS/RT group in the third clinical trial exhibited evoked movements during intraoperat ive stimulation compared to 100% in the phase I and 42% in the phase II clinical tr ial (Plow et al., 2009). The portion of participants in the phase III trial that did show evoked movements during surgery also responded to CS/RT by exhibiting greater motor improvements compared to participants receiving RT (Plow et al., 2009). There are several other issues concerning the translation of CS/RT from animal studies to clinical studies that were not addressed in the pres ent experiments. For example the most effective versions of t he timing of stimulation during CS/RT, the amount of CS/RT given daily and the intensity of CS/R T given across days are not known (Plow et al., 2009). It is likely that information from noninvasive brain stimulation studies such as TMS and tDCS can guide CS/RT studies (Plow et al., 2009). Additionally, many of the st imulation parameters used in CS/RT such as polarity (Kleim et al., 2003; Adkins et al., 2006) and frequen cy (Adkins-Muir and Jones, 2003; Teskey et al., 2003) of stimulation were identified in rodent models of stroke. The stimulation parameters for CS/RT in the clinical trials have deviat ed from those recommended by the animal work without providing rationales for the parameters selected (Brown et al., 2006; Huang et al., 2008; Levy et al., 2008). Las tly, individual characteristics of each person with stroke may impact responsiveness to CS/RT therapy. Malcolm et al. (2007) demonstrated the importance of individual responses to brai n stimulation in a report where only a subgroup of stroke patients demonstrated the desired effects of rTMS in the affected hemisphere. No animal studies of CS/RT have investigated effects in

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167 females and human clinical trials of CS/RT have not reported sex as a covariate. There is some evidence that sex is predictive of poor functional outcome after stroke even after correction for prognosti c factors (Gargano et al., 2 007; Gray et al., 2007). The sexes may also respond differently to st roke treatments (Saini and Shuaib, 2008). Finally, there appears to be a genetic co mponent to cortical plasticity and responsiveness to brain stimulation. For ex ample, the BDNF val66met polymorphism is associated with less alleviation of drug resistant depression by rTMS (BocchioChiavetto et al., 2008). The val66met polymorphism is also associated with lower training-induced cortical reorganization (Kleim et al., 2006). These individual factors may also be limiting overall effectiveness of CS/RT in clinical studies. Improvements in translating CS/RT from animal studies to human clinical trials as well as appreciating individual differences in responsiveness to br ain stimulation will li kely improve CS/RT therapeutic efficacy in i ndividuals with stroke. Conclusion The variables associated with neural recove ry/compensation after stroke are well understood. An understanding of the mechanisms that produce motor improvements after stroke will lead to more effective treat ments strategies. Afte r brain ische mia, the motor cortex has the capacity to reorganize in response to rehabilitative experience. Adjuvant therapies such as cortical stimul ation have the potential to magnify cortical reorganization and behavioral improvements after stroke. The present studies investigated the importance of several parameters of CS/RT. Distributed configurations of CS/RT electrodes resulted in e nhanced motor improvements and motor map plasticity that were not found with focal conf igurations of CS/RT. Among different types of distributed CS/RT, the num ber of independent contact site s did not affect CS/RTs

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168 ability to augment motor improvements followi ng cortical ischemia. Early rehabilitative training after cortical ischemia did not affe ct CS/RTs ability to augment improved motor performance. In contrast, lesion locati on predicted CS/RT outcome. CS/RT produced enhancements in motor performance in two separat e models of cortical stroke that were absent in a model of subcortical stroke involving white matter damage. The present data support that a more detailed understanding of how to translate CS/RT findings from preclinical animals studies to its clinic al application in human stroke patients will improve its use in this stroke population. These and future studies will provide a greater understanding of how the brain repairs itself after injury that will guide the development of more effective post-stroke treatment inte rventions and ultimately improve the quality of life for stroke victims.

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197 BIOGRAPHICAL SKETCH Jeffery A. Boychuk was born and raised in Lethbridge, Alberta, Canada. He graduated from Lethbridge Collegia te Institute in 2001. He then eared his Bachelor of Science in neuroscience from the University of Lethbridge (U of L) in May of 2005. During his time at the U of L, he worked as a research assistant in Professor John Vokeys laboratory studying cognitive processes such as the psychophysics of fingerprint identification. In the spring of 2005, he joined Professor Jeffrey Kleims laboratory to investigate mechanisms of stro ke recovery. Upon receiving his bachelors degree, he moved with Dr. Kleim to the University of Florida (UF) in Gainesville. In the fall of 2005, he began pursuit of a doctoral degr ee in the UF College of Medicines Interdisciplinary Program in Biomedical Sciences.