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1 IDENTIFYING INFLAMMATORY BIOMARKERS OF COGNITIVE AGING THROUGH RELATIONSHIPS BETWEEN MEASURES OF INFLAMMATION, NEUROGENESIS AND COGNITION IN AGED RATS By RACHEL BROOKE SPEISMAN A DISSERTATION PRESENTED TO THE GRADUATE SCH OOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
2 2013 Rachel Brooke Speisman
3 To my loving family
4 ACKNO WLEDGMENTS The path to a doctoral degree is filled with bumps and detours. I am extremely grateful for the people that have accompanied me on this academic journey. While I do not have enough pages to list everyone, I am indebted to you all for your suppor t. I am most grateful to have had Lawrence Scheinert, my husband and the love of my life, stand by my side through the last five years. I thank him for being patient and understanding, despite the miles between us. He is my rock. His love and support mean the world to me. I would also like to thank my parents, Michael and Renee Speisman. With their guidance every obstacle becomes an opportunity. I appreciate every long phone call, the escapes to the beach and the plate of dinner left in the refrigerator. I am fortunate to have wonderful role models as parents, who have taught me the value of both education and kindness. Additionally, I would like to thank Jordan for being the best big brother and my grandparents Nathan and Elisabeth Weinberger and Rose Speis man for instilling in me the power of perseverance and showering me with unconditional love. I thank Dr. Brandi K. Ormerod for her guidance in the laboratory. She not only taught me how to ask a solid research question, but also how to design an experim ent to answer it. I would also like to thank my fellow lab mates, Aditya Asokan, Vikram Munikoti, Lan Hoang Minh and Crystal Stephens for their camaraderie and expertise. I have also had the pleasure of working with many undergraduate students and would li ke to particularly thank Christian Lee, Jamie Severance and Jessica Pastoriza for their contributions. My dissertation experiments were done in collaboration with Dr. Thomas C. Foster and his laboratory in the McKnight Brain Institute. I thank Dr. Foster f or his
5 generous support and guidance. I am grateful to have had the opportunity to work with the Foster laboratory and especially thank Dr. Ashok Kumar and Asha Rani for all of their efforts. This material is based upon work supported by the National Scien ce Foundation Graduate Research Fellowship program under grant DGE 0802270, National Institute of Health (grants: AG014979, AG036800 and AG037984), the McKnight Brain Research Foundation and the Broad Foundation for Biomedical Research. Finally, this diss ertation would not have been possible without the direction provided by my doctoral committee, Dr Brandi K. Ormerod, Dr. Thomas C. Foster, Dr. Paul R. Carney and Dr. Kyle D. Allen.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 A REVIEW OF COGNITIVE AGING ................................ ................................ ....... 14 Age r elated Cognitive Decline ................................ ................................ ................ 15 The Human Hippocampus and Age related Cognitive Decline ......................... 15 A Rodent Model of Age related Cognitive Decline ................................ ........... 16 Diminished Hippocampal Neurogenesis with Age ................................ .................. 18 The Relationship between Neurogenesis and Cognition ................................ ........ 21 Systemic Regulators of Neurogenesis and Cognition ................................ ............. 23 Stress Hormo nes ................................ ................................ .............................. 24 Inflammation and Neuroinflammation ................................ ............................... 29 Differential Experience ................................ ................................ ............................ 30 Environmental Enrichment ................................ ................................ ............... 30 Physical Exercise ................................ ................................ ............................. 31 Social Interaction ................................ ................................ .............................. 32 2 ENVIRONMENTAL ENRICHMENT RESTORES NEUROGENESIS AND RAPID ACQUISITION IN AGED RATS ................................ ................................ .............. 34 Introduction ................................ ................................ ................................ ............. 34 Methods ................................ ................................ ................................ .................. 36 Subjects ................................ ................................ ................................ ............ 36 Differential Experience: Environmental Enrichment and Individual Housing .... 36 Water Maze Training and Testing ................................ ................................ .... 38 BrdU Injections and Histology ................................ ................................ .......... 39 Immunohistochemistry ................................ ................................ ...................... 40 Cell Quantification ................................ ................................ ............................ 41 Statistical Analyses ................................ ................................ .......................... 43 Results ................................ ................................ ................................ .................... 44 Daily Enrichment Partially Reverses the Effect of Age on Spatial Ability .......... 44 Enrichment enhances spatial learning in aged rats ................................ .... 44 Enrichment enhances cue discrimination learning in aged rats ................. 47 The Effect of Enrichment Overcomes the Effect of Age on Neurogenesis ....... 48 Enriched environment reverses the effect of age on total new cell number ................................ ................................ ................................ ... 48
7 Enriched environment does not reverse the effect of age on n euronal differentiation ................................ ................................ .......................... 49 Enriched environment increases net neurogenesis ................................ ... 51 Higher Rates of Neurogenesis Relate to Better Wate r Maze Performance in Aged Rats ................................ ................................ ................................ ..... 52 Discussion ................................ ................................ ................................ .............. 53 3 INFLAMMATORY BIOMARKERS INDICATIVE OF COGNITIVE AGE .................. 59 Introduction ................................ ................................ ................................ ............. 59 Methods ................................ ................................ ................................ .................. 60 Subjects ................................ ................................ ................................ ............ 60 Water Maze Training and Testing ................................ ................................ .... 6 2 Sample Collection and Protein Harvest ................................ ............................ 64 Bio Plex Quantification of Cytokine s ................................ ................................ 64 Correlation Cluster Analysis ................................ ................................ ............. 67 Biological Age Regression Analysis ................................ ................................ 68 Statistical Analyses ................................ ................................ .......................... 69 Results ................................ ................................ ................................ .................... 70 Water Maze Visible Platform Task Performance Deteriorates with Age ........... 70 Hidden Platform Training on the Water Maze is Impaired with Age ................. 73 Impaired Probe Trial Performance in Aged Rats Begins to Appear in Middle Age ................................ ................................ ................................ ................ 75 Circulating and Central Inflammatory Biomarker Profiles are Modified with Age ................................ ................................ ................................ ................ 76 Correlation Analyses Reveal Cytokine Clu sters Altered with Age .................... 81 Biomarker Profiles Differ for Memory impaired Versus Memory unimpaired Rats Despite Age ................................ ................................ .......................... 83 Potential S erum and Hippocampal Biomarkers Predict Biological Age of Cognitively Impaired Aging Rats ................................ ................................ ... 86 Discussion ................................ ................................ ................................ .............. 88 4 DAILY EXERCISE IMPR OVES MEMORY, STIMULATES HIPPOCAMPAL NEUROGENESIS AND MODULATES IMMUNE AND NEUROIMMUNE CYTOKINES IN AGING RATS ................................ ................................ ............... 97 Introduction ................................ ................................ ................................ ............. 97 Methods ................................ ................................ ................................ ................ 100 Subjects ................................ ................................ ................................ .......... 100 Water Maze Training and Testing ................................ ................................ .. 102 Inhibitory Avoidance Training and Testing ................................ ...................... 104 Bromodeoxyuridine Injections ................................ ................................ ........ 105 Histology ................................ ................................ ................................ ......... 105 Protein Harvest from Brain Tissue ................................ ................................ .. 107 Immunohistochemistry ................................ ................................ .................... 107 Cell Quantification ................................ ................................ .......................... 109 Multiplex Quantification of Cytokines ................................ .............................. 111
8 Cytokine Cluster Analysis ................................ ................................ ............... 113 Stat istical Analyses ................................ ................................ ........................ 114 Results ................................ ................................ ................................ .................. 115 Aging Rats that Run Daily Locate a Visible Platform as Well as Controls but Swim Faster ................................ ................................ ................................ 115 Daily Exercise Improves Spatial Ability in Aging Rats ................................ .... 118 Aging Rats that Exercise Exhibit Better Memory for the Platform Location on Probe Trials ................................ ................................ ............................ 120 Inhibitory Avoidance Scores ................................ ................................ ........... 123 Daily Exercise Increases Neurogenesis in Aged Rats by Increasing New Cell Nu mber ................................ ................................ ................................ 123 Distinct Cytokine Relationships were Detected in Serum, Hippocampal and Cortical Compartments ................................ ................................ ............... 127 Measures of Behavior and Neurogenesis Relate to Concentrations of Cytokines Modulated by Running ................................ ................................ 129 Discussion ................................ ................................ ................................ ............ 136 Implications ................................ ................................ ................................ ........... 144 5 CONCLUSION ................................ ................................ ................................ ...... 146 Major Experimental Findings ................................ ................................ ................ 146 A Novel Relationship Between Hi ppocampal Neurogenesis and Hippocampus dependent Task Performance in Aged Rats ........................ 147 Identification of Prognostic and Diagnostic Biomarkers of Age related Cognitive Decline ................................ ................................ ........................ 148 Hypothesis driven Pathway Analysis Reveals Inflammatory Prognostic and Diagnostic Biomarkers of Hippocampal Neurogenesis and Integrity ........... 150 Lifest yle Changes can Restore Hippocampal Neurogenesis and Hippocampal Integrity, Possibly through Modulation of Inflammatory Mediators ................................ ................................ ................................ .... 151 Applications and Implications ................................ ................................ ................ 153 Development of a Biomarker Assay for the Detection of Age related Cognitive impairments ................................ ................................ ................. 154 Data Compilation, Pathway Analysis and Machine Learning .......................... 156 LIST OF REFERENCES ................................ ................................ ............................. 159 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 185
9 LIST OF TABLES Table page 4 1 Some hippocampal (pg/mg), cortical (pg/mg) and circulating (pg/mL) cytokines are modulated by daily exercise in aging rats ................................ ... 128 4 2 Spe arman rank correlation coefficients ( r s ) between cytokine pairs detected ... 131 4 3 Measures of several variables significantly modulated by daily exercise in aging rats correlate ................................ ................................ ........................... 134
10 LIST OF FIGURES Figure page 1 1 Adult hippocampal neurogenesis ................................ ................................ ........ 19 1 2 Cytokines, chemokines, growth factors and hormones. ................................ ..... 26 1 3 Cytokines, chemokines, growth factors and hormones are modified by age. ..... 27 1 4 Cytokines, ch emokines, growth factors and hormones modulate neurogenesis and neural plasticity. ................................ ................................ .... 28 2 1 Experiment timeline. Rats were housed individually (n = 7 young, n = 7 aged) or in pairs and exposed t o an enriched environment daily (n = 7 young, n = 9 aged) for 10 weeks ................................ ................................ ............................. 37 2 2 Exposure to an enriched environment subdues age dependent impairments on hidden and visible platform trials ................................ ................................ ... 45 2 3 Exposure to an enriched environment reversed the effects of age on neurogenesis ................................ ................................ ................................ ...... 49 2 4 Fewer new cells expressed mature neurona l phenotypes in the dentate gyri of aged rats ................................ ................................ ................................ ......... 50 2 5 Net neurogenesis declines with age but is increased by exposure to enrichment whereas age dependent increases in gliogenesis are unaffected by enrichment ................................ ................................ ................................ ..... 52 2 6 New neuron number correlates with measures of spatial ability ......................... 54 3 1 Experiment timeline ................................ ................................ ............................ 61 3 2 Water maze visible platform task performance declines with age ...................... 71 3 3 Water maze hidden platform performance is impaired with age ......................... 74 3 4 Impaired probe trial performance in aged rats begins to appear in middle age .. 75 3 5 Circulating and central cytokine profiles are modified with age .......................... 78 3 6 Cytokine clusters reveal analytes that synergistically change with age .............. 82 3 7 Memory impaired rats hav e a distinct cytokine profile ................................ ........ 85 3 8 Potential serum and hippocampal biomarkers predict biological age ................. 88 4 1 Experiment timeli ne. ................................ ................................ ......................... 101
11 4 2 Runners and controls perform similarly on the visible platform task ................. 117 4 3 Conditioned runners outperformed control s on the water maze hidden platform task ................................ ................................ ................................ ..... 119 4 4 Conditioned runners exhibit better memory in the water maze and on an inhibitory avoidance task ................................ ................................ .................. 122 4 5 Conditioned running potentiated hippocampal neurogenesis in aging rats ....... 125 4 6 Probe trials scores relate to measures of neurogenesis in aging rats .............. 126 4 7 Cytokine clusters detected in the serum, hippocampal and cortical samples obtained from aging rats ................................ ................................ ................... 132 4 8 Some cytokines are modulated in a coordinated fashion by conditioned running in aging rats and relate to measures of hippocampus dependent behavior and hippocampal neurogenesis ................................ ......................... 135
12 Abstract of Dissertation Presented to the Graduate School of the Univers ity of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IDENTIFYING INFLAMMATORY BIOMARKERS OF COGNITIVE AGING THROUGH RELATIONSHIPS BETWEEN MEASURES OF INFLAMMATION, NEUROGENESIS AND COGNITION IN AGED RATS By Rachel Brooke Speisman May 2013 Chair: Brandi K. Ormerod Major: Biomedical Engineering The extent and cost of care required for our burgeoning elderly population will greatly depend upon their level of cognitive ability Chronological a ge is not a direct predictor of this cognitive decline, which can vary in degree of impairment without any to dysregulated expression of pro and anti inflammatory cytoki nes, among other proteins. Senescent rats can be characterized as memory unimpaired or memory impaired using behavioral tasks such as the water maze. Interestingly, these memory impairments begin to appear in middle age, when inflammatory gene expression i s also increased. The protein compliment of these gene products could comprise a unique biomarker array to predict and/or diagnose age related cognitive decline Here, young (4 8 mo), middle aged (12 mo) and aged (18 22 mo) Fisher 344 rats were behaviorall y characterized, hippocampal neurogenesis was immunohistochemically evaluated using light and confocal microscopy potential biomarkers were identified in the blood serum and brain using Bio Plex technology and correlation analyses were used to reveal the relationships between these measures and create cytokine pathway clusters. The data
13 presented in this dissertation demonstrate 1) hippocampal neurogenesis correlates with learning and memory task performance in aged rats, 2) memory impaired rats have a dis tinct inflammatory cytokine profile, 3) inflammatory cytokine expression correlates with hippocampal neurogenesis and learning/memory and 4) simple lifestyle changes, including environmental enrichment and physical exercise, can also alter cytokine express ion and preserve hippocampal plasticity and integrity across age. Identification of biomarkers capable of predicting cognitive impairment may lead to the engineering of an assay panel that can be used to monitor blood samples of the aging human population.
14 CHAPTER 1 A REVIEW OF COGNITIVE AGING Approximately 10,000 Baby Boomers turn 65 each day, a trend that will continue for the next 18 years resulting in a significant increase in the elderly population as it grows faster than any other age group ( Cohn and Taylor 2010; Passel and Cohn 2008; U.S. Census Bureau 2011 ) As this aged population grows so does the number of people suffering from age related cognitive decline In fact, worldwide there are approximately 36 million people currently sufferi ng from dementia and without neuromodulatory intervention this number may rise to an estimated 115 million within the next 40 years (World Health Organization, 2012) The aging, demographics and memory study (ADAMS) is the first population based study of dementia in the United States to employ a single standardized diagnost ic protocol to measure cognition in aging subjects from all regions of the country. The ADAMS estimates that 14% of the se, vascular dementia and Lewy Body dementia (Langa et al., 2005) However, the majority of the elderly population will experience age related cognitive decline without any known pathology (Colsher and Wallace, 1991; Small et al., 1999) This C hapter will review age related cognitive decline, the relationship between age related cognitive decline and age related changes in hippocampal neurogenesis, regulators of both measures, such as stress and inflammation and the beneficial effects of differential experience.
15 Age r elated Cognitive Decline The Human Hippocampus and Age r elated Cognitive Decline With the aging population growing faster than all other age groups, we must bett er understand how to prepare for and control the burden and cost associated with age related cognitive decline Recent work has highlighted that c hronological age is not an accurate predictor of age related cognitive decline. Instead, the senescent populat with age appropriate impairments with lower memory, reasoning, processing speed and general cognitive capacity scores than their age and education matched norms. Working memory (Hultsch et al., 1990; Salthouse et al., 1989) and the acquisition and retrieval of episodic information (Zec, 1995) are often compromised in unsuccessful or i mpaired agers whereas crystallized int elligence, general knowledge and well learned skills a ppear to remain less affected in these individuals (Cattell, 1963) In fact, the correlation between fluid intelligence s crystallized intelligence scores on the Wechsler Adult Intelligence Scale weaken with age (Cunningham et al., 1975) Of course, age related changes in human cognition identified in cross sectional studies spanning multiple generations versus longitudinal studies may appear robust, but may also be confounded by changin g generational variables, such as legislated shifts in education level and access to nutrition and medical care (Schaie, 1980; Schaie and Strother, 1968; Schaie and Willis, 1993) However, reliable age related chang es in longitudinal studies are being reported more frequently For example, longitudinal studies reliably show that healthy individuals can exhibit impaired hippocampus dependent memory scores on the Selective Reminding Test while language (Boston Naming T est), visuospatial (Benton Visual Retention and
16 Rosen Drawing Test) and abstract reasoning ability (Wechsler Adult Intelligence Scale) scores remain intact across age (Small et al., 1999) Interestingly, decreased hippocampal volumes have also been linked to age related cognitive decline in longitudinal studies (Driscoll et al., 2009; Golomb et al., 1994; Grundman et al., 2002; Lupien et al., 1998) These age related decreases in hippocampal volume are most often attributed to atrophy or cell death (West, 1993) but could be aggravated b y reduced regenerative capacity. Indeed, post mortem hippocampal samples of patients exhibiting profound memory impairments are typically devoid of signs of neurogenesis (Coras et al., 2010; Correa et al., 2004; Cros sen et al., 1994; Monje et al., 2007; Roman and Sperduto, 1995; Siffert and Allen, 2000) Thus, diminished levels of neurogenesis could contribute to decreased hippocampal volume and cognitive impairments on tasks that rely upon hippocampal integrity in a ged individuals. While the technology and availability to study in vivo neurogenesis in the human brain does not yet exist, rodent models allow us to more readily explore this relationship. In fact, Chapters 2 and 4 illustrate that measures of hippocampal neurogenesis and performance in a hippocampus dependent water maze task are related in aged rats and suggest that strategies restoring neurogenesis to the levels observed in young rats may improve memory. A Rodent Model of Age related Cognitive D ecline S imilar to aged humans, aged rodents can exhibit impaired performances on cognitive tasks ( Alexander et al., 2012 ) Hidden water maze platform tasks promote an spatial cues surrounding th e maze to learn the location of an escape platform over several training sessions Pr evious experiments have shown that an intact hippocampus is required to perform
17 this task (Morris et al., 1982) and several studies have shown that aged rats exhibit impaired performances on this task when compared to younger rats suggesting that hippocampal integrity can become compromised with age ( Gage et al., 19 84a; Gage et al., 1984c; Gallagher et al., 1993; Markowska, 1999; Markowska et al., 1989 ). Like aged dependent tasks, such as the Morr is water maze (Dobrossy et al., 2003; Drapeau et al., 2003; Drapeau et al., 2007; Markowska, 1999; Markowska et al., 1989) Identifying prognostic or predictive and diagnostic or mechanistic biomarkers (that could i nclude hippocampal neurogenesis) in these subgroups of aged animals may lead to novel strategies for predicting and treating age related cognitive decline. Foster and colleagues have previously demonstrated that a rapid acquisition hidden platform water maze t ask is both proficient and efficient at identifying age related cognitive impairments in rats (Blalock et al., 2003; Carter et al., 2009; Fugger et al., 1998; Norris and Foster, 1999) In this task, rats are t rained to locate a hidden platform in a single training session and memory probes can be administered after a delay. Age related performance deficits revealed in this task relate to age related impairments in measures of hippocampal integrity in the object memory and the inhibitory avoidance tasks (Blalock et al., 2003; Foster and Kumar, 2007; Foster et al., 2003) Consistent with studies conducted in water maze tasks with more distributed training trials (Bizon and Gallagher, 2003; Bizon et al., 2004; Blalock et al., 2003; Gage et al., 1984a; Gage et al., 1984b; Gallagher et al., 1993; Merrill et al., 2003; Rasmussen et al., 1996) aged r ats typically exhibit longer pathlength s than young rats on training trials and poorer
18 discrimination index scores on probe trials administered immediately after training to test strength of learning or after a delay to test memory (Blalock et al., 2003; Carter et al., 2009; Fugger et al., 1998; Norris and Foster, 1999) Recent studies have demonstrated that new, specifically maturing, neurons are integral for pattern separation (Deng et al., 2010; McHugh et al., 20 07) We therefore suspect that the rapid water maze task may tax hippocampus dependent cognition more heavily than distributed tasks and could therefore reveal novel relationships between new neuron number and hippocampal integrity. In Chapters 2 4 we use this task to show the relationship between these and other measures in aged rats as well as to identify other biomarkers of cognitive age. Diminished Hippocampal Neurogenesis with Age The hippocampus not only mediates the detection, memory and recall of people, places and events and spatial ability but is neurogenic as well In fact, the hippocampus and olfactory bulbs are the only two brain regions that add significant numbers of new neurons throughout life in mammals from rodents to humans (Altman, 1969; Altman and Das, 1965; Alvarez Buylla and Garcia Verdugo, 2002; Eriksson et al., 1998; Gould et al., 1999b) Neural progenitor cells (NPCs) residing in the subgranular zone (SGZ) of the hippocampal dentate gyr i div ide and the daughter cells are thought to either retain a multipotent NPC phenotype or differentiate into new granule neurons or glia ( Ming and Song 2011 ) Note that the identity and potential of hippocampal NPCs a re still under debate, with some data suggesting that they are unipotent progenitors that can make either neurons or glia (reviewed in Ming and Song, 2011). Those daughter cells that acquire neuroblast phenotypes are thought to migrate deeper into the gran ule cell layer
19 (Seki et al., 2007) before rapidly extending dendrites and then an axon (H astings and Gould, 1999) Figure 1 1. Adult hippocampal neurogenesis. Neural progenitor cells located in the subgranular zone of the hippocampal dentate gyrus divide. New daughter cells then begin to differentiate into specific neural cells types (neuro nal and glial). As new neurons mature they begin to extend axons to communicate with other cells, particularly pyramidal cells in the CA3 region. Synapses are formed with cornu ammonis (CA) 3 region within 4 10 days and after four weeks new neurons are mo rphologically and functionally indistinguishable from other granule cell neurons (Cameron and McKay, 2001; Hastings and Gould, 1999; van Praag et al., 1999a) In fact, Cameron and colleagues have reported that withi n the large pool of granule cell neurons there is significant (~%50) new cell death beginning approximately six days after the birth of new progeny that remains constant for up to 28
20 days, at which point cells that do survive remain active for at least mon ths (Dayer et al., 2003) Since cells that survive this cycle of cell death and mature into functional neurons may be more likely to contribute to hippocampal integrity, we measured basal levels of neuro genesis 3 4 weeks after injecting the cell synthesis marker bromodeoxyuridine (BrdU) in Chapters 2 4. Like performance in some cognitive tasks adult hippocampal neurogenesis declines with age (Lemaire et al., 2000 ; Lichtenwalner et al., 2001; Nacher et al., 2003; Seki and Arai, 1995) Age related decreases in adult hippocampal neurogenesis appear to be primarily mediated by increasing quiescence among SGZ NPCs because uptake of the division marker BrdU and the exp ression of proliferation markers, such as Ki67 is significantly reduced in aged animals (Cameron and McKay, 1999a; Hattiangady and Shetty, 2008; Kuhn et al., 1996) Some studies employing extended differential exper ience protocols (i.e. extended living in environmental and social enrichment) also suggest that neuronal differentiation and the survival of new neurons is compromised by age, potentially because the expression of growth factors that support neuronal diffe rentiation and survival can decrease with age (Kempermann et al., 2002; Kempermann et al., 1997, 1998; Kuhn et al., 1996) We examine both differentiation and survival of new cells in Chapters 2 and 4 where we emplo y differential experience to rescue diminished neurogenesis in aged rats. Specifically, in Chapter 2 we demonstrate that diminished hippocampal neurogenesis in aged rats is due to a decrease in the number of cells expressing both the proliferative marker B rdU and neuronal differentiation markers. However, we show that extended exposure to environmental enrichment restored the number of BrdU + cells to levels observed in
21 young rats and thus rescued overall neurogenesis (total number of new neurons), despite h aving no effect on differentiation. Similarly, in Chapter 4 we show that wheel running amplifies the production of new cells again contributing to an increase in neurogenesis. The Relationship between Neurogenesis and Cognition In young animals, levels of hippocampal neurogenesis generally relate to their ability in hippocampus dependent tasks. Early work that ablated neurogenesis chemically with the m ethylating agent methylazoxymethanol acetate, show ed that performance in hippocampus dependent tasks such as trace eyeblink conditioning and spatial navigation were impaired whereas performance in non hippocampus dependent tasks such as delay eyeblink conditioning and cued navigation were intact suggesting that neurogenesis supports performance on hippocampus dependent tasks (Shors et al., 2001) Similarly, radial maze and contextual fear conditioning performance are impaired in animals exposed to hippocampus targeted gamma i rradiation that ablates hippocampal neurogenesis (Winocur et al., 2006) In fact, this strategy was employed to show that young (4 28 days old) granule neurons are necessary for long term spatial memory in adult rats (Snyder et al., 2005) and mice (R ola et al., 2004) Recently, transgenic mice (Dupret et al., 2008) and other genetic strategies (Saxe et al., 2006) that diminish (but not ablate) neurogenesis garnered further support to show that hippocampus dependent learning and memory are linked to neurogenesis in young animals. The idea that hippocampus dependent behavioral tr aining could influence hippocampal neurogenesis was first suggested by work performed in young adult rats by Gould and colleagues in 1999 (Gould et al., 1999a) They reported that although
22 similar numbers of new cells were generated in the hippocampi of rats that engaged in hippocampus dependent versus non hippocampus dependent tasks, more new neurons survived in the former group (Gould et al., 1999a). Drapeau and colleagues expand ed upon this work by showing that participation in early phase water maze training trials improved the survival of young neurons but compromise d neuron production, while participation in later phase training trials did not impact neurogenesis (Drapeau et a l., 2007). Interestingly, the number of young ( doublecortin (DCX) labeled) neurons quantified four weeks after behavioral testing relates to performances of female Fisher 344 x Brown Norway hybrid rats across age (3, 12 and 24 mo) and across hippocampus de pendent tasks (Driscoll et al., 2006) While both hippocampal neurogenesis and hippocampal integrity measured on behavioral tasks decline with age the relationship between their measures remains debated. While man y studies conducted across animal species have revealed correlations between measures of hippocampal neurogenesis and hippocampus dependent task performance in aged animals (Aizawa et al., 2009; Drapeau et al., 2003; Driscoll et al., 2006; Lemaire et al., 2000; Siwak Tapp et al., 2007) a handful conducted with rats using standard water maze tasks have not (Bizon and Gallagher, 2003; Bizon et al., 2004; Merrill et al., 2003) W hile no correlation was reported between new cell production and spatial ability in aged (25 mo) rats (Bizon and Gallagher, 2003) higher basal levels of neurogenesis were noted in aged rats with poorer spatia l learning in a subsequent study (Bizon et al., 2004) Bizon and colleagues measured basal neurogenesis by injecting male Sprague Dawley rats one week after behavioral testing to minimize the potentially confounding effects of training
23 on neurogenesis. In order to highlight the possible relationship between n eurogenesis the variability that we expected within each measure by exposing our aged and young cohorts to differential experience for extended durations. Indeed, Chapte rs 2 and 4, reveal novel relationships between total new neuron number and measures of learning and memory (average pathlength to hidden platform on final training trial and immediate and 24 h probe DI scores) in aged rats Systemic Regulators of Neuroge nesis and Cognition Hippocampal neurogenesis could be compromised concomitantly with hippocampus dependent behavior by age through a systemic factor that is expressed in variable concentrations across age. For example, gonadal hormones affect neurogenesis and cognition and level s of sex steroids decrease with age (Galea et al., 2006; Galea et al., 2008; Ormerod et al., 2003, 2004; Spritzer and Galea, 2007; Tanapat et al., 1999) Furthermore, long term stress and ass ociated corticosterone levels can reduce neurogenesis and impair performance on behavioral tasks (Cameron and McKay, 1999a; Gould et al., 1997; Gould et al., 1998; Lemaire et al., 2000; Mohapel et al., 2006; Montaron et al., 2006; Tanapat et al., 1998) I nflammation can also disrupt hippocampal neurogenesis and hippocampus dependent task performance (Ekdahl et al., 2003; Monje et al., 2003) Interestingly, regulation of both s tress and inflammation can become dysregulated with age. Therefore, investigatin g link s between stress, inflammation and neurogenesis may help to elucidate the mechanisms behind age related cognitive decline and are the focus of Chapters 3 and 4. Chapter 3 reveals age related changes in inflammatory cytokines, chemokines, growth factors and stress hormones and whether these proteins can be used as prognostic or diagnostic
24 biomarkers of age related cognitive decline. Chapter 4 examines whether physical exerc ise induced improvements in neurogenesis and behavior could be mediated by modulation of circulating and/or central biomarkers in aged rats. A review of inflammatory cytokines, stress hormones and other regulators of neurogenesis that may change with age a re presented in Figure 1 2 1 4 Stress Hormones The hypothalamic pituitary stress, among other functions, through the feedback and release of corticotropin releasing hormone (CRH), adrenocorticotropic h ormone (ACTH) and cortisol in humans or corticosterone (CCS) in many animals. CCS also regulates immune activation and inflammation (Munck et al., 1984) Under an acute stressful situation CRH is released from the hypothalamus, which stimulates production of ACTH in the anterior pituitary thus stimulating the adrenal cortex to produce C CS. CCS is transported in the blood and passively diffuses across the blood brain barrier where it can bind to receptor proteins in the limbic system, including the hippocampus. Binding to receptors in the hypothalamus halts the production of both CRH and ACTH through negative feedback. However, under chronic stress, hormone concentrations are unable to return to homeostatic levels and may impair receptor controlled n egative feedback leading to HPA axis dysregulation. Dysregulation of the HPA axis with ag e has been reported in humans (Born et al., 1995; Lupien et al., 1998; Lupien et al., 1999; McEwen et al., 1999) and rodents alike (Brett et al., 1983; DeKosky et al., 1984; M eaney et al., 1988; Sapolsky et al., 1983a) with characteristic increased levels of ACTH and CCS (Sencar Cupovic and Milkovic, 1976) This dysregulation is thought to manifest in shunted negative feed back
25 that may be produced by a loss of glucocorti coid receptor expression in the hippocampus (McEwen et al., 1999; Meaney et al., 1988; Sapolsky et al., 1983b) The hippocampus expresses mineralocorticoid and glucoc orticoid receptors that are thought to be critical for providing negative feedback to the HPA axis (Jacobson and Sapolsky, 1991) A ged rats with elevated HPA axis activity exhibit impaired performances across hippocampus dependent tasks (Montaron et al., 2006 ; Sandi and Touyarot, 2006) and aging humans that with higher cortisol levels exhibit lower memory scores and have smaller hippocampi than aging humans with lower cortisol levels (Lupien et al., 1998) (for review see (Lupien et al., 2009) and Table 1 2). Ind eed, dampening glucocorticoid dysregulation with mid life adrenalectomy rescues memory impairments in aged rats and slows neuron loss (Landfield et al., 1981) Interestingly, long term stress and elevated CCS levels can also decrease hippocampal neurogenesis (Cameron and McKay, 1999a; Gould et al., 1997; Gould et al., 1998; Lemaire et al., 2000; Montaron et al., 2006; Tanapat et al., 1998) Cameron and colleagues showed that normalizing stress hormone level s with midlife adrenalectomy could also restore proliferating NPC numbers (and therefore neurogenesis) in the hippocampi of aged rats as well as restore Morris water maze performances (Cameron and McKay, 1999b; Mon taron et al., 2006; Montaron et al., 1999)
26 Figure 1 2. Cytokines, chemokines, growth factors and hormones.
27 Figure 1 3 Cytokines, chemokines, growth factors and hormones are modified by age.
28 Figure 1 4 Cytokines, chemokines, growth factors and hormones modulate neurogenesis and neural plasticity.
29 Inflammation and Neuroinflammation We and others have shown that lipopolysaccharide (LPS), an endotoxin found in the outer cell wall of gram negative bacteria both impair memory and decrease neurogenesis in young rodents (Asokan, 2010; Ekdahl et al., 2003; Mohapel et al., 2006; Monje et al., 2003; Ormerod B.K. 2013; Tanapat et al., 1998) Peripherally administered LPS produces an acute inflam matory and then neuroinflammatory response that activates microglia and blocks the differentiation of progenitor cell progeny into neurons (Ekdahl et al., 2003; Monje et al., 2003; Ormerod B.K. 2013 ) The effects o f LPS on both neurogenesis and memory can be blocked by non steroidal anti inflammatory treatment (Ekdahl et al., 2003, Monje et al., 2003, Ormerod et al., 2013). These data suggest that conditions associated with chronic neuroinflammation are likely to im pact hippocampus dependent behavior potentially through their effects on neurogenesis. Our collaborator, Dr. Thomas C. Foster and his colleagues h a ve shown that inflammatory genes are one of the five largest functional categories of genes to be upregulat ed with age while genes associated with signaling and neuro plasticity are downregulated (Blalock et al., 2003) A few inflammatory markers have been shown to increase with age (Chung et al., 2009) and are linked to m emory decline in both rodents and humans (Blalock et al., 2003; De Martinis et al., 2005; Foster, 2006; Magaki et al., 2007; Rafnsson et al., 2007; Solfrizzi et al., 2006; Villeda et al., 2011) For review see Table 1 3. For example in humans, inflammatory markers like C reactive protein (CRP), IL 6 and TNF are upregulated in middle age (Gimeno et al., 2008) and IL 6 and TNF are elevated in older individuals that exhibit impaired memory (Krabbe et al., 2009) We employ a commercially available panel of cytokines, chemokines and growth factors
30 t o examine the concentrat ion across age in Chapter 3 and in response to physical exercise in Chapter 4. We then explore the relationship between inflammatory analytes that may impact neurogenesis and cognition using hypothesis driven pathway analyse s. Differential Experience W hy some individuals are successful cognitive agers, living to a robust old age with intact memory while unsuccessful agers suffer mild to severe dementia with unknown pathology is unclear. conveys the common conception that brai n exercise may combat age related cognitive decline. In an experimental setting, the effect of d ifferential experience in the form of physical exercise, environmental enrichment and even social interaction on neuroplasticity, cognitive ability and hippocam pal neurogenesis has been tested and generally shown to improve measures of each variable. Environmental Enrichment Daily exposure to environmental enrichment stimulates neurogenesis primarily by increasing the probability of neuronal fate decisions and survival among NPC progeny and improves performances on cognitive tasks (Kempermann et al., 2002; Kempermann et al., 1997, 1998; Leal Galicia et al., 2008; Segovia et al., 2006) For example, Gage and colleagues f ound that adult (21 day old) mice in an enriched environment had an increased number of new cells four weeks after administering BrdU (Kempermann et al., 1997) and these new cells were more likely to adopt a neuronal phenotype in both young (8 mo) and aged (20 mo) mice (Kempermann et al., 1998) Longer term enrichment beginning in middle age (10 mo) increased the number of new neurons in age d (20 mo) mice (Kempermann et al., 2002) Interestingly, one week old neurons are in the process of generating synapses, (Hastings and Gould, 1999;
31 Hastings et al., 2002) and synaptophysin, a protein necessary for synaptic transmission, is also upregulated in rats exposed to environmental enrichment (Lambert et al., 2005) These findings suggest that differential experience may attenuate age related deficits in hippocampal neurogenesis on multiple fronts, but perhaps rescuing just one of these me asures may lead to improve hippocampal integrity. Therefore, understanding how an enriched environment may impact measures of hippocampus dependent task performance and hippocampal neurogenesis with age, along with the relationship between these two measur es is the focus of Chapter 2. Physical Exercise Physical exercise stimulates hippocampal neurogenesis primarily by increasing the proliferating NPC pool and improves performances on hippocampus dependent tasks and measures of hippocampal synaptic plasti city in rodents (Brown et al., 2003; Farmer et al., 2004; Kronenberg et al., 2003; Lambert et al., 2005; Lugert et al., 2010; Steiner et al., 2008; Suh et al., 2007; van et al., 1999; van Praag et al., 2002) Recent ly, van Praag and colleagues demonstrated that physical exercise might impact neurogenesis by upregulating brain derived neurotrophic factor (BDNF) (Kobilo et al, 2011). If a link between age related changes in neurogenesis and cognition could be drawn, the n circulating neurogenic molecules could also potentially be used as biomarkers of cognitive age. In Chapter 4 we explore the impact of long term physical exercise on memory tasks and hippocampal neurogenesis. We also measure immune and neuroimmune signali ng to elucidate whether changes in measures of hippocampal plasticity and integrity are modulated by changes in inflammatory biomarkers.
32 Social Interaction Social enrichment alone may promote hippocampal neurogenesis (Fowler et al., 2002; Lu et al., 2003) and potentiate the effects of environmental enrichment and running on hippocampal neurogenesis (Leasure and Decker, 2009; Madronal et al., 2010; Stranahan et al., 2006b) I n fact, individual housing induced decrease s in new neuron number and learning deficits can be reversed by housing rats in social groups (Lu et al., 2003) suggesting that simple lifestyle changes could stimulate hippocampal plasticity, neurogenesis and improved cognition later in life. Conversely, s ocial grouping and exposure to a novel environment each day may be perceived as stressful and animals with prolonged elevated CCS levels have been shown to hamper neurogenesis (Cameron and McKay, 1999a; Gould et al. 1997; Gould et al., 1998; Montaron et al., 2006; Tanapat et al., 1998) Lon g er term exposure to differential experience may com bat an initially stressful response Indeed, Leal Galicia and colleagues (2008) have previously found that aged rats given lo ng term enrichment since youth also had improved recognition memory and increased levels of neurogenesis (Leal Galicia et al., 2008) Therefore we pair housed rats in our environmental enrichment experiment, in Chap ter 2, for 10 weeks to augment positive effects and took this into consideration when analyzing the effects of individually housed rats in our 18 week physical exercise experiment in Chapter 4. Overall, physical activity, environmental enrichment and soc ial intera ction have been shown to promote neuroplasticity among other health benefits in both the young and aging brain in rodents and humans alike. However, testing whether the preserved cognition observed among humans who engage in lifelong physical and mental exercise (Christensen and Mackinnon, 1993; Churchill et al., 2002; Erickson et al., 2010; Kramer
33 et al., 2004) is related to preserved neurogenesis awaits the development of technologies that can measure neu rogenesis in vivo Meanwhile, our studies presented in Chapter 2 and 4 examining the effects of differential experience across age present data supporting long term environmental enrichment and physical activity as possible interventions for curbing age re lated cognitive decline, even when begun later in life.
34 CHAPTER 2 ENVIRONMENTAL ENRICHMENT RESTORES NEUROGENESIS AND RAPID ACQUISITION IN AGED RATS Introduction Altered hippocampal function likely contributes to age related changes in cognitive ability because hippocampus dependent tasks are sensitive to age related cognitive decline (Foster, 1999) Decades ago, the standard Morris water maze task re vealed impaired performances among some senescent rats (Gage et al., 1984b; Rapp et al., 1987) More recent behavioral assessments have sought to increase task sensitivity to age related cognitive decline ( Kennard and Woodruff Pak 2011 ) so that the deficits and their underlying mechanisms can be better characterized and potentially manipulated. Here we employ a rapid water maze task sensitive to age related cognitive decline to test whether daily exposure to an enriched environment can reverse the effects of age on hippocampal function concomitantly with hippocampal neurogenesis. Neurogenesis is a striking form of neural plasticity that persists throughout life in the hippoca mpus and olfactory bulbs of all mammals investigated, including humans (Altman and Das, 1965; Cameron et al., 1993; Eriksson et al., 1998) Although the precise role that new neurons play in hippocampal integrity is debated, new neuron number in young animals generally correlates with their performance measures in hippocampus dependent tasks [ (Deng et al., 2010) but see (Epp and Galea, 2009) ] Manipulations that attenuate neurogenesis chronically a ssociate with impaired performance (Madsen et al., 2003; Raber et al., 2004; Saxe et al., 2006; Shors et al., Reprinted with permission from Speisman, R.B., Kumar, A., Rani, A., Pastoriza, J.M., Severance, J.E., Foster, T.C., Ormerod, B.K., 2013 Environmental enrichment restores neurogenesis and rapid acquisition in aged rats. Neurobiol. Aging
35 2002; Snyder et al., 2005; Winocur et al., 2006) while those that potentiate neurogenesis associate with better performance (Dalla et al., 2009; Ormerod et al., 2004; van Praag et al., 2005) Postmortem signs of hippocampal neurogenesis in human patients who exhibited profound memory impairments are scarce (Coras et al., 2010; Correa et al., 2004; Crossen et al., 1994; Monje et al., 2007; Roman and Sperduto, 1995; Siffert and Allen, 2000) Hippocampal neurogenesis declines with age in rodents primarily because neural progenitor c ells (NPCs) become increasingly quiescent and NPCs that do divide may be less likely to produce surviving neuronal progeny (Cameron and McKay, 1999a; Hattiangady and Shetty, 2008; Kempermann et al., 1997; Kuhn et al. 1996; Lichtenwalner et al., 2001; Nacher et al., 2003) While several studies have related new neuron number and cognitive measures in aged rats (Drapeau et al., 2003; Drapeau et al., 2007; Driscoll et al., 2006; Lemaire et al., 2000) dogs (Siwak T app et al., 2007) and nonhuman primates (Aizawa et al., 2009) the strength of this relationship among aged rats tested in the water maze varies. For example, new neuron number appears unrelated to the performance of aged rats in water maze tasks th at distribute training across 8 10 days (Bizon and Gallagher, 2003; Bizon et al., 2004; Merrill et al., 2003) but related in protocols that mass training across 2 3 days (Drapeau et al., 2003; Driscoll et al., 2006) Moreover, new neuron survival in the hippocampi of aged rats is enhanced by their participation in early but not later trials of the distributed water maze protocol (Drapeau et al., 2007) These results suggest t hat the strength of the relationship between neurogenesis and water maze performance in aged rats may depend upon the speed of learning demanded by the task.
36 In aged rodents, daily exposure to environmental enrichment primarily stimulates neurogenesis by i ncreasing the probability that new neurons survive to maturity (Kempermann et al., 2002; Kempermann et al., 1997, 1998; Leal Galicia et al., 2008; Segovia et al., 2006) and improves the rapid acquisition of spatial information in a condensed water maze task ( Kumar et al., 2011 ) Here we tested the hypothesis that daily exposure to environmental enrichment would reverse age related impairments in rats' abilities to rapidly acquire a spatial search strategy concomitantly with ongoing rates of neurogenesis. Methods S ubjects Young (5 8 months old) and aged (20 22 months old) sexually naive male F344 rats obtained fr om the National Institute of Aging colony at Harlan Sprague Dawley (Indianapolis, IN, USA) were treated in accordance with University of Florida and federal policies regarding the ethical use of animals for experimentation. Rats exhibiting signs of aggress ion (bites and scratches) or age related health problems (poor grooming, hunching, excessive porphyrin secretion, weight loss, and tumors) were euthanized humanely. Differential Experience: Environmental Enrichment and Individual Housing For the 10 week e xperiment, the rats were housed in a 12:12 hour light cycle with access to food and water ad libitum either individually (n = 7 young [YI] and n = 7 aged [AI]) or pair housed with 2 3 hours of access daily to an enriched environment (n = 7 young [YE] and n = 9 aged [AE]). The goal of the differential experience protocol was to provide opportunities for the enriched group to engage in a variety of hippocampus dependent behaviors while limiting them for the individually housed
37 group. The enriched environment consis ted of a large wooden box, empty water maze tank, or large wire cage containing assorted 3 dimensional toys (e.g., plastic tubes, balls, and various objects), food, and water. Figure 2 1 Experiment timeline. Rats were housed individually (n = 7 young, n = 7 aged) or in pairs and exposed to an enriched environment daily (n = 7 young, n = 9 aged) for 10 weeks. In the 5th week, rats were trained and tested on hidden platform trial s and then visible platform trials 3 days later. Beginning 1 week after testing, rats were injected daily with bromodeoxyuridine (BrdU; 50 mg/kg) over 5 days and then perfused 4 weeks later to quantify neurogenesis. The environment and toys were randomly rotated daily to maintain novelty. Daily exposure to this environment modifies hippocampal electrophysiology and facilitates the rapid acquisition of a spatial search strategy in aged rats (Foster and Dumas, 2001; Kumar et al., 2011; Kumar et al., 2007) B ehavioral testing commenced in the 4 th week of differential experience and bromodeoxyuridine (BrdU) injections commenced 1 week after behavioral testing was completed. The rats were perfused 4 weeks after the final BrdU injection to quantify neurogenesis ( Fig ure 2 1 )
38 Water Maze Training and Testing A black water maze tank (1.7 m diameter) filled with water (27 2 C) to a depth of 8 cm below its rim was housed in a well lit room. A Columbus Instruments (Columbus, OH, USA) tracking system recorded escape l atencies (seconds) and path lengths (cm; see Figure 2 2 ). Hidden and cued platform training consisted of 5 blocks (15 minute inter block interval) of 3 60 second trials (20 second inter trial interval) administered in a single session. This massed protocol is sensitive to both age related cognitive decline (Carter et al., 2009; Foster and Kumar, 2007; Foster et al., 2003) and the effects of differential experience on cognition in aged rats (Kumar et al., 2012). Rats were dried between blocks. Hidden platform trials. After 4 weeks of differential experience, rats were trained over a single session to locate a platform (29 cm diameter) hidden approximately 1 cm below the water surface in the northeast quadrant of the po ol in the presence of highly visible extra maze cues. Rats were first habituated to the pool by being given 3 opportunities to climb onto the platform from different directions. On the subsequent hidden platform trials, the rats were released randomly from north, south, west, or east start locations and given 60 seconds to locate the hidden platform before being guided. Probe trial. A 60 second free swim probe trial during which the platform was removed from the pool was conducted 15 minutes after the last hidden platform training block. The ra ts were released from the quadrant opposite the goal quadrant and quadrant and t(G) is time spent in the goal quadrant, served as our strength of learning measure.
39 Cued trials Three days after the hidden platform training session, rats were trained to locate the now flagged platform that protruded approximately 1.5 cm above the water in water maze tank now surrounded by a black curtain to mask distal cues. The rats were guided to the flagged platform if they failed to escape the maze within 60 seconds. The north, south east, and west release points and the location of the flagged platform were changed on each trial. BrdU Injections and Histology BrdU was dissolved in fresh 0.9% sterile saline (20 mg/mL wt/vol) and injected intraperitoneally (2.5 mL/kg or 50 mg/kg) once per day over 5 days, starting 1 week after behavioral testing to minimize the well known effects of learning on neurogenesis (Epp et al., 2010; Gould et al., 1999a) This BrdU dose safely and effectively labels dividing NPCs in the hippocampus of young and aged adult rodents (Cameron and McKay, 2001; Drapeau et al., 2003; Ko lb et al., 1999) Approximately 4 weeks after the final BrdU injection, the rats were anesthetized with 90 mg/kg ketamine and 10 mg/kg xylazine (Webster Veterinary, Sterling, MA, USA) and perfused transcardially with ice cold isotonic saline and 4% parafo rmaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA). By 4 weeks, many new cells express mature neuronal proteins and are relatively permanent (Cameron and McKay, 2001; van Praag et al., 2002) Extracted bra ins were stored overnight in perfusate, equilibrated in 30% sucrose (approximately 4 days) at 4 C and then sectioned intervals using a freezing stage sledge microtome (A merican Optical Corp., Buffalo, NY, USA). Sections were stored at 20 C in 30% ethylene glycol, 25% glycerin, and 45% 0.1 M sodium phosphate buffer (vol/vol/vol) until immuno stained.
40 Immunohistochemistry Free floating sections were stained immunohistoche mically to quantify 28 32 day old BrdU + cells and confirm their neuronal or glial phenotypes as described previously (Ormerod et al., 2004; Palmer et al., 2000) Sections were washed repeatedly between steps in Tris buffered saline (TBS; pH 7.4). Enzyme substrate i mmunostaining. BrdU + cells were revealed enzymatically on every 12th section through the dentate gyrus of each rat and counted under light microscopy to estimate total new cell numbers ( Figure 2 3). Section s were incubated in 0.3% H2O2 for 10 minutes to quench endogenous peroxidase, rinsed in 0.9% NaCl and then incubated in 2M HCl for 20 minutes at 37 C to denature DNA. The sections were then blocked in a solution of 3% normal donkey serum and 0.1% Triton X in TBS and incubated overnight in blocking solution containing rat anti BrdU (1:500; AbD Serotec, Raleigh, NC, USA) at 4 C and then for 4 hours in biotinylated secondary anti rat IgG (Jackson ImmunoResearch, West Grove, PA, USA; 1:500) at room temperatur e (RT). Next, the sections were incubated in avidin biotin horseradish peroxidase (Vector Laboratories, Burlingame, CA, USA) and then reacted in a solution of 0.02% 3,3' diaminobenzidine tetrahydrochloride (DAB; Sigma Aldrich, St. Louis, MO, USA) and 0.5% H2O2. Sections were mounted on glass slides, dried overnight, dehydrated in an alcohol series and then cover slipped under Permount (Fisher Scientific, Pittsburgh, PA, USA). Fluorescent immunostaining. The percentage of BrdU + cells expressing neuronal or g lial protein was quantified on sections that were immuno stained using fluorescent secondary antibodies under confocal microscopy ( Figure 2 4). The sections were blocked in a solution of 3% normal donkey serum and 0.1% Triton X in TBS and
41 then incubated ov ernight at 4C in blocking solution containing the mature neuronal marker mouse anti Neuronal Nuclei (NeuN, 1:500; Chemicon, Temecula, CA, USA) and the immature neuronal marker goat anti doublecortin (DCX, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, U SA) or the oligodendrocyte precursor marker rabbit anti chondroitin sulfate proteoglycan (NG2, 1:500; Chemicon) and the astrocyte marker chicken anti glial fibrillary acidic protein (GFAP, 1:750; EnCor Biotech, Alachua, FL, USA). The next day, sections wer e incubated in maximally cross adsorbed fluorescein isothiocyanate (FITC) conjugated anti mouse and cyanine 5 (Cy5) conjugated anti goat secondary antibodies to reveal neurons or FITC conjugated anti rabbit and Cy5 conjugated anti chicken secondary antibod ies to reveal glia for 4 hours at RT (all secondary antibodies 1:500; Jackson ImmunoResearch, Westgrove, PA, USA). Sections were then fixed in 4% paraformaldehyde, rinsed in 0.9% NaCl, incubated in 2 M HCl and then incubated overnight at 4C in rat anti Br dU (1:500; AbD Serotec, Raleigh, NC, USA) and then Cy3 conjugated anti rat secondary for 4 hours at RT the next day before being incubated in 4',6 diamidino 2 phenylindole (DAPI; 1:10,000; Calbiochem, San Diego, CA, USA) for 10 minutes and then mounted on glass slides under 2.5% diazabicyclooctane (in TBS with 10% polyvinyl alcohol and 20% glycerol). Cell Quantification Total new cell number. BrdU + cells distributed through the subgranular zones and granule cell layers (GCL; Figure 2 3 A) were counted on se ts of every 12th section (9 10 sections per rat) through the rostral caudal extent of the hippocampal dentate gyrus under a 40 objective on a Zeiss Axio Observer Z1 inverted microscope (Thornwood, NY, USA) and optical fractionator principles (Kempermann et al., 2002; West et al., 1991) The first section of each rat's set was randomly selected from the 1 st
42 11 th section of dentate gyrus. Because BrdU + cells are typically distributed irregularly, we counted all new ce lls (mean standard error of the mean [SEM] total BrdU + cells: YI that could represent cells in adjacent cell sections and multiplied that number by 12 (the section in terval) to generate a stereological estimate of total cell number (see Figure 2 3) without fractionating section thickness (Kempermann et al., 2002) Because age or enrichment related changes in BrdU + cell nucleus diameter could affect cell estimates estimated this way, we confirmed that nuclear diameters of approximately 10 20 BrdU + cells completely contained within 1 of these sections in 3 4 rats per group were F (1,9) = 2.82], enrichme nt effect [ F (1,9) = 0.29], interaction effect [ F (1,9) = 0.28]). Because exposure to enriched environments can increase hippocampal volumes, we measured GCL and subgranular zone areas (in mm 2 ) under a 20 objective using AxioVision software (version 4.8, Ze iss, Thornwood, NY, USA) and then calculated volumes using a truncated cone formula that accurately predicts the volume of many biological regions (Galea et al., 2000; Seifert et al., 2010; Uylings et al., 1986) : V olume = 1 / 3 I ( h 1 h 1 h 2 + h 2 ) where I volumes between are calculated are h 1 and h 2 Although neither age nor differential experience affected BrdU + cell nuclei diameters, B rdU + cells per mm 2 as well as total cell estimates are reported because of expected effects of enrichment on dentate volumes.
43 New cell phenotypes. To determine whether new cells differentiated into neurons or glia, we examined at least 100 BrdU + cells on q uadruple fluorescent stained sections (2 4 in young rats and 4 6 in aged rats) randomly selected from a set of every 12th section through the dentate gyrus for the co expression of neuronal and glial proteins using a Zeiss meta LSM 710 fully spectral laser scanning confocal microscope with 405, 488, 543 and 633nm laser lines (Thornwood, NY, USA) under a 40 objective (and 2.3 digital zoom). BrdU + cells were considered co + nucleus was unambiguous ly associated with DCX and/or NeuN, NG2, or GFAP. The percentage of BrdU + cells expressing each protein was calculated ( Figure 2 4). Statistical Analyses Statistical analyses were performed using Statistica software (Version 10; Statsoft, Tulsa, OK, USA). Analyses of variance (ANOVA) explored the effects age (young, aged) and experience (individually housed, enriched group housed), on cognitive (latencies, path lengths, and probe trial discrimination index scores), health (body mass, swim speeds), and neuro genesis (new cell numbers, percentage and total new neurons and glia) measures and Newm an Keuls post hoc tests revealed group 2 tests revealed the number of animals that performed at or above chance on probe trials and Pearson product moment correlations ( r ) tested relationships between neurogenesis and behavioral measures. The set at 0.05
44 Results Daily Enrichment Partially Reverses the Effect of Age on Spatial Ability Enrichment enhances spatial learning in aged rats Because measures of path length and latency over trials were correlated p ositively ( r (29) = 0.82; p < 0.0001), we report only path lengths to avoid redundancy. An ANOVA exploring the effects of age (young vs. aged), training block (blocks 1 5), and differential experience (individually housed vs. enriched) on path length ( Figur e 2 2 A) revealed significant effects of age ( F (1,26) = 15.65; p < 0.001) and training block ( F (4,104) = 5.85; p < 0.001) and significant age environment ( F (1,26) = 5.57; p < 0.05), age training block ( F (4,104) = 4.94; p < 0.001) and age environment training block ( F (4,104) = 3.24; p < 0.05) interaction effects. All rats improved their perform ance across blocks (block 1 > 3 5 and 3 > 5; p values < 0.005) but, as expected, young rats outperformed aged rats. Across all blocks, AI rats performed more p oorly than AE rats ( p < 0.05) and young rats in either group ( p values < 0.001) while YI and YE rats' performance improved equally rapidly across training blocks ( p values > 0.1). Specifically, AI rats performed significantly more poorly on training blocks 3, 4, and 5 ( p values < 0.01) and AE rats performed significantly more poorly only on training block 5 relative to YI and YE rats ( p values < 0.01). An ANOVA on swim speeds (mean [SEM] cm/second = 26.06 1.07 [YI], 29.92 2.07 [YE], 19.45 0.94 [AI], and 18.68 1.64 [AE]) revealed a significant effect of age ( F (1,26) = 33.49; p < 0.001) and a significant environment training block interaction ( F (4, 104) = 2.68; p < 0.05). Young rats swam significantly faster than aged rats across all training blocks and enriched rats swam significantly faster during the first training block relative to all other training blocks ( p values < 0.05).
45 Figure 2 2 Exposu re to an enriched environment subdues age dependent impairments on hidden and visible platform trials. The rats were trained on hidden platform trials (A), tested on a probe trial administered 15 minutes after the last hidden platform trial (B) and cued pl atform trials 3 days later (C). Line graphs depict group means ( standard error of the mean) of measures obtained from the young rats housed individually (YI; white circles), young rats housed in an enriched environment (YE; dark gray circles), aged rats housed individually (AI; light gray squares) and aged rats housed in an enriched environment (AE; black squares) groups. (A) Enrichment enhanced the ability of aged rats to rapidly acquire a spatial search strategy. On all training blocks combined, young r ats swam more directly to the hidden pla tform than aged rats. AI rats swam more circuitous routes to the hidden platform than either AE rats or young rats in either group. (B) Probe trial discrimination index (DI) scores 2 tests confirmed that the percentage of rat s that performed above or below chance (discrimination index (DI) score = 0, dashed line) decreased with age but increased with exposure to enrichment. Specifically, 69% of enriched rats performed above chance whereas only 50% of individually housed rats p erformed above chance. (C) Previous experience influences performance on visible platform trials. Young rats outperformed aged rats on all training blocks, including the initial training block, likely because they retained procedural information from the s patial task and AE rats outperformed AI rats. The effects of age o n swim speed are unsurprising because aged rats were heavier than young rats (mean [ SEM] g = 336.83 6.38 [YI], 347.37 4.44 [YE], 419.21 10.78 [AI], and 406.74 8.21 [AE]; F (1,26) = 78.87; p < 0.001). The effect of enrichment on the performance of aged rats on hidden trials is likely related to cognition because enrichment neither affected swim speeds nor body mass in aged rats.
46 An ANOVA exploring the effects of age, training block, and environment on the percent of time spent in the outer annulus during hidden platform trials (mean [ SEM] % = 56.07 4.47 [YI], 43.00 4.15 [YE], 81.98 0.92 [AI], and 64.32 3.13 [AE]) revealed significant effects of age ( F (1,26) = 46.63; p < 0.0001), environment ( F (1,26) = 19.76; p < 0.0001), training block ( F ( 4,104) = 3.32; p < 0.05), and a significant age training block interaction effect (F (4,104) = 3.95; p < 0.01). Overall, the percentage of time spent in the outer annulus significantly decreased in young versus aged rats, in enriched versus individually h oused rats and on later versus earlier training blocks block 1 and 2 > 5; p values < 0.05). While all young rats decreased their time spent in the outer annulus across blocks (block 1 and 2 > 3 5; p values < 0.01), aged rats maintained their time across bl ocks ( p values > 0.78). An ANOVA revealed a significant effect of age on probe trial discrimination index scores ( F (1,26) = 8.40, p values < 0.01) but no effect of environment (Figure 1 2 B). Because one YI and one YE rat performed at chance (i.e., discrim ination index = 0) and 2 tests on the percentage of rats performing 2 = 78.55, p < 2 = 14.44, p < 0.0005), with 6 9% of enriched rats performing above chance and only 50% of individually housed rats performing above chance. The effect of differential experience was mainly due to an effect of 2 = 71.59, p < 0.0005). Take n together, these data confirm that although young rats outperformed aged rats, enrichment enhanced the ability of aged rats to rapidly acquire a spatial search strategy.
47 Enrichment enhances cue discrimination learning in aged rats An ANOVA exploring the e ffects of age, training block, and environment on path length for the cued discrimination task revealed significant effects of age ( F (1,26) = 62.37; p < 0.001) and training block ( F (4,104) = 9.92; p < 0.001) but not enrichment. Post hoc tests confirmed tha t young rats swam more directly to the visible platform than aged rats but that all groups exhibited improved performance across training blocks (block 1 > 2 > 3, 4, and 5; p group indica ted a significant effect of training in 3 groups (YE: F (4,24) = 5.68; p < 0.005; AE: F (4,32) = 3.66; p < 0.05; AI: F (4,24) = 3.28; p < 0.05), with a tendency ( p = 0.07) for a training effect in YI animals. This tendency was due, in part, to near asymptotic performance on the first training block. Indeed, young rats exhibited shorter path lengths on the first block of cue training relative to the first block of spatial training ( Figure 2 2 A and C) indicating a carryover effect of prior training on the spati al task. Finally, age tended to interact with environment ( p < 0.10) and post hoc tests indicated that AE rats swam shorter path lengths than AI rats ( p = 0.05; Figure 2 2 C). An ANOVA exploring the effects of age, training block, and environment on averag e swim speed across visible platform trials (YI = 27.88 1.04 cm/s, YE = 26.28 1.33 cm/s, AI = 20.69 1.10 cm/s, and AE = 22.74 1.31 cm/s) confirmed that young rats swam significantly faster than aged rats ( F (1,26) = 18.82; p < 0.001), but there was no effect of differential experience. Age tended to interact with training block ( F (4,104) = 2.32; p = 0.062) such that young rats increased their swim speeds (block 1 < 4 5; p values < 0.05, while aged rats maintained their slower swim speeds ( p > 0.74) a cross all blocks.
48 An ANOVA exploring the effects of age, training block, and environment on the percentage of time spent in the outer annulus during cued platform training (YI = 36.00 2.94%, YE = 28.90 2.94%, AI = 75.14 4.08%, and AE = 64.15 4.69%) revealed significant effects of age ( F (1,26) = 88.64; p < 0.0001) and differential experience ( F (1,26) = 5.24; p < 0.05). Less time was spent in the outer annulus by young versus aged rats and by enriched versus individually housed rats. While all rats de creased the time they spent in the outer annulus across blocks ( F (4,104) = 12.57; p < 0.0001; training blocks 1 and 2 > 3 5; p < 0.05), young rats ventured from the maze wall in early trials (block 1 > 2 > 3 5; p values < 0.001) whereas aged rats ventured from the wall only in later training blocks (block 3 > 4 and 5; p values < 0.05; age block interaction effect: F (4,104) = 8.18; p < 0.001). The Effect of Enrichment Overcomes the Effect of Age on Neurogenesis Enriched environment reverses the effect of a ge on total new cell number An ANOVA revealed significant effects of age ( F (1,26) = 4.26; p < 0.05) and environment ( F (1,26) = 11.14; p < 0.01) on the total number of new (BrdU + ) cells produced and/or surviving 4 wee ks in young and aged rats ( Figure 2 3). More new cells were found in the dentate gyri of young versus aged rats and in enriched versus individually housed rats ( Figure 2 3 C). Enrichment similarly increased the number of new cells in the dentate gyri of both young and aged rats. This effect of e nrichment appears robust because we found similarly increased (mean [SEM] new cell densities enriched: 1345.13 204.12 cells/mm 3 vs. individually housed: 881.10 156.82 cells/mm 3 F (1,26) = 5.83; p < 0.05) despite increased GCL volumes (enriched: 3.97 0.31 mm 3 vs. individually housed: 3.25 0.17 mm 3 ; F (1,26) = 7.81; p < 0.01). Neither cell
49 density nor GCL volume was affected by age or the interaction between age and environment. Enriched environment does not reverse the effect of age on neuronal diff erentiation We calculated the proportion of BrdU + cells that coexpressed markers for immature (DCX + ), transitioning (DCX/NeuN + ), or mature (NeuN + ) neurons, or GFAP + astrocytes, or NG2 + oligodendrocyte precursors ( Figure 2 4 A C). Figure 2 3 Exposure to an enriched environment reversed the effects of age on neurogenesis. Rats were given 5 daily injections of br omodeoxyuridine (BrdU) beginning 1 week after behavioral testing and perfused 4 weeks later. The total number of new cells surviving 4 weeks was estimated stereologically using BrdU + cell counts obtained under light microscopy from every 12th section throu gh the dentate gyrus (DG). The bar graph depicts group means ( standard error of the mean) of total new cell number in the dentate gyri of young rats housed individually (YI; white bars), young rats housed in an enriched environment (YE; dark gray bars), aged rats housed individually (AI; light gray bars) and aged rats housed in an enriched environment (AE; black bars). (A) Coronal view of the rat brain. The dentate granule cell layer (GCL) is highlighted in turquoise. (B) Photomicrograph of new (BrdU + ) ce lls in the DG of an aged rat. Representative examples of 4 5 week old cells labeled with BrdU (in brown) revealed enzymatically with 3,3' diaminobenzidine tetrahydrochloride (DAB). (C) Total new cell numbers declined with age but were potentiated by enrich ment, regardless of age. More new cells survived approximately 4 weeks in the dentate gyri of young versus aged and enriched versus individually housed rats. p p An ANOVA exploring the eff ects of age and environment on the percentage of new cells expressing each phenotype revealed significant effects of age ( F (1,26) = 7.99; p < 0.01)
50 and phenotype ( F (1,104) = 532.30; p < 0.001) and a significant age phenotype interaction effect ( F (1,104) = 17.18; p < 0.001; Figure 2 4 D). Figure 2 4. Fewer new cells expressed mature neuronal phenotypes in the dentate gyri of aged rats. At least 100 bromodeoxyuridine (BrdU) + cells per rat were examined under confocal microscopy (40 objective with 2.3 digital zoom) to calculate proportions expressing markers of immature (doublecortin [DCX] + ), transition ing (DCX/NeuN) + or mature (Neuronal Nuclei [NeuN] + ) neurons, as well as glial fibrillary acidic protein (GFAP) + astrocytes or chondroitin sulfate proteoglycan (NG2) + oligodendrocyte precursors, which were revealed using fluorescent immunohistochemistry. A ll BrdU + cells stained with the nuclear marker 4'6 Diamidino 2 Phenylindole Dihydrochloride (DAPI). (A and B) Confocal images of new neurons and astrocytes in the dentate gyrus of an adult rat. Representative images of approximately 4 week old BrdU + cells (in red) that express the neuronal markers DCX (in blue) and/or NeuN (in green; A) or the glial markers GFAP (in blue) or NG2 (in green; B). (C) The proportion of new cells expressing neuronal phenotypes decreased with age and was unaffected by enrichment regardless of age. Mean ( standard error of the mean) percentage of new cells in the dentate gyri of young rats housed individually (YI; white bars), young rats housed in enriched environment (YE; dark gray bars), aged rats housed individually (AI; light gray bars), and aged rats housed in an enriched environment (AE; black bars) expressing neuronal and glial phenotypes are shown. In all rats, the majority of approximately 4 week old cells expressed mature neuronal phenotypes. However, a lower percentage o f new cells expressing mature neuronal phenotypes and a higher percentage of new cells expressing glial phenotypes was detected in aged versus young rats. No effect of differential experience on the percentage of new cells expressing either phenotype was d etected in young or aged rats. p p
51 Consistent with the extended survival period of the study, the majority of new cells expressed a mature neuronal phenotype ( p < 0.0001 vs. all other phenotypes). Of the < 10% of BrdU + cells expressing glial or immature neuronal phenotypes, astrocyte phenotypes were expressed most fr equently ( p < 0.01 vs. immature and transitioning neurons). Significantly fewer BrdU + cells expressed a mature neuronal phenotype in aged versus young rats ( p < 0.0001) and this effect was not reversed by enrichment. In fact, a higher proportion of BrdU + c ells in aged versus young rats did not express the markers of differentiation employed in this study (YI = 13.80 3.79, YE = 15.94 5.07, AI = 27.85 8.10, AE = 33.05 4.39% BrdU + cells; F (1,26) = 8.00; p < 0.01). Enriched environment increases net ne urogenesis We next determined the total number of new neurons (immature, transitioning, and mature neurons combined) and new glia (oligodendrocytes and astrocytes) by multiplying the estimated total number of BrdU + cells by the proportion of BrdU + cells co expressing each phenotype ( Figure 2 5). An ANOVA exploring the effects of age and environment on total new neuron number revealed statistically significant effects of age ( F (1,26) = 10.32; p < 0.01) and environment ( F (1,26) = 7.18; p < 0.05). More new ne urons were found in the dentate gyri of young versus aged rats and in enriched versu s individually housed rats ( Figure 2 5 A). Importantly, no age environment interaction was observed indicating that enrichment increased net neurogenesis similarly in you ng and aged rats. An ANOVA exploring the effects of age and environment on total new glia revealed a statistically significant effect only for age ( F (1,26) = 4.26; p = 0.05), such that more new glia (primarily astrocytes) were found in the dentate gyri of aged versus young rats ( Figure 2 5 B). However, the reliability of this effect requires replication in future work because of the low frequency in which new glia were observed.
52 Figure 2 5. Net neurogenesis declines with age but is increased by exposure to enrichment whereas age dependent increases in gliogenesis are unaffected by enrichment. Net neurogenesis and gliogenesis was calculated by multiplying total new cell numbers ( Figure 2 3) by percentage of new cells expressing neuronal and glial phenotypes ( Figure 2 4), respectively. The bar graphs depict group mean ( standard error of the mean) numbers of neurons (A) or glia (B) in the dentate gyri of young rats housed individually (YI; white bars), young rats housed in an enriched environment (YE; dark gray bars), aged rats housed individually (AI; light gray bars), and aged rats housed in an enriched environment (AE; black bars) rats. (A) Net neurogenesis declines with age but increases with enrichment, independent of age. Neurogenesis declin ed with age and was potentiated by exposure to an enriched environment regardless of age. A few weeks of exposure to an enriched environment, therefore, returned levels of hippocampal neurogenesis in aged rats to those observed in young individually housed rats. (B) Age dependent increases in gliogenesis are unaffected by exposure to enrichment. We detected a small but significant increase in gliogenesis in aged versus young rats that was unaffected by differential experience. p p Higher Rates of Neurogenesis Relate to Better Water Maze Performance in Aged Rats Pearson product moment correlations were employed to measure the relationships between ongoing neurogenesis and measures of water maze performanc e (mean path length and discrimination index) in each age group. Note that longer path lengths on hidden platform trials are indicative of more circuitous routes and therefore poorer performance whereas higher discrimination index scores indicate better di scrimination between the target and opposite quadrants on probe trials and therefore
53 better performance. New neuron number correlated significantly with average path length across hidden platform t rials ( r p < 0.05; Figure 2 6 B) and probe trial discrimination index s cores ( r = 0.59; p < 0.05; Figure 2 6 D), in aged but not young rats. Discussion In the current study, we confirmed that hippocampal neurogenesis and spatial learning are compromised by age and that exposure to environmental enrichment potentiates neurogenesis, regardless of age. We found that environmental enrichment improves the performance of aged but not young rats on a water maze task in which the hidden platform location is learn ed in a single day. We propose that this task requires the ability to rapidly acquire and flexibly use spatial information that appears intact and therefore unaffected by enrichment in young rats but compromised and improved by enrichment in aged rats. We also reveal a novel age specific relationship between total new neuron number and indexes of ability in a rapid water maze task. Decreased hippocampal neurogenesis is characteristic of aging (Cameron and McKay, 1999a; Kuhn et al., 1996; Nacher et al., 2003) Although our single experiment end point cannot disentangle the effects of age on NPC proliferation versus new cell survival, our effect is consistent with the well known effects of age o n NPC proliferation. Fewer BrdU + cells expressed neuronal markers and more BrdU + cells were devoid of differentiation markers in the hippocampi of aged versus young rats, which is consistent with some reports that neuronal differentiation is compromised by age (Kempermann et al., 1998) Our finding that gliogenesis increased with age has been noted by others (Bizon et al., 2004) but because so few new glia were detected in the dentate gyri of either young or aged rats, the reliability of this effect should be tested in future work.
54 Overall, our findings support published work show ing age related decreases in neurogenesis are mediated by increasing NPC quiescence across life and because fewer NP C progeny adopt neuronal fates. Figure 2 6. New neuron number correlates with measures of spatial ability. Graphs depict total neuron number plotted agai nst mean path lengths across hidden platform training blocks (A and C) or against probe trial discrimination index scores (B and D) for young rats housed individually (YI; white circles), young rats housed in an enriched environment (YE; dark gray circles) aged rats housed individually (AI; light gray squares), and aged rats housed in an enriched environment (AE; black squares). Mean path lengths correlated negatively with total new neuron number in aged rats ( r shorter path lengths indicate better performance. (D) The number of new neurons and discrimination index score are correlated positively in aged rats ( r = 0.59). p
55 Environmental enrichment increases neurogenesis in aged rodents by potentiating neuronal differentiation and new cell survival (Kempermann et al., 2002; Kempermann et al., 1998; Leal Galicia et al., 2008; Segovia et al., 2006) Indeed, we found similar enrichment induced increases in the number of new cells surviving 4 5 weeks in the dentate g yri of young and aged rats ( Figure 2 3). However, enrichment neither reversed the effects of age on the proportion of BrdU + cells that expressed neuronal phenotypes nor increased the proportion in young rats ( Figure 2 4). Other studie s showing that exposure to enriched environments potentiates neuronal differentiation in young and aged have employed running wheels, larger social groups, and earlier more extended exposures to enriched environments, which could each potentiate different components of neurogenesis and probably each require more detailed investigation ( Lazarov et al., 2010; Lugert et al., 2010 ) Overall, we show that just a few weeks of exposure to environmental enrichment can increa se net neurogenesis ( Figure 2 5) in the hippocampus of aged rats by robustly enhancing new cell survival to the extent that it overcomes the effects of age on NPC proliferation ( Figure 2 3) and neuronal fate choice ( Figure 2 4). We expanded upon work showi ng that exposure to environmental enrichment enhances the ability of aged rats to discriminate the spatial location of a platform hidden in water maze tasks that distribute training across days (Fernandez et al., 200 4; Frick and Fernandez, 2003; Lores Arnaiz et al., 2006) by confirming that it also enhances their ability to discrimi nate a platform spatially ( Figure 2 2 A and B; ( Kumar et al., 2011 ) ) and visually ( Figure 2 2 C) in a water maze task that masses training sessions into a single day. We did not observe the anticipated beneficial effect of enrichment on water
56 performance in our young rats (Leggio et al., 2005; Schrijver et al., 2002) However, the effects of weeks rather than longer exposures to enrichment on spatial ability may be sex dependent and only observable in water maze protocols that distribute trainin g across days (Frick et al., 2003; Harburger et al., 2007) The rapidly asymptotic performances of YE and YI rats across hidden platform training blocks is consistent with the notion that the rapid water maze task m ay be sensitive to performance impairments but not enhancements in young rodents and precludes a meaningful evaluation of the relationship between their measures of ne urogenesis and spatial ability. Our data showing that AE and AI rats exhibited similar an xiety levels (percentage of time spent in the outer annulus), fitness (swim speeds and body mass), and perhaps visual acuity (similar performance was exhibited on early visible platform blocks), suggests that enrichment reverses age related changes in syst ems mediating spatial and visual discrimination, independent of overt effects on sensorimotor ability. Exposure to enrichment improves cerebellar, in addition to hippocampal function (Camel et al., 1986; Greenough an d Volkmar, 1973; Kumar et al., 2011 ) which could improve both spatial and visual discrimination. In addition, our unpublished data and previous research (Gerlai, 2001; Ormerod and Beninger, 2002) suggests that tra ining on sequential tasks (including spatial vs. visual discrimination) may beneficially or detrimentally affect performance on the second task. Indeed, young rats appeared to readily employ procedural information they acquired on spatial discrimination tr ials about escaping the water maze on early vis ual discrimination blocks ( Figure 2 2 A vs. C).
57 Exposure to an enriched environment produces many effects in the hippocampus that could relate to improved spatial discrimination in aged rats. For example, expo sure to an enriched environment increases hippocampal and vascular volumes as well as morphological and electrophysiological measures of plasticity in aged rats (Hattiangady and Shetty, 2008; Kumar et al., 2011; Leve nthal et al., 1999; Palmer et al., 2000) In support of other work employing water maze protocols with massed training schedules (Drapeau et al., 2003; Driscoll et al., 2006) measures of neurogenesis and spatial a b ility correlated strongly ( Figure 2 6). This rapid task may be more sensitive to the relationship than distributed training water maze protocols (Bizon and Gallagher, 2003; Bizon et al., 2004; Merrill et al., 2003) because it taxes the hippocampus by requiring faster acquisition and more flexible use of spatial information ( Foster 2012 ) We also may have simply increased the variability within our measures enough to detect the relationship by exposing aged r ats to differential experience. We cannot conclude that neurogenesis mediates spatial ability from our correlation data. However, our dat a do suggest that neurogenesis may be a marker of spatial ability and hippocampal integrity in aged rats because aged rats with higher ongoing rates of neurogenesis exhibited better spatial ability than those with lower rates. Indeed, environmental enrichm ent increases the expression of factors associated with enhanced spatial ability and neurogenesis, such as brain derived neurotrophic factor (Lee et al., 2002; Obiang et al., 2011) and stimulates the production of f actors that are downregulated with age and are known to be neurogenic, such as fibroblast growth factor 2, vascular endothelial growth factor, and insulin growth factor 1 (Shetty et al., 2005) Our data do suggest t hat future work investigating the relationship between
58 neurogenesis and hippocampal function across age may provide insight into the etiology and potential interventions for age related cognitive decline. In summary, we found that several weeks of daily ex posure to an enriched environment partially reverses the effects of age on the rapid acquisition of a spatial search strategy in the water maze, potentially through its effects on neurogenesis because we found higher ongoing rates of neurogenesis in aged r ats that exhibited better performance in the task. Our data suggest that engaging in mentally and physically stimulating activity could reverse some aspects of age related cognitive decline perhaps by potentiating neurogenesis.
59 CHAPTER 3 INFLAMMATORY BIOMARKERS INDICATIVE OF COGNITIVE AGE Introduction Chronological age is not a direct predictor of cognitive ability in aged individuals, which can be compromised in the absence of a direct pathology (Brayne, 2007; Foster, 2006) Unsuccessful cognitive aging has been linked to dysregulated gene expression, hormone levels and circulating cytokines (Blalock et al., 2003) Interestingly, memory deficits often appear by middle ag e, a time when gene expression is particularly variable (Foster, 2007) and circulating analytes, especially those associated with inflammation, could be used as biomarkers to help better predict and prevent age related cognitive decline. Aging rats show large variability in their performances on behavioral tasks across age. We (Foster, 2007) have used the water maze task to demonstrate that middle aged and aged rats can be classified as memory unimpaired (MU) or memory impaired (MI). Performance impairments among MI rats in these tasks begin to appear around 13 mo (Blalock et al., 2003) and are maximal at ~18 mo (Foster, 2007; Markowska, 1999) Consistent with other work, our results show that retention of recently acquired information is compromised in aged animals (Foster, 1999; Markowska, 1999; Speisman et al., 2013b ) Inflammato ry m arkers increase with age (Chung et al., 2009) and have been linked to memory decline in rodents and humans alike (Blalock et al., 2003; De Martinis et al., 2005; Foster, 2006; Magaki et al., 2007; Rafnsson et al., 2007; Solfrizzi et al., 2006; Vil leda et al., 2011 ) In particular, IL 6 and CRP are elevated in middle age and correlate with poor performance on cognitive tasks (Gimeno et al., 2008) Stress
60 hormones may also increase with age in humans and have been associated with impaired memory and hippocampal atrophy (He et al., 2008; Issa et al., 1990; Lupien et al., 1998; McEwen, 1998) In fact, we have shown that the expression of genes associated with hypothalamic pituitary adrenal axis activity and i nflammation increase with age in rats as well (Blalock et al., 2003) We, and others, have also found that hippocampal neurogenesis and other forms of plasticity decrease following neuroinflammatory insult, stress a nd with age (Casolini et al., 2002; Montaron et al., 2006; Ormerod et al., 2013; Sandi and Touyarot, 2006) P reviously we demonstrated a link between concentrations of central and circulating inflammatory cytokines in aged rats, with readouts of neurogenesis and cognitive ability and reversed age related decline with exercise ( Speisman et al., 2013a ) Here, we examine whether there are prognostic/diagnostic inflammatory bioma rkers of age related cognitive decline. We used water maze performance to identify age related memory impairments and measured the concentrations of circulating and central inflammatory cytokines and stress hormones in order to identify a useful biomarker to help predict biological age in comparison to chronologic age. While the readings taken from serum may offer a non invasive method of estimating cognition, hippocampal biomarker profiles offer potential mechanisms behind this biological process. Methods Subjects Young (8 mo; n=13), middle aged (14 mo; n=41) and aged (20 mo; n=24) male Fischer 344 rats were purchased from the National Institute of Aging colony at Harlan
61 Figure 3 1. Experiment timeline. Young (8 mo; n=14), middle aged (14 mo ; n=39) and aged (20 mo; n=27) male F344 rats were trained and tested on a rapid acquisition water maze task. Rats were given one day of visible platform training, followed three days later by one day of hidden platform training. Two probe trials were adm inistered to assess strength of learning and memory, one immediately following training and one 24 h later. Rats were sacrificed two weeks later. Blood serum was collected and brains were quickly extracted and dissected to obtain the hippocampus and cortex which were flash frozen for later quantification of proteins using Bio Plex technology. Sprague Dawley (Indianapolis, IA) where they were pair housed and given a NIH 31 diet and water ad libitum Similarly, in the University of Florida colony room rats were pair housed in shoebox cages on a 12:12 h light:dark cycle at 24 1 C and provided with Harlan Teklad irradiated food and reverse osmosis filtered water ad libitum Rats were routinely inspected for signs of aggression (bites and scratches) and age related health problems (poor grooming, hunched posture, excessive porphyrin around the eyes and nose, weight loss and tumors). Unhealthy rats were euthanized humanely. Every effort was made to minimize the number of rats used for the experiments and their suffering. All rats were treated in accordance with the policies set forth by the University of Florida Institutional Animal Care and Use Committee and the National
62 Institutes of Health regarding the ethical use of animals for experimentation. The experim ent timeline is depicted in Figure 3 1. Water Maze Training and T esting Rats were trained and tested on a rapid acquisition version of the Morris water maze consisting of a single day of visible platform training, followed three days later with a single d ay of hidden platform training on which the rats were tested immediately and 24 hours after with the platform removed. We have previously used this massed training paradigm to characterize age related cognitive decline in rats (Blalock et al., 2003; Carter et al., 2009; Fugger et al., 1998; Norris and Foster, 1999; Speisman et al., 2013a; Speisman et al., 2013b ) Water maze training and testing were conducted in a well lit room containing several large observable cue s located around a black cylindrical tank (1.7 m diameter). The tank was filled with water (27 2 C) to a depth of either 1.5 cm below a flagged black platform (29 cm diameter) on visible trials or 1.5 cm above a submerged platform (8 cm below the rim of th e tank) on hidden trials and probe trials for which the platform was removed. Pathlengths (cm), escape latencies (s), quadrant search times (s) and platform crossings were recorded throughout the tasks using a Columbus Instruments tracking system (Columbus OH). Between blocks, rats were warmed and dried and returned to their cages. Visible platform trials. The visible platform task gauges sensorimotor ability and visual acuity while also habituating the rats to the pool and creating the association between the platform and escape. For this non spatial task all visual extra maze cues were removed. Rats were given five blocks of three 60 s trials (20 s inter trial interval and 15 min inter block interval) to locate a flagged platform 1.5 cm above the water
63 su rface to escape the pool (Morris et al., 1982; Morris, 1981) Release and goal quadrants were randomly assigned for each trial. Failure to find the platform within 60 s prompted the experimenter to gently guide the rat to the platform before removal from the pool. Latency (s), pathlength (cm) and swim speed (cm/s) were recorded. Hidden platform trials. Three days after the visible platform task, ra ts began training on the hidden platform task which evaluates spatia l navigation, specifically using robust extra maze cues around the room to locate a platform hidden 1.5 cm below the surface of the water. Rats were released randomly from one of the three quadrants of the pool not containing the platform over five blocks of three 60 s trials (20 s inter trial interval and 15 min inter bock interval). If a rat failed to locate the platform within 60 s the experimenter gently guided the rat to the platform before removing him from the pool. Latency (s) and pathlength (cm) ac ross trials served as measures of learning while swim speeds (cm/s) were indicative of sensorimotor ability. Probe trials. The escape platform was removed from the water maze pool for two probe trials; the first was conducted immediately fo llowing hidden platform training to assess strength of learning and immediate memory and the second 24 h later to measure longer term memory. Rats were released from the quadrant opposite the previous location of the escape platform for both probes and allowed to swim fr eely around the pool for 60 s. The time (s) and distance (cm) spent in each quadrant, number of platform crossings, as well as a discrimination index (t(G) t(O)/t(G)+t(O), where t(G) is time spent in the goal quadrant and t(O) is time spent in the opposite quadrant) were recorded. Immediately after the first probe trial a block of hidden
64 platform training was given to reinforce the association between finding the platform and escape from the pool. Sample Collection and Protein H arvest Two weeks following th e last water maze probe trial rats were deeply anesthetized with isoflurane (Halocarbon Laboratories, River Edge, NJ) prior to decapitation. Collected trunk blood was kept at 4 C for 24 h before centrifugation at 1,000 x g for 10 min, after which blood ser um was collected and stored at 86 C until Bio Plex analysis. Hippocampal and cortical tissue were quickly dissected from both hemispheres of the extracted brain and immediately flash frozen and stored at 86 C until protein harvest. Tissue samples were ma intained at 4C throughout protein harvest. Tissue was suspended in 0.1 M tris buffered saline (TBS) containing 0.1% Igepal and 1 L/mL of two protease inhibitor cocktails added immediately before use: 1) 0.5 M phenylmethylsulfonyl fluoride, 5 mg pepstatin A and 1 mg chymostatin/1 mL DMSO and 2) 1 M G aminocapfroic acid, 1 M P aminobenzidine, 1 mg leupeptin and 1 mg aprotinin/mL sterile water. Tissue was mashed manually and then sonicated with a dismembrator (ThermoFisher Scientific; Pittsburgh, PA) before centrifugation at 12,000 rpm for 10 min at 4C to isolate protein. Protein concentrations were measured using a Bradford protein assay and Bio Rad SmartSpec Plus Spectrophotometer (Hercules, CA). Tissue supernatant was stored at 86C until further analysi s. Bio Plex Quantification of C ytokines A Bio Rad Bio Plex 2000 suspension array system and EMD Millipore Rat Cytokine/Chemokine kits (Cat No. RCYTO 80K PMX; Billerica, MA) and Rat Stress Hormone kits (Cat No. RSH69K) were used to quantify the concentra tions of immune
65 cytokines/chemokines and stress hormones in the blood serum and hippocampal and cortical supernatant samples according to kit instructions and has been described previously ( Speisman et al., 2013a ) A cytokine/chemokine kit simultaneously measures the following 24 analytes in a single sample: eotaxin (3.27 20,000 pg/mL), G CSF (1.31 20,000 pg/mL), GM CSF (13.11 20,000 pg/mL), GRO/KC (2.06 20,000 pg/mL), IFN (4.88 20,000 pg/mL), IL 20,000 pg /mL), IL 20,000 pg/mL), IL 2 (3.67 20,000 pg/mL), IL 4 (2.30 20,000 pg/mL), IL 5 (2.89 20,000 pg/mL), IL 6 (9.80 20,000 pg/mL), IL 9 (12.85 20,000 pg/mL), IL 10 (5.41 20,000 pg/mL), IL 12 (4.13 20,000 pg/mL), IL 13 (23.2 20,000 pg/mL), IL 17 (1.61 20,000 pg/mL), IL 18 (4.78 20,000 pg/mL), IP 10 (3.78 20,000 pg/mL), leptin (21.50 100,000 pg/mL), MCP 1 (3.81 20,000 pg/mL), MIP 20,000 pg/mL), RANTES (54.42 20,000 pg/mL), TNF 20,000 pg/mL) and VEGF (4.93 20,000 pg/mL). The stress hormo ne kit measures: ACTH (3.8 4,000 pg/mL), corticosterone (10,834 400,000 pg/mL) and melatonin (897 400,000 pg/mL). Briefly, all samples were prepared on ice and serum and tissue samples were run on separate plates. Standards were prepared by serial dilutio n with kit assay buffer for the cytokine/chemokine assay (with expected concentrations of 20,000, 5,000, 1,250, 312.5, 78.13, 19.53 and 4.88 pg/mL of each analyte except leptin which had expected concentrations of 100,000, 25,000, 6250, 1,562.5, 390.63, 97 .66 and 24.41 pg/mL) and the hormone assay (with expected concentrations of 400,000, 133,333, 44,444, 14,814, 4,938 and 1,646 pg/mL of each analyte except ACTH which had expected concentrations of 4,000, 1,333, 444.4, 148.1, 49.4 and 16.5 pg/mL). Tissue su pernatant samples were used neat while serum samples were diluted with kit assay
66 buffer (1:5 for cytokine/chemokine assay or 1:3 for hormone assay). of each standard, vendor supplied control and sample were loaded in duplicate into a 96 well filter plate (EMD Millipore; Billerica, MA). All wells were brought to a final volume of 50L with the addition of either 25 L assay buffer to a ll sample wells or 25 L kit serum matrix or extraction buffer to standard and control wells in the serum or tissue quantification plates respectively. A mixture of 24 different polystyrene beads with unique color addresses and capture antibodies for the c ytokine/chemokine kit and three different color addresses and capture antibodies for the stress hormone kit were added to all wells before overnight incubation with agitation at 4 C. Numerous vacuum filtration washes using kit wash buffer were repeated bet ween steps. Biotinylated detection antibodies were then adsorbed to the appropriate bead. Following several washes, a streptavidin phycoerythrin reporter was added. The bead complexes were washed and resuspended in sheath fluid (Bio Rad; Hercules, CA) to b e run through the dual laser Bio Rad Bio Plex 2000 system with Luminex xMAP technology (Bio Rad; Hercules, CA). Bead wavelength emission identified each analyte by its unique color address and concentrations were quantified by phycoerythrin emission intens ity. Data were compiled using Bio Plex Manager Software version 4.1. All standard, control and sample protein concentrations were acquired from readings from more than 35 beads that passed within the double discriminator gated region to exclude broken or aggregated beads. A five parameter logistic non linear regression model was used to generate a standard curve for each analyte based on the average of duplicate observed concentrations. A single standard was used if one of the duplicate observed standard c oncentrations was > 10% CV (coefficient of variation) or if
67 the percent recovery (observed/expected concentration) fell outside of the accepted 70 130%. Provided positive control concentrations were confirmed to fall within the expected ranges before sampl e concentrations were determined according to their median fluorescent intensity using the appropriate standard curve. Cytokine concentrations that fell below the threshold of detection were set at 0 and those that exceeded the maximum expected concentrati on were set to the maximum expected concentration. If the % CV for a set of duplicate sample concentrations was > 10% and a concentration fell 2 standard deviations from the group mean the outlying concentration was discarded. Final duplicate sample conce ntrations were averaged and reported in pg/mL for serum while hippocampal and cortical tissue concentrations were normalized using tissue mass and thus reported as pg/mg of hippocampal or cortical tissue. Correlation Cluster Analysis Spearman Rank correl ations were used to examine the relationships between cytokines within the same compartment (blood, hippocampus or cortex) or across compartments. The level of significance was adjusted using Bonferroni correction for multiple comparisons of analytes (27 a nalytes in 3 compartments produces 3,240 comparisons) to p = 0.0000154. Analyte pairs with significant Spearman r values were plotted in descending order as previously described (Baron and Kenny, 1986; Erickson and B anks, 2011; Speisman et al., 2013a ) modification of (Baron and Kenny, 1986; Erickson and Banks, 2011) an analyte was only connected to an existing cluster if it significantly c orrelated to all analytes in the existing cluster. For example, if analytes A and B are already plotted (A B) and analyte C correlates significantly to A, then it must also correlate with B before it can be plotted
68 in connection to A (C A B). If C does not correlate with B, then a new analyte pair will be drawn, A C in addition to A B. When an analyte pair was indirectly connected by previous connections (through mediators) then the residual correlation was calculated by subtracting the product of all media ting correlations ( r values) from the correlation in question. Only if the residual correlation was still significant then the pair would be connected thus creating a loop. Biological Age Regression Analysis Using multiple linear regression analyses a bio logical age based on serum and hippocampal biomarkers of age related cognitive decline was generated for chronologically 14 months old middle aged and 20 months old aged rats. Spearman rank correlation were used to examine the relationships between analyte concentrations and immediate and 24 h DI scores in order to identify potential biomarkers ( r > 0.20). Ranked analyte concentrations were then used as independent parameters in linear combination to outline a linear relationship to the specific chronologic al ages used in the experiment (young: 8 mo, middle aged: 14 mo, and aged: 20 mo) such that a biological age could then be calculated. For analysis of biomarkers capable of predicting learning impairments on the immediate water maze probe rats were divided into two groups: learning unimpaired (DI > 0.25) or learning impaired (DI < 0.25). Similarly to assess memory impairments rats were classified as memory unimpaired (DI > 0) or memory impaired (DI < 0) on the 24 h water maze probe. In order to assess the goodness of fit the statistical significance of a t test of the individual parameter, biological age, for each set of possible biomarkers was examined comparing impaired to unimpaired groups. We also report the percent accuracy for which the model predicts the correct classification (unimpaired or impaired) and the percent of type I (false negatives) and
69 type II (false positives) errors. The best biomarker or biomarker combination produced a significant difference between cognitively impaired and cognitivel y unimpaired rats and furthermore classified rats based on biological with the highest percent accuracy. Individual analyses were conducted using either serum or hippocampal biomarkers to predict learning or memory impairments for either middle aged or age d rats. Statistical A nalyses STATISTICA software (Version 10; StatSoft; Tulsa, OK) was used for all statistical analyses. Analyses of variance (ANOVA) explored the effect of the independent variable age on measures of general health (body mass and swim s peeds), water maze performance ( visible and hidden platform trial latencies and pathlengths, time spent in each quadrant, probe trial discrimination indices and number of platform crossings). Using the 24 hr probe discrimination index (DI) middle aged and aged rats were classified as either memory unimpaired (MU) if DI > 0 or memory impaired (MI) if DI < 0. Note, all young rats were classified as MU since all scores were > 0. The resulting classifications were used as an independent variable to further anal yze cytokine concentrations. Newman Keuls post hoc tests were used to examine significant group differences. All data are represented as the group average ( S.E.M.) except the probe trial discrimination indices, which plot individual scores, and was stat istically analyzed with a Mann levels for all statistical tests was 0.0000154 using Bonferroni correction for multiple comparison.
70 Results Water Maze V isible Platform Task Performance Deteriorates with A ge Since path lengths and escape latencies correlated positively across both the visible ( r values 0.68, p values < 0.001) and hidden ( r values 0.84, p values < 0.001) platform tasks, we report path l engths to avoid redundancy. An ANOVA exploring the effects of age and training block on visible platform path lengths (Figure 3 2 A) found an effect of age ( F (2,75) = 9.57; p < 0.001), training block ( F (4,300) = 26.51; p < 0.001), and an interaction betwee n the two ( F (8,300) = 2.70; p < 0.01). A Newman Keuls post hoc analysis revealed that young rats swam shorter distances to the visible platform to escape the pool than both middle aged ( p < 0.01) and aged rats ( p < 0.001), with middle aged rats tending to swim shorter distances than aged rats ( p = 0.06). Combined, all rats swam increasingly more directly to the platform on successive training blocks (block 1 > 2 > 3 > 4, p values < 0.05) except the last block compared to block 4 ( p > 0.05). Specifically, y oung rats swam more directly to the platform on all blocks compared to the first ( p values < 0.05) as well as block 4 and 5 < block 2 ( p values < 0.01). Middle aged rats also improved across blocks (1 and 2 > 4 and 5; 3 > 5; p values < 0.05). However, aged rats showed little improvement with rats tending to have shorter pathlengths on block 4 ( p = 0.052) and 5 ( p = 0.077) compared to block 2. Since aged rats have been shown to have shallow le arning curves on this task further analysis compared the first two blocks (block 1 and 2) and the last two blocks (block 4 and block 5).
71 Figure 3 2. Water maze vis ible platform task performance declines with age. Data are shown as group means ( S.E.M.). White circles represent young, gray triangles represent middle aged and black squares represent aged rats. (A) Young rats swam shorter distances to the visible escap e platform than both middle aged ( p < 0.05) and aged rats ( p < 0.001). Middle aged rats also swam shorter pathlengths than aged rats ( p < 0.05). All rats swam more directly to the platform on successive training blocks (1 > 2 > 3 > 4, p values < 0.05) exce pt the last block ( p > 0.05). Specifically, young rats: 1 and 2 > 3, 4 and 5 ( p values < 0.05), middle aged rats: 1 > 3, 4 and 5 ( p values < 0.05), 2 > 4 and 5 ( p values < 0.01) and 3 > 5 ( p < 0.01), and aged rats: 2 > 4 ( p < 0.05). (B) Swim speed decrease d with age (young > middle aged > aged, p values < 0.01). All rats combined swam the slowest on the first training block compared to all other blocks ( p values < 0.001) and slower on block 2 compared 5 ( p < 0.05). Specifically, for young rats block 1 < 3, 4 and 5, and block 2 < 5, while middle aged rats only swam more quickly on block 3 compared to block 1, and for aged rats 1 < 2, 3 and 5 (all p values < 0.05). (C) All rats spent less time around the edge of the tank, a measure of anxiety, on blocks 3, 4 a nd 5 compared to blocks 1 and 2 ( p values < 0.01). Anxiety decreased as training commenced for young rats on blocks 3, 4, 5 < 1, 2 ( p values < 0.05), and for middle aged rats 5 < 1, 2 and 4 ( p values < 0.05), but aged rats failed to show any improvement ac ross training ( p values > 0.05). An ANOVA similarly found an effect of age ( F (2,75) = 8.54 ; p < 0.001) with young rats swimming faster than both middle aged and aged rats ( p values < 0.05) and middle
72 aged rats tending to swim faster than aged rats ( p = 0.051). There was also an effect of block ( F (1 ,75) = 107.80 ; p < 0.001) with all rats swimming more directly to the platform on the last two blocks compare d to the first two blocks. A significant age and block interaction ( F (2,75) = 4.97 ; p < 0. 01) revealed that young, middle aged and aged rats ( p values < 0.001) all found the platform more directly at the end of training compared to the start, thus all rats learned the task. An ANOVA examining the effects of age and training block on swim speed (pathlength (cm)/latency (s); Figure 3 2 B) uncovered an effect of age ( F (2,75) = 16.26; p < 0.001) and training block ( F (4,300) = 9.03; p < 0.001) with a noted tre nd in the age x training block interaction ( F (8,300) = 1.82; p = 0.07). All rats combined swam the slowest on the first training block compared to all other blocks ( p values < 0.01) and slower on the second compared to the last ( p < 0.05). Swim speed decre ased significantly with age (young > middle aged > aged, p values < 0.01). Slower swim speeds with age may be due to body mass (young: 415.18 11.95 g, middle aged: 462.62 7.72 g, aged: 457.89 6.30 g), which changes with age ( F (2,75) = 6.07; p < 0.01) as young rats in this experiment weighed significantly less than both middle aged and aged rats ( p values < 0.01). T he percent of time spent in the outer annulus of the water maze tank (Figure 3 2 C) was assessed as a measure of anxiety using an ANOVA to detect an effect of age ( F (2,37) = 3.64; p < 0.05), training block ( F (4,148) = 13.67; p < 0.001) and age x block interaction ( F (8,148) = 3.22; p < 0.01). Newman Keuls post hoc analysis revealed that aged rats spent significantly more time around the outer edge of the tank compared to young rats ( p < 0.05) while this was only a tendency for middle aged rats ( p = 0.08). All
73 rats spent less time around the edge of the tank on blocks 3, 4 and 5 compared to blocks 1 and 2 ( p values < 0.01). Specifically, young rats showed decreased anxiety as training commenced (blocks 3, 4, 5 < 1; and 4, 5 < 2 ( p values < 0.05) and middle aged rats spent less time around the tank edge on block 5 compared to blocks 1, 2 and 4 ( p values < 0.05), but aged rats failed to show signi ficant improvement across training (all p value s > 0.05). Hidden Platform Training on the Water Maze is Impaired with A ge An ANOVA exploring the effect of age and training on pathlength (Figure 3 3 A) to find a hidden platform found an effect of age ( F (2, 75) = 5.54; p < 0.01) and training block ( F (4,300) = 49.34; p < 0.001) but no interaction effect ( F (8,300) = 0.94; p > 0.05). Newman Keuls post hoc analysis revealed that young rats swam more directly to the hidden platform than both middle aged and aged r ats ( p values < 0.01). All rats combined had shorter pathlengths on all subsequent blocks compared to the initial block and on blocks 4 and 5 compared to blocks 2 and 3 ( p values < 0.001). Furthermore, a planned comparison looking at the last block c ompare d to the first block showed that young middle aged and aged rats all learned this spatial task ( p values < 0.001). The effects of age and training block on swim speeds (Figure 3 3 B) were analyzed using an ANOVA to reveal an effect of age ( F (2,75) = 13.76; p < 0.001) and training block ( F (4,300) = 3.88; p < 0.01) but not the interaction between ag e and training block ( F (8,300) = 1.23; p > 0.05). Swim speed was found to significantly decr ease with age (young > middle aged > aged; p values < 0.05) after Newman Keuls post hoc analysis. With all rats combined, swim speeds on hidden platform training we re faster on blocks
74 2, 3 and 4 compared to the first block ( p values < 0.05) and on the last block compared to the second block. Figure 3 3. Water maze hidden platform performance is impaired with age. Data are shown as group means ( S.E.M.). White circles represen t young, gray triangles represent middle aged and black squares represent aged rats. (A) Young rats swam more directly to the hidden platform than both middle aged and aged rats ( p values < 0.05) and all rats combined had shorter pathlengths on all subsequ ent blocks compared to the initial block and on blocks 4 and 5 compared to blocks 2 and 3 ( p values < 0.001). Planned comparison of the last block to the first block showed that young, middle aged and aged rats all learned ( p values < 0.001). (B) Young rat s swam faster than middle aged ( p < 0.05) and aged rats ( p < 0.001) and middle aged rats swam faster than aged rats ( p < 0.01). Swim speeds on hidden platform training were faster on training blocks 2 and 3 compared to the first block ( p values < 0.05) wit h all aged groups combined. (C) Middle aged and aged rats spent more time around the tank outer annulus compared to young rats ( p values < 0.001) and all rats collectively spent less time around the edge on blocks 3, 4 and 5 compared to blocks 1 and 2 ( p v alues < 0.05). An ANOVA examining the effect of age and training block on the percent time spent in the outer annulus of the water maze tank during hidden platform training (Figure 3 3 C) revealed an effect of age ( F (2,37) = 11.87; p < 0.001), and training block ( F (4,148) = 11.60; p < 0.001) but no age x training block interaction ( F (8,148) = 0.93; p >
75 0.05). Newman Keuls post hoc analyses conf irmed that middle aged and aged rats spent more time around the tank edge compared to young rats ( p values < 0.001) and all rats collectively spent less time around the outer annulus on later blocks (3, 4 and 5) compared to earlier blocks (1 and 2; p value s < 0.01 ). Impaired Probe Trial Performance in Aged Rats Begins to Appear in Middle A ge Immediately following the fifth block of hidden platform training the platform was removed to administer a 60 s probe trial to assess strength of learning and perhaps short term memory. Figure 3 4. Impaired probe trial performance in aged rats begins to appear in middle age. Each data point represents an individual animal coded as white circles for young, gray triangles for middle aged and black squares for aged rats. Immediately followi ng training and 24 h later, 60 s probe trials were administered to assess strength of learning and memory. The time spent in each quadrant of the water maze was recorded and a discrimination indices were calculated form the time spent in the goal quadrant minus the time spent in the opposite quadrant divided by the time spent in both quadrants (t(G) t (O))/(t(G) + t (O)). (A) On the immediate probe trial, there was a noted trend of age affecting discrimination index ( p = 0.08) with performance worsening w ith age. (B) On the 24 h probe both middle aged and aged rats had worse discrimination indices than young controls ( p values < 0.01). The time spent in each goal quadrant and the number of times a rat crossed directly over the original location of the platform was recorded and a discrimination index was
76 calculated form the time spent in the goal quadrant minus the time spent in the opposite quadrant divided by the time spent in both quadrants (t(G) t (O))/(t(G) + t (O)). An A NOVA exploring the effect of age on discrimination index noted an effect age ( F (2,75) = 5.12; p < 0.01) with middle aged and aged rats having significantly lower discrimination indices than young rats ( p values < 0.01; Figure 3 4 A). The number of platform crossings, a measure of precision memory, was also affected by age (young: 6.00 0.64, middle aged: 3.49 0.32, aged: 3.17 0.36; F (2,75) = 9.52; p < 0.001). Newman Keuls post hoc analysis revealed that young rats crossed the exact platform location si gnificantly more times than middle aged or aged rats ( p values < 0.001). A second probe was administered 24 h after the first to assess longer term memory. An ANOVA examining the effect of age on the 24 h probe trial uncovered an effect of age ( F (2,75) = 7.46; p < 0.01) such that both middle aged and aged rats were found to have significantly worse discrimination indices than young controls following Newman Keuls post hoc analysis ( p values < 0.01; Figure 3 4 B). Similarly, age significantly affected the n umber of platform crossings as well (young: 3.85 0.61, middle aged: 2.20 0.23, aged: 1.71 0.28; F (2,75) = 8.08; p < 0.001) with young rats crossing the precise location the hidden platform had been located for training more than middle aged and aged rats ( p values < 0.01). Circulating and Central Inflammatory Biomarker Profiles are Modified with A ge ANOVAs were used to explore the effect of age on the serum concentration of each analyte (Figure 3 5 A). Note that GM CSF was below the minimum level of detection and therefore is not reported. Leptin and RANTES, however, were expressed at significantly higher concentrations than all other analytes ( p <0.001). Age was found
77 to significantly affect the levels of corticosterone ( F (2,69) = 4.68; p < 0.05), e otaxin ( F (2,71) = 5.47; p < 0.01), GRO KC ( F (2,74) = 3.79; p < 0.05), IFN ( F (2,74) = 8.49; p < 0.001), IL 1 ( F (2,69) = 4.55; p < 0.05), IL 1 ( F (2,74) = 8.37; p < 0.001), IL 2 ( F (2,73) = 3.47; p < 0.05), IL 4 ( F (2,74) = 7.84; p < 0.001), IL 5 ( F (2,72) = 6.21; p < 0.01), IL 6 ( F (2,72) = 5.32; p < 0.01), IL 13 ( F (2,72) = 5.18; p < 0.01), IL 17 ( F (2,73) = 5.04; p < 0.01), IL 18 ( F (2,72) = 8.25; p < 0.001), IP 10 ( F (2,72) = 3.76; p < 0.05), leptin ( F (2,75) = 5.07; p < 0.01), MCP 1 ( F (2,72) = 8.43; p < 0.001) MIP 1 ( F (2,72) = 4.49; p < 0.05) and RANTES ( F (2,71) = 3.75; p < 0.05). While age also tended to affect the serum concentrations of ACTH ( F (2,72) = 2.63; p = 0.08), IL 10 ( F (2,70) = 2.54; p = 0.09) and VEGF ( F (2,72) = 2.61; p = 0.08), there was no effec t of age on G CSF ( F (2,73) = 2.31; p > 0.10), IL 9 ( F (2,47) = 0.14; p > 0.10), IL 12 ( F (2,47) = 2.07; p > 0.10), MLT ( F (2,57) = 0.58; p > 0.10) nor TNF ( F (2,74) = 1.30; p > 0.10). Newman Keuls post hoc analyses revealed that corticosterone and leptin wer e upregulated in middle age rats ( p values < 0.01) while the concentrations of eotaxin, GRO KC, IFN IL 1 IL 1 IL 2, IL 4, IL 5, IL 6, IL 13, IL 17, IL 18, IP 10, leptin, MCP 1, MIP 1 and RANTES were all elevated in aged rats when compared to serum levels in young rats ( p values < 0.05). Interestingly, the serum concentration of IL 10 was increased in both middle aged and aged rats ( p values < 0.05). The concentrations of total protein in the hippocampal and cortical tissue samples were used to norma lize analyte concentrations for each rat. Hippocampal protein concentrations (young: 5.95 0.24 mg, middle aged: 5.88 0.22 mg, aged: 5.67 0.20 mg) were similar across age ( F (2,75) = 0.31; p > 0.05). However, cortical protein concentrations (young: 25.63 1.16 mg, middle aged: 19.08 0.57 mg, aged: 18.80 0.96 mg) were influenced by age ( F (2,75) = 13.97; p < 0.001), with significantly diminished
78 total protein in middle age and aged rats compare d to young controls ( p values < 0.001). After normalization, ANOVAs were used to examine the effect of age on analyte expression in the hippocampus and cortex. Age significantly affected the hippocampal levels of IL 5 ( F (2,72) = 7.93; p < 0.001), IL 9 ( F ( 2,55) = 4.88; p < 0.05), IL 12 ( F (2,42) = 3.22; p < 0.05), IL 18 ( F (2,72) = 3.01; p < 0.06), MIP 1 ( F (2,74) = 4.57; p < 0.05), RANTES ( F (2,74) = 7.69; p < 0.001) and TNF ( F (2,71) = 3.36; p < 0.05). Age tended to affect hippocampal levels of CCS ( F (2,40) = 2.76; p = 0.08), G CSF ( F (2,72) = 2.38; p = 0.10), IL 1 ( F (2,73) = 2.47; p = 0.09), and IL 10 ( F (2,73) = 2.43; p = 0.10) but had no affect on ACTH( F (2,73) = 1.12; p > 0.10), GM CSF ( F (2,75) = 1.51; p > 0.10), GRO KC ( F (2,72) = 0.98; p > 0.10), eotaxin ( F (2,74) = 0.45; p > 0.10), IFN ( F (2,74) = 2.35; p > 0.10), IL 1 ( F (2,73) = 0.41; p > 0.10), IL 2 ( F (2,73) = 1.38; p > 0.10), IL 4 ( F (2,74) = 1.56; p > 0.10), IL 6 ( F (2,72) = 2.17; p > 0.10), IL 13 ( F (2,71) = 2.16; p > 0.10), IL 17 ( F (2,74) = 0.93; p > 0.10), IP 10 ( F (2,71) = 1.62; p > 0.10), leptin ( F (2,75) = 0.11; p > 0.10), MCP 1 ( F (2,73) = 0.20; p > 0.10), MLT ( F (2,52) = 1.79; p > 0.10), and VEGF ( F (2,74) = 0.50; p > 0.10). F igure 3 5. Circulating and central cytokine profiles are modified with age. The group average ( S.E.M.) concentration of each analyte is presented for young, middle aged and aged rats quantified in serum, hippocampus and cortex. Significant ( p < 0.05) inc reases (shades of green) or decreases (shades of red) are denoted as fold changes compared to young baseline concentrations (black). (A) Within serum, leptin was upregulated in middle aged rats, and eotaxin, GRO KC, IFN IL 1 IL 1 IL 2, IL 4, IL 5, I L 6, IL 13, IL 17, IL 18, IP 10, MCP 1, MIP 1 and RANTES were upregulated with age. Note that GM CSF was below the minimum level of detection and therefore is not reported. Leptin and RANTES, however, were expressed at significantly higher concentrations than all other analytes ( p <0.001). (B) Hippocampal levels of IL 9, MIP 1 and RANTES were elevated in aged rats while IL 18 was elevated in both middle aged and aged rats. However, TNF in middle aged rats, IL 12 in aged rats, and IL 5 expression in both midd le aged and aged rats were down regulated. (C) Cortical concentrations of IL 5 and leptin were elevated in middle aged rats, and GRO KC, eotaxin, IL 10 and RANTES in aged rats. IL 1 IL 4, IL 6, IL 18, MCP 1, MIP 1 and VEGF were upregulated in both m iddle aged and aged rats.
80 Post hoc analyses confirmed aged rats had elevated levels of IL 9 ( p < 0.05), MIP 1 ( p < 0.01), RANTES ( p < 0.01) as well as IL 18 ( p < 0.05), which also tended to be elevated in middle aged rats ( p = 0.06). However, compared to young controls, the concentration of TNF in middle aged rats ( p = 0.056), IL 12 in aged rats ( p < 0.0 5), and IL 5 expression in both middle aged and aged rats ( p values < 0.01) was significantly reduced (Figure 3 5 B). Cortical concentrations influenced by age included GRO KC ( F (2,73) = 10.23; p < 0.001), eotaxin ( F (2,73) = 6.53; p < 0.01), IL 1 ( F (2,74 ) = 7.42; p < 0.01), IL 4 ( F (2,74) = 6.01; p < 0.01), IL 5 ( F (2,73) = 6.88; p < 0.01), IL 6 ( F (2,71) = 5.71; p < 0.01), IL 9 ( F (2,57) = 4.44; p < 0.05), IL 10 ( F (2,71) = 3.07; p < 0.05), IL 18 ( F (2,74) = 9.70; p < 0.001), leptin ( F (2,73) = 3.65; p < 0.05), MCP 1 ( F (2,74) = 8.08; p < 0.001), MIP 1 ( F (2,73) = 8.61; p < 0.001), RANTES ( F (2,73) = 9.05; p < 0.001) and VEGF ( F (2,72) = 4.34; p < 0.05). However, age did not affect levels of ACTH ( F (2,72) = 1.79; p > 0.10), CCS ( F (2,61) = 2.07; p > 0.10), G CSF ( F ( 2,72) = 2.12; p > 0.10), GM CSF ( F (2,75) = 1.08; p > 0.10), IFN ( F (2,73) = 0.21; p > 0.10), IL 1 ( F (2,72) = 1.29; p > 0.10), IL 2 ( F (2,74) = 1.92; p > 0.10), IL 12 ( F (2,41) = 0.97; p > 0.10), IL 13 ( F (2,72) = 2.17; p > 0.10), IL 17 ( F (2,74) = 1.70; p > 0.10), IP 10 ( F (2,70) = 1.05; p > 0.10), MLT ( F (2,56) = 2.15; p = 0.10) or TNF ( F (2,72) = 1.98; p > 0.10). Newman Keuls post hoc analyses revealed elevated levels of IL 5 ( p < 0.01) and leptin ( p < 0.05) in middle aged rats, while leptin tended to be ele vated in aged rats as well ( p = 0.08). Similarly, IL 1 IL 4, IL 6, IL 9, IL 18, MCP 1, MIP 1 and VEGF were significantly upregulated in both middle aged and aged rats ( p values < 0.05). While eotaxin and IL 10 tended to be increased in middle aged rats, they were significantly elevated in aged rats along with levels of GRO KC and RANTES ( p values < 0.05; Figure 3 5 C).
81 Correlation Analyses Reveal Cytokine Clusters Altered with A ge In order to examine if cytokines that change with age (identified in Figu re 3 5) were altered in a correlated fashion we ran a pathway analysis. Using Spearman rank correlations we plotted cytokine pairs that remained significant after Bonferroni correction ( p values < 0.000071, Table 3 1) in accordance to the method described in section 2.5. to create analyte clusters as shown in Figure 3 6 Within the serum there were 15 cytokine clusters. Many analytes, and analyte pairs, appear repeatedly in multiple clusters. IFN and IL 18 are most frequently represented within 6 clusters, IL 13 and MI P both appear 5 times, IP 10 is in 4 clusters, IL 2, IL 10, IL 1 MCP 1 and eotaxin were repeated in three clusters, and IL 5 was only connected twice, while leptin, IL 17 and IL 4 were found to have only one relationship. One cytokine pair crossed comp artments into the hippocampus (serum IL 2 correlated positively with hippocampal MIP 1 ). While there were only three clusters found solely in the hippocampus (IL 5, IL 12 and TNF ; IL 9 to TNF ; and MIP 1 to IL 18) three other clusters crossed compartm ents into the cortex as well (1: hippocampal IL 12 correlated negatively with cortical MIP 1 2: hippocampal MIP 1 and RANTES correlated negatively with cortical IL 5 and 3: hippocampal RANTES and cortical MIP 1 and RANTES all correlated positively). Wi thin the cortex there were 16 cytokine clusters again with many cytokines and cytokine pairs frequently repeating. IL 10 was plotted most frequently in eight clusters, followed by MCP 1 in 7 clusters, and IL 6, MIP 1 and IL 4 in 6 clusters. RANTES was in 5 different clusters, while eotaxin, IL 9, and GRO KC were each plotted four times and VEGF, leptin, IL 1 and IL 5 were all plotted twice. One additional cluster
82 crossed from the cortex into the serum compartment (cortical IL 18 and MIP 1 correlated pos itively with serum MCP 1). Figure 3 6. Cytokine clusters reveal analytes that synergistically change with age. The Spearman rank correlations between cytokines that were modified with age in Table 3 1 that remained significant after Bonferroni correction ( p values < 0.000071) were plotted in descending order to create analyte clusters. Analytes are colored based on their predominant functions such that pink is pro inflammatory, blue is anti inflammatory, gray is recruitment and trafficking, yellow is a growth factor and purple is a hormone. Green lines denote a positive correlation while red lines denote a negative correlation. Note that many analytes, and even analyte pairs, are repeated fr equently and there are a few significant cross compartment relationships.
83 Biomarker Profiles Differ for Memory impaired Versus Memory unimpaired Rats Despite A ge To assess analyte levels that were modified with memory impairment across age test compared the analyte concentrations in seru m, hippocampal and cortical samples from rats that were behavioral characterized as either memory unimpaired (MU; DI > 0) or memory impaired (MI; DI < 0) on the 24 h water maze probe trial (Figure 3 4 B). Changes in analyte concentration are denoted in Fig ure 3 7. First, all rats, young middle aged and aged were compared. With all ages combined, MI rats had significantly increased circulating levels of GRO KC ( t (75) = 2.54; p < 0.05) IL 4 ( t (74 ) = 2.75; p < 0.01) IL 6 ( t (73 ) = 2.24; p < 0.05) MCP 1 ( t (73 ) = 2.50; p < 0.05) and RANTES ( t (72 ) = 2.05; p < 0.05) and tended to have higher levels of IL 1 ( t (75) = 1.78; p = 0.08) and IL 18 ( t (73 ) = 1.74; p = 0.09) compared to MU rats. We then examined middle aged rats alone to identify potential prognos tic biomarkers in the blood serum. MI middle aged rats tended to have increased levels of IL 4 ( t (39 ) = 2.01; p = 0.05) IL 12 ( t (28 ) = 1.84; p = 0.08) IL 13 ( t (38 ) = 1.70; p = 0.10) and TNF ( t (39) = 1.92; p = 0.06) but decreased levels of IL 5 ( t (3 8 ) = 1.78; p = 0.08) There was no difference in analyte expression in MI compared to MU aged rats, possibly due to already elevated levels with age (See Figure 3 5) creating too little variability to pick up difference with memory impairment. However, whe n middle aged and aged rats were combined, GRO KC ( t (62 ) = 2.17; p < 0.05) and IL 4 ( t (62 ) = 2.02; p < 0.05) were again significantly elevated while RANTES ( t (60 ) = 1.86; p = 0.07) tended to be increased in MI rats. In the hippocampus, we hoped to find mainly diagnostic biomarkers that could also help identify potential mechanism to explain age related cognitive decline. With all ages combined, hippocampal IL 2 ( t (74 ) = 2.14; p < 0.05) was decreased, while levels of GM
84 CSF ( t (76 ) = 2.72; p < 0.01) IL 9 ( t (56 ) = 2.74; p < 0.01) IL 18 ( t (73 ) = 2.03; p < 0.05) and IP 10 ( t (72 ) = 2.68; p < 0.01) were elevated with a tendency for CCS ( t (41 ) = 1.79; p = 0.08) MIP 1 ( t (75 ) = 1.74; p = 0.09) and VEGF ( t (75 ) = 1.90; p = 0.06) to be up regulated as well in rats with memory impairments compared to those without. When middle aged and aged rats were examined separately different cytokine profiles emerged. I n middle age hippocampal IL 2 ( t (38 ) = 2.98; p < 0.01) and IL 10 ( t (37 ) = 3.00; p < 0.01) are downregulated with a declining trend noted in IFN ( t (38 ) = 1.86; p = 0.07) and IL 5 ( t (38 ) = 1.83; p = 0.08), while MCP 1 ( t (37 ) = 2.28; p < 0.05) and VEGF ( t (38 ) = 2.08; p < 0.05) are upregulated in MI rats. In aged MI rats, hippocampal levels of CCS ( t (9 ) = 2.28; p < 0.05), GM CSF ( t (22 ) = 3.48; p < 0.01), IL 9 ( t (15 ) = 2.76; p < 0.01) and RANTES ( t (21 ) = 2.18; p < 0.05) are also elevated with a tendency for IP 10 ( t (21 ) = 1.93; p = 0.07) to be higher but significantly decreased IL 12 ( t (9 ) = 2.37; p < 0.05). When middle aged and aged rats were combined, MI rats ag ain had significantly higher levels of GM CSF ( t (63 ) = 2.21; p < 0.05), IL 9 ( t (44 ) = 2.53; p < 0.05), IP 10 ( t (60 ) = 2.00; p < 0.05) with a noted trend of decreased IL 2 ( t (61 ) = 1.91; p = 0.06) and IL 5 ( t (60 ) = 1.79; p = 0.08) along with increased RA NTES ( t (62 ) = 1.86; p = 0.07) when compared to MU rats. Memory impaired rats had elevated cortical levels of IL 1 ( t (75) = 2.97; p < 0.01), IL 9 ( t (58 ) = 2.14; p < 0.05), IP 10 ( t (71 ) = 2.19; p < 0.05), MCP 1 ( t (75) = 2.14; p < 0.05) and RANTES ( t (7 4) = 2.65; p < 0.01) and tended to have higher levels of IL 18 ( t (75 ) = 1.91; p = 0.06) and VEGF ( t (73 ) = 1.74; p = 0.09) when all ages were combined.
85 Figure 3 7. Memory impaired rats have a distinct cytokine profile. Rats w ere classified as either memory unimpaired (MU) or memory impaired (MI) based on their 24 h probe performance ( see Figure 4). The concentration of each analyte measured in the blood serum, hippocampus and cortex was then examined combines all young, middle aged and aged rats together whil denotes only middle aged and aged rats together. The color black indicates that there was no difference from baseline of MU rats while shade s of green indicate an increase in an analyte with memory impairment and red a decrease in an analyte with memory impairment. Note the darker colors represent a tendency (0.05 < p < 0.10) and brighter colors denote a significant difference ( p < 0.05).
86 Interestingly, in middle aged MI rats there was a predominant loss of G CSF ( t (37 ) = 2.09 ; p < 0.05), IL 10 ( t (37 ) = 2.14; p < 0.05) and MIP 1 ( t (38 ) = 2.16; p < 0.05) while GRO KC ( t (37 ) = 1.75; p = 0.09) and IL 18 ( t (38 ) = 1.84; p = 0.07) also tended to be lower and ACTH ( t (38 ) = 1.96; p = 0.06) higher. However, in aged MI rats IL 1 ( t (21 ) = 2.52; p < 0.05) and IL 18 ( t (22 ) = 3.02; p < 0.01) were significantly increased compared to MU rats, while eotaxin ( t (22 ) = 1.84; p = 0.08), RANTES ( t (20 ) = 1.74; p = 0.10) and VEGF ( t (21 ) = 1.75; p = 0.09) also showed an increased trend. Combining middle age d and aged MI rats revealed a trend of increased expression of IL 1 ( t (62 ) = 1.68; p = 0.10) and RANTES ( t (61) = 1.70; p = 0.10). Potential Serum and Hippocampal Biomarkers Predict Biological Age of Cognitively Impaired Aging Rats In order to examine w hether these potential serum and hippocampal biomarkers could predict a biological age that could divide cognitively unimpaired and impaired aging rats regression analyses of circulating and central analytes and discrimination index scores on the immediate (learning) and delayed 24 h (memory) probes were performed. Here, we defined learning unimpaired as having a DI > 0.25 on the immediate probe and memory unimpaired as DI > 0 on the 24 h probe. Subsequently, learning impaired rats had a DI < 0.25 on immedi ate probe and memory impaired rats had a DI < 0 on the 24 h probe. All results are presented in Figure 8. Serum analytes identified in middle age may serve as potential prognostic biomarkers while those found in aged rats may serve as diagnostic biomarke rs. In chronologically 14 mo middle aged rats, the combin ation of GRO KC, RATNES, and TNF an average biological age of 36.6 6.6 mo for learning impaired compared to 6.8 3.1 mo for learning unimpaired rats ( t (37) = 4.40; p < 0.0001) with 77% accuracy (type I
87 error: 50%, type II error: 4%). Similar ly using serum levels of GRO KC, RANTES, and IL 4 to formulate biological age (MU: 9.3 1.6 mo, MI: 16.6 3.0 mo; t (37) = 2.26; p < 0.05) memory performance could be predicted in middle aged rats with 74% accuracy (type I error: 27%, type II error: 25% ). No serum analytes were capable of generating a biological age predictive of learning ability in aged rats. However, circulating levels of MCP 1, GRO KC and MLT were used to most accurately (85% with 0% type I errors and 31% type II errors) classify memory unimpaired (12.2 1.4 mo) and memory impai red (25.0 1.7 mo; t (24) = 4.99; p < 0.0001) in chronologically 20 months old aged rats. Hippocampal biomarkers may most effectively be used for potential diagnostic function. In middle aged rats (14 mo) MCP 1 was used to reveal a significant biological age difference for learning unimpaired (8.9 3.1 mo) and learning impaired (23.1 5.7 mo) rats ( t (37) = 2.20; p < 0.05; 69% accuracy with 50% type I and 8% type II errors). In regards to memory performance, hippocampal IL 2 and IL 6 generated significan tly different biological ages (MU: 14.2 0.6 and MI: 15.9 0.4 mo; ( t (37) = 2.26; p < 0.05; 67% accuracy, 42% type I and 20% type II errors). Hippocampal levels of RANTES only tended to predict a biological age difference with learning impairment in age d rats ( learning unim paired: 17.4 3.2 mo, learning impaired: 26.7 1.8 mo; t (24) = 1.88; p = 0.07; 62% accuracy, 56% type I and 0% type II errors). However, RANTES was alternatively used to successfully classify (81% accuracy, 18% type I and 25% type I I errors) chronologically a ged 20 mo rats as either memory unimpaired with a biologica l age of 9.8 5.0 mo or memory impaired with a biological age of 25.8 1.2 mo ( t (24) = 4.01; p < 0.001).
88 Figure 3 8 Potential serum and hippocampal biomarkers pre dict biological age. The relationship between circulating and central analytes and discrimination index scores on the immediate (learning) and delayed 24 h (memory) probes were examined using multiple regression analyses. Analytes identified in middle age may serve as potential prognostic biomarkers while those found in aged rats may serve as diagnostic biomarkers. Hippocampal biomarkers may most effectively be used for diagnostic capability as well. The relationship between the biomarkers and biological ag e, unimpaired (DI > 0.25 on immediate probe and DI > 0 on the 24 h probe) and impaired (DI < 0.25 on immediate probe and DI < 0 on the 24 h probe) are shown as the group mean S.E.M. with significance noted as p < 0.05, ** p < 0.01 and *** p < 0.001. Th e predictive capability of these biomarkers is expressed as percent accuracy taking into account the spread between type I and type II errors. Discussion In the current study, we confirmed that performan c e on a rapid acquisition water maze task declines with age and based on this performance middle aged and aged rats can be characterized as either memory unimpaired (MU) or memory impaired (MI). We also found that central and circulating inflammatory cytoki nes, chemokines, growth factors and stress hormones are modulated with age. Interestingly, some analyte concentration s change in a correlated fashion creating cytokine clusters. Furthermore,
89 we found that MI rats have a distinct cytokine profile when compa red to MU rats implicating potential biomarkers of age related cognitive decline and these biomarkers can be used to generate a biological age predictive of cognitive impairments As expected, performance on both the visible and hidden platform tasks was impaired with age, which has been discussed in length previously (Foster, 1999; Gage et al., 1984b; Rapp et al., 1987; Speisman et al., 2013a; Speisman et al., 2013b ) Interestingly, we found that these impairments begin to appear in middle age. This is most clear when comparing the immediate and 24 h probe trial scores, where all rats were able to recall the location of the escape platform immediately after training, however, the following day all young rats retain this information while about half of the middle aged rats and majority of the aged rats exhibit memory impairments (Figure 4). Middle age is also a time when gene expression is and changes in inflammatory biomarkers have been noted (Blalock et al., 2003; Gimeno et al., 2008) Figure 4 also highlights the fact that age related memory impairments are not inescapable as some middle aged and aged rats perform as well as young cohorts on the 24 h probe. Variability in cog nitive ability has been noted previously and allows for the population to be divided into two groups: memory unimpaired and memory impaired (Dobrossy et al., 2003; Drapeau et al., 2003; Drapeau et al., 2007; Gage et al., 1984a; Gage et al., 1984c; Gallagher et al., 1993; Markowska, 1999; Markowska et al., 1989) Here we set a threshold such that rats with a DI < 0 were deemed MI while all young rats, about half of the middle aged and very few aged rats were MU. With the distinction between MU and MI rats we were then able to compare potential prognostic and diagnostic
90 inflammatory biomarkers. Therefore middle age may be a crucial time to identify pending decline and begin preventative neuromodulatory measures. Biomar kers of inflammation (Blalock et al., 2003; De Martinis et al., 2005; Gimeno et al., 2008; Krabbe et al., 2004; Magaki et al., 2007; Rafnsson et al., 2007; Solfrizzi et al., 2006; Villeda et al., 2011) and stress (Issa et al., 1990; Lupien et al., 1998; McEwen, 1998) increase with age in rodents and humans alike. Indeed, using Bio Plex technology we identified circulating and central inflammatory biomarkers that were modified with age. Particularly, in blood serum we noted a robust age induced increase in the basal level of both known and novel analytes. We confirmed that compared to young rats, eotaxin, IL 6, IL 10, IL 18, IP 10, MCP and RANTES were elevated in aged rats. Spec ifically, increased eotaxin was recently found in aged rats with impaired cognition and neurogenesis (Villeda et al., 2011) Similarly, levels of IL 10 in mice (Zhao et al., 2 010) and in humans IP 10 (Palmeri et al., 2011) RANTES (Gerli et al., 2000) IL 18 (Gangemi et al., 2003) IL 6 (Krabbe et al., 2009; Mariani et al., 2006; Palmeri et al., 2011) and MCP 1 (Gerli et al., 2000; Mariani et al., 2006; Seidler et al., 2010) were elevated. However, MCP 1 (Kim et al., 2011) and IL 4 (Palmeri et al., 2011) have conversely been noted to decrease with age. We also detected novel age induced increases in GRO KC, I FN IL 2,IL 5, IL 13, IL 17 and MIP Interestingly, corticosterone, a hormone mainly associated with stress, and leptin, a hormone that influences metabolism and energy, were elevated significantly in middle age and, while not statistica lly significant, remained elevated in aged rats. Mid life long term stress can lead to cognitive impairments as the rat ages (Sandi and Touyarot, 2006) and elevated corticosterone levels in aged animals has been linked t o impaired
91 cognition and decreased neuroplasticity including hippocampal neurogenesis (DeKosky et al., 1984; He et al., 2008; Issa et al., 1990; Landfield et al., 1978; Montaron et al., 2006; Tanapat et al., 1998) We have previously shown that serum levels of leptin also correlated with measures of cognition and neurogenesis in aged rats ( Speisman et al., 2013a ) and due to its response to inflammatory assault (Mastronardi et al., 2005; Sarraf et al., 1997) and ability to influence inflammatory molecules in the brain (Hosoi et al., 2002a, b) leptin has been implicated as a mediator between the inn ate and adaptive immune systems. In this experiment we also measured basal levels of inflammatory markers across age in the hippocampus and cortex. We were specifically interested in the hippocampus, as changes in this region may aid in elucidating the me chanism behind age related cognitive decline, as we have previously linked changes in hippocampal neuroimmune signaling with measures of cognition and neurogenesis ( Speisman et al., 2013a ) In the hippocampus we not ed a decrease in IL 5 in middle age which continued into old age along with the modulation of a few other cytok ines: I L 9, IL 12, IL 18, MIP and RANTES. To the best of our knowledge, this is the first report of higher levels of hippocampal IL 9 or IL 12 reported in aged rats. Previously, increased hipp ocampal concentrations of MIP and RANTES (Felzien et al., 2001) were reported in aged mice and elevated IL 18 correlates with impaired spatial learning ability and long term potentiation ( LTP ) (Griffin et al., 2006; Mawhinney et al., 2011) While the age induced cortical infla mmatory analyte profile was low grade with only ~2 fold increase in most analytes (compared to >10 up to 100 fold change when stimulated with an inflammatory assault like LPS; (Asokan, 2010) ), the amplified levels
92 are of note as they mainly began in middle age and included more than half of the anal ytes measured. This overall inflammatory profile, which includes elevated levels of both pro and anti inflammatory cytokines, may indicate an overall dysregulation of the cytoki nes during an assault, may become dystrophic leading to eventual degeneration with age causing a disruption of feedback communication with the neurons they normally protect and aid in regenerating (Streit, 2006; Stre it et al., 2008; Streit et al., 2004) Our data also demonstrates that basal serum, hippocampal and cortical analyte concentrations are not uniform nor are they modified with age in a similar fashion. Because we did not perfuse the brain before measuring tissue analyte concentration it would seem plausible that central concentrations may be due to circulating levels, especially since many of these analytes are also capable of crossing the blood brain barrier. However, similar to our previous findings ( Speisman et al., 2013a ) we do not believe this to be the case since we did not find that individual analytes concentrations in the blood and brain corresponded nor did we find that these values directly correlated acr oss compartments (see Table 3 1 and Figure 3 6). The differences in hippocampal and cortical values suggest that age induced changes are region specific, which is central for designing prevention and/or treatment targeting a specific molecule. In order to see if cytokines that are modulated with age are altered in a correlated fashion we ran a pathway analysis using Spearman rank correlations. We plotted cytokine pairs that remained significant after Bonferroni correction and identified cytokine clusters t hat synergistically change with age. This cluster analysis allowed us
93 to confirm known relationships like between serum IL 6 and IL 13 (de Waal Malefyt et al., 1993) and reveal novel pathways like the relationship b etween h ippocampal IL 4, IL 12 a nd TNF which we have previously noted in an earlier experiment ( Speisman et al., 2013a ) and have been studied in other cell types (Levings and Schrader, 1999) We also ide ntified neuroimmunomodulatory targets like Eotaxin, which Tony Wys s C lab recently found to be elevated in aged mice with impaired neurogenesis and cognitive ability (Villeda et al., 2011) We then expanded w ork showing age related decline in cognitive ability and increase in inflammatory markers by demonstrating that inflammatory biomarker profiles differ for memory impaired versus memory unimpaired rats despite age. Using the 24 h probe scores to separate ou r rats into MU and MI groups, we re examined the biomarker data. First, looking at serum, all MI (middle aged and aged) rats ha d higher levels of GRO KC, IL IL 4, IL 6, IL 18, MCP 1, and RANTES when compared to all (young, middle aged and aged) MU rats Looking at middle aged rats we hoped to find potential prognostic biomarkers and in aged rats diagnostic biomarkers. However, since analyte levels were consistently increased with age we found too little variability to pick up significant difference betw een levels expressed MU and MI rats. We then analyzed middle aged and aged rats together and again GRO KC and IL 4 were revealed as potential biomarkers of age related cognitive decline. In order to identify potential diagnostic biomarkers and perhaps mec hanism underlying age related cognitive decline we examined differences in the hippocampal inflammatory profiles of MI versus MU rats. Again, MI rats had higher levels of many central analytes including CCS, GM CSF, IL 2, IL 18, IP 10, MIP and VEGF.
94 Int erestingly, in middle age rats alone, a loss of many importa nt inflammatory cytokines (IFN IL 2, IL 5 and IL 10) was noted with memory impairment. Once more, novel and known molecules were identified like CCS, which is known to be elevated with age and linked to memory impairment due to chronic stress (DeKosky et al., 1984; He et al., 2008; Issa et al., 1990; Landfield et al., 1978; Montaron et al., 2006; Tanapat et al., 1998) To further examine the predictive a bility of these identified potential biomarkers we used multiple linear regression analyses. Potential biomarkers that correlated with water maze probe performance were used to create a biological age for each chronologically middle aged or aged rat. Learn ing or memory impaired rats were found to have significantly higher biological ages than their unimpaired cohorts. Circulating GRO KC, MCP 1 and MLT were prognostic of mnemonic ability in aged rats. Interestingly, we have previously found levels of GRO KC and MCP 1 change in a correlated fashion in aged rats and were both negatively correlated with immediate water maze probe DI score, not 24 h DI score as we found in this study ( Speisman et al., 2013a ) While GRO KC and MCP 1 were both found to be elevated in aged rats, melatonin (MLT) was not significantly altered. However, the variability in melatonin expression in aged rats allowed for the negative correlation with 24 h probe score. Melatonin has been previously r eported to inhibit spatial learning and memory possibly through the inhibition of hippocampal LTP in the CA1 region (Feng et al., 2002) inflammatory function, particularly the ability to downregulate chemokine expression ( Cuesta et al., 2010; Kireev et al., 2012; Li et al., 2008) and ( Min et al., 2012 ) a critical regulator of
95 transcription in inflammatory response, suggests that MLT levels may rise in effort to neutralize elevated MCP 1 and GRO KC expression in rats with inflammation induce cognitive impairments. Ag a in serum levels of GRO KC in combination with RANTES and TNF predicted learning ability or with RANTES and IL 4 predicted mnemonic ability in middle aged rats. TNF inflammatory cytokine that has been notoriously associated with systemic acute phase response and cancer (Sethi et al., 2008) but also chronic inflammation like that seen with aging (Krabbe et al., 2009) TNF relationship to cognitiv e impairment with age (Krabbe et al., 2009) may be linked to its ability to alter neural progenitor cell (NPC) differentiation ( Keohane et al., 2009 ) and inhibit long term po tentiation (Cunningham et al., 1996) Interestingly, hippocampal expression of RANTES alone could significantly forecast memory impairment in aged rats. RANTES, which stands for Regulated on Activation, Normal T cell Expressed and Secreted, is an inflammatory chemokine (Aizawa et al., 2009) The CCR1 and CCR5 among other receptors for RANTES can be found on hippocampal neurons and addition of RANTES to hippocampal neur ons in culture alters calcium signaling and promotes survival by blocking apoptosis (Meucci et al., 1998) Here, the relationship between hippocampal RANTES and learning/memory in aging rats appears to be novel. How ever, the hippocampal biomarkers IL 2 and IL 6, which were predictive of memory in middle aged rats, have often been noted in relation to cognitive impairment and aging (Gimeno et al., 2008; Hanisch and Quirion, 1995 ; Krabbe et al., 2009; Sparkman and Johnson, 2008) Taken together, these observations
96 offer insight into possible biomarkers that can be used to predict, diagnose or treat age related cognitive decline. In summary, we found that memory impairments begi n to appear in middle age, a time when inflammatory biomarkers (cytokine, chemokines and growth factors) expression is particularly variable. Furthermore, inflammatory biomarkers that are suggesting that these molecules work in a synergistic fashion. We also demonstrate that rats suffering from memory impairment across age have a distinct inflammatory biomarker profile that revealed possible immunomodulatory mechanisms behin d ag e related cognitive decline. Predictive analysis using these biomarkers to generate a biological age that corresponded to cognitive health expanded upon the knowledge necessary to develop a biomarker assay to predict such decline.
97 CHAPTER 4 DAILY EXE RCISE IMPROVES MEMORY, STIMULATES HIPPOCAMPAL NEUROGENESIS AND MODULATES IMMUNE AND NEUROIMMUNE CYTOKINES IN AGING RATS Introduction Developing novel strategies to protect cognition in our burgeoning elderly population is critical for managing the burden and cost of its care. Hippocampal neurogenesis is a form of plasticity that declines significantly with age in rodents (Bizon et al., 2004; Dupret et al., 2008; Kuhn et al., 1996) dogs (Siwak Tapp et al., 2007) and non human primates (Aizawa et al., 2009; Gould et al., 1999b) primarily because the neural progenitor cell (NPC) precursors of new neurons and glia become increasingly quiescent with age (Cameron and McKay, 1999a) The abundance of neurons added daily to the young mammalian hippocampus (Cameron and McKay, 2 001) suggests that neurogenesis contributes to hippocampal integrity and indeed, measures of neurogenesis and ability in hippocampus dependent tasks generally relate in young mammals ( (Deng et al., 2010) but see (Epp et al., 2011; Gould et al., 1999a) Measures of neurogenesis have been related to measures of performance in hippocampus dependent tasks among aged dogs (Siwak Tapp et al., 2007) aged non human primates (Aizawa et al., 2009) and when an experimental manipulation introduces enough vari ability into both measures to detect the relationship in aged rats (Bizon et al., 2004; Dupret et al., 2008; Kempermann et al., 2002; Speisman et al., 2013b ) Combined, these data suggest that protecting hippocampal neurogenesis from the effects of age may also protect some forms of cognition. Reprinted with permission from Speisman, R.B., Kumar, A., Rani, A., Foster, T.C., Ormerod, B.K., 2013 D aily exercise improves memory stimulates hippocampal neurogenesis and modulates immune and neuroimmune cytokines in aging rats. Brain Behav Immun
98 Experimental manipulations that produce neuroimmune responses can impair hippocampal neurogenesis and cognition. For example, systemic or central bacterial lipopolysaccharide ( LPS) injections activate microglia, potently block neuronal differentiation (Ekdahl et al., 2003; Monje et al., 2003) and disrupt the integration of young neurons into existing hippocampal circuitry ( Belarbi et al., 2012 ) Of the cytokines known to be stimulated by LPS (Erickson and Banks, 2011 ) only a handful have been shown to affect in vivo or in vitro neurogenesis (Ben Hur et al., 2003; Buckwalter et al., 2006; Grotendorst et al., 1989; Liu et al., 2009; Monje et al., 2003; Qin et al., 2008; Turrin e t al., 2001; Vallieres et al., 2002; Villeda et al., 2011 ) In humans, experimental LPS impairs verbal and non verbal memory (Reichenberg et al., 2001) but confirming its effects on neurogenesis awaits technology that permits the visualization of neurogenesis in the living brain. However little, if any, evidence of hippocampal neurogenesis is detected in the post mortem tissue of patients who exhibited profound irradiation therapy, which also st imulates neuroimmune signaling (Coras et al., 2010; Correa et al., 2004; Crossen et al., 1994; Monje et al., 2007) The irradiation on hippocampal neurogenesis in rodents can be bloc ked by non steroidal anti inflammatory treatment (Monje et al., 2003; Rola et al., 2008; Tan et al., 2011 ) confirming a role for downstream immune and/or neuroimmune signaling cascades in mediating the effects of t hese treatments on neurogenesis. In aged rodents, systemic or central LPS administration stimulates exaggerated microglial responses, cytokine levels and memory impairment (Barrientos et al., 2006; Chen et al., 2008; Godbout et al., 2005; Xu et al., 2010) In fact, the transcription of
99 neuroimmune molecules is upregulated categorically with age but most robustly in aged rodents that exhibit impaired performances across hippocampus dependent tasks (Blalock et al., 2003; Kohman et al., 2011b ) Whole brain preparations have revealed that the concentrations of some cytokines that increase with age in rodents also associate negatively with measures of long term potentiation and spat ial ability (Felzien et al., 2001; Griffin et al., 2006; Prechel et al., 1996; Ye and Johnson, 1999) In aged and aging humans, increased circulating immune cytokine concentrations have been linked to cognitive impa irments (Gimeno et al., 2008; Krabbe et al., 2009; Krabbe et al., 2004; Magaki et al., 2007; Rachal Pugh et al., 2001; Rafnsson et al., 2007; Weaver et al., 2002) In a recent study, Villeda and colleagues elegantly narrowed a list of 17 potential circulating cytokines (of 66 examined) down to 6 that related to age impaired in neurogenesis and cognition. They then showed that increased circulating eotaxin concentrations alone compromise neurogenesis, synaptic plastic ity and memory across hippocampus dependent tasks ( Villeda et al., 2011 ) These data highlight that the systematic testing of circulating and central cytokine biomarker correlates of neurogenesis and cognition can r eveal mechanistic candidates. Importantly, these candidates can include hypoactive or senescent immune and neuroimmune cytokine signaling, particularly in aged rats (Conde and Streit, 2006; Ziv et al., 2006) Elderl y humans who exercise regularly exhibit better scores on cognitive tests and have larger hippocampal volumes relative to sedentary elderly humans (Christensen and Mackinnon, 1993; Churchill et al., 2002; Colcombe and Kramer, 2003; Erickson et al., 2010) Young and aged rodents that exercise daily on a running wheel exhibit enhanced measures of plasticity that include neurogenesis and long term
100 potentiation and better performances on hippocampus dependent tasks (Brown et al., 2003; Creer et al., 2010; Kronenberg et al., 2003; Kumar et al., 2011; Lambert et al., 2005; Lugert et al., 2010; Madronal et al., 2010; Steiner et al., 2008; Suh et al., 2007; van Praag et al., 1999a; van P raag et al., 2002; van Praag et al., 2005) In young rats that run voluntarily, increased levels of neurogenesis are associated with reduced hippocampal IL (Chennaoui et al., 2008; Farmer et al., 2004; Lea sure and Decker, 2009; Stranahan et al., 2006a) suggesting that physical activity may stimulate plasticity and improve cognition by modulating neuroimmune signaling pathways. There is even evidence in aged mice that cognition and immune system signaling can be modulated by physical exercise ( Kohman et al., 2011a; Kohman et al., 2011b ) Therefore, we tested the effects of conditioned wheel running on the rapid acquisition and retention of a water maze hidden platfor m location, inhibitory avoidance acquisition and retention, hippocampal neurogenesis and 24 immune and neuroimmune cytokine concentrations in aging F344 rats. We expected that conditioned runners would exhibit better learning and memory indices and have hi gher rates of neurogenesis than control rats. We also expected that conditioned runners might have altered levels of immune and/or neuroimmune cytokines that may relate to measures of hippocampal integrity and/or hippocampal neurogenesis. Methods Subjects All rat subjects were treated in accordance with University of Florida and federal policies regarding the humane care and use of laboratory animals. Upon arrival, sexually nave male Fischer 344 rats (18 mo; n = 12) purchased from the National Institute of Aging colony at Harlan Sprague Dawley Laboratories (Indianapolis, IA) were
101 housed individually in corn cob bedding lined hanging shoebox cages loc ated in a colony room maintained on a 12:12 h light:dark cycle at 24 1C. Fig ure 4 1. Experiment timeline. Male F344 rats (18 mo) were assigned randomly to either a conditioned running group that voluntarily ran for food for the entire 18 weeks long experiment or a sedentary control group fed ad libitum All rats underwent water m aze training and testing during the 13 th week followed by inhibitory avoidance training and testing during the 14 th week. During the 16 th week, the rats were BrdU injected (50 mg/kg/day; i.p.) daily for 5 days and then killed at the end of the 18 th week to quantify 24 immune and neuroimmune cytokine simultaneously with hippocampal neurogenesis. The rats were given access to Harlan Teklad Rodent Diet #8604 and water ad libitum. All rats were weighed weekly and checked daily to ensure that they did not exhibit age related h ealth problems including (but not limited to) poor grooming, reduced food and water intake, excessive porphyrin secretion or weight loss. One week after arrival, the rats were assigned randomly to the conditioned runner or control group (n = 6 per group). Control rats were maintained individually in standard laboratory cages with access to food and water ad libitum for the 18 weeks long duration of the experiment while runners were conditioned to run for food to prevent the well documented decreases in runn ing behavior exhibited by aged rats across weeks of an experiment (Cui et al., 2009; Holloszy et al., 1985; Kumar et al., 2011 ) Therefore, runners were housed individually in a chamber containing a running
102 wheel (m odel H10 38R, Coulbourn Instruments, Allentown, PA) on which they could run for unlimited food ( Kumar et al., 2011 ) A Graphic State Notation comput er program (Version 3.02, Coulbourn Instruments, Allentown, PA) recorded wheel rotations and was programmed to deliver 45 mg food pellets (Harlan Teklad Rodent Diet #8604) based upon wheel rotations. The frequency of 45 mg food pellet delivery was decrease d from 1 pellet per rotation at the beginning of conditioning to 1 pellet per 3 4 m by 4 weeks. By the 8 th week of conditioning, all runners consistently ran 4 km per week. If a conditioned runner lost more than 10% of the weight expected based on their pre conditioning baseline and the weight changes of the control rats, the number of wh eel rotations required for food delivery was reduced. Note that the body masses of conditioned runners (418.52 5.12 g) were similar to controls (414.26 5.26 g) at the beginning of the experiment ( t (10) p = 0.66) and tended to be smaller (357.9 7 12.79 and 417.50 33.41 g, respectively) at the end of the experiment ( t (10) = 1.97; p = 0.08). The experiment timeline is depicted in Figure 4 1. Water Maze Training and Testing Each rat was trained and tested in a black water maze tank (1.7 m diamet er) housed in a well lit room. The tank was filled with water (27 2C) to a depth of 8 cm below the tank rim. A Columbus Instruments tracking system (Columbus, OH) was used to record latencies (s), pathlengths (cm), % time spent in the outer annulus of t he maze and platform crossings. Rats were initially habituated to the pool on three trials during which they were released from different pool locations and allowed to climb onto a visible platform. Rats were dried with towels and warm air between blocks a nd before being returned to their home cages.
103 Visible platform t raining Beginning the 13th week of the experiment, the rats were trained in 5 blocks of 3 60 s visible platform trials (15 min inter block interval [IBI] and 20 s inter trial interval [ITI]) that require intact procedural and sensorimotor ability (Vorhees and Williams, 2006) The flagged platform (29 cm diameter) protruded 1.5 cm from the water surface and the pool was surrounded by a black curtain to mask distal cues. The platform location and N, S, E and W release points were randomized across trials. Ra ts failing to locate and climb onto the platform within the allotted 60 s were guided to the platform by the experimenter. One control rat was removed from the experiment after failing to locate the visible platform on 2 trials over the last 2 blocks. Lat encies (s) and pathlengths (cm) served as measures of procedural and sensorimotor ability, % time spent in the outer annulus served as a measure of anxiety and swim speed (cm/s) served as a measure of locomotor ability. Hidden platform t raining. Three days after visible platform training, the rats were trained on five blocks of 3 60 s hidden platform trials (15 min IBI and 20 s ITI) that require intact spatial ability (Vorhees and Williams, 2006) This rapid water maze training protocol is sensitive to age related cognitive decline and the effects of differential experi ence on spatial ability in aged rats (Carter et al., 2009; Foster and Kumar, 2007; Foster et al., 2003; Kumar et al., 2011; Speisman et al., 2013b ) The platform was hidden 1.5 cm below the water surface in the cent er of the NE quadrant of the water maze now surrounded by highly visible distal cues. N, S, E and W release points were randomized across each trial. Rats that failed to locate and climb onto the platform within the allotted 60 s were guided to the platfor m by the experimenter before being removed from the maze. Latencies (s) and pathlengths (cm) served as measures
104 of spatial ability, % time spent in the outer annulus served as a measure of anxiety and swim speed (cm/s) served as a measure of locomotor abil ity. Imm ediate and delayed probe t rials. The escape platform was removed from the water maze in probe trials administered immediately or 24 h after the last hidden platform training trial to test strength of learning and memory, respectively, for the platf orm location. In both probe trials rats were released from the quadrant opposite to the goal quadrant for a 60 s free swim. A hidden platform trial block was administered after the first probe trial to reinforce the association between the platform localiz ation and escape from the pool. The time (s) spent in each quadrant, platform location time spent in the opposite quadrant and t(G) is time spent in the goal quadran t] served as measures of strength of learning and memory in probe trials. DI scores take into memory index for aged rats that frequently make wide sweeping turns while navigating by swimming. Inhibitory Avoidance Training and Testing Beginning the 14 th week, the rats were trained and tested in an inhibitory avoidance apparatus (Coulbourn Instrumen ts, Allentown, PA) consisting of dark and lighted chambers with a shockable metal grid floor separated by a sliding door. During acquisition, the rat was placed in the lighted compartment for 90 s before the sliding door opened and latency to enter the dar k compartment was recorded. Upon entry to the dark compartment, the door closed and a mild foot shock (0.21 mA for 3 s) was
105 confirmed that they had experienced the shoc k. The rat was then returned its home cage before being returned to the lighted chamber for 90 s both 1 and 24 h later, and the time taken to enter the dark side after the door opened was recorded as a measure of memory. Retention latencies were set at 900 s for rats not entering the dark compartment within 15 min. Door opening, shock delivery and data acquisition was computer controlled. Bromodeoxyuridine Injections We waited 15 weeks after the experiment onset and 3 weeks after spatial learning before labeling dividing NPCs with the DNA synthesis marker bromodeoxyuridine (BrdU; Sigma Aldrich, St. Louis, MO) to measure the effects of long term daily exercise on neurogenesis to minimize the well known effects of spat ial behavior on neurogenesis (Epp et al., 2010; Gould et al., 1999a) NPC proliferation is unaffected when BrdU is administered at the end of hippocampus dependent learning (Gould et al., 1999a) and any latent effects of hippocampus dependent behavior on new neurons produced 3 weeks earlier are possible but unexpected. Rats were injected intraperitoneally once per day over 5 days beginning 1 6 weeks after the experiment onset to label dividing cells. BrdU was dissolved in freshly prepared 0.9% isotonic sterile saline at a concentration of 20 mg/ml (w/v) just prior to use at a volume of 2.5 mL/kg (50 mg/kg/injection). This dose of BrdU labels d ividing hippocampal NPCs safely and effectively in adult rodents (Cameron and McKay, 2001; Kolb et al., 1999) Histology At the end of the 18th week (21 d after the first BrdU injection), the rats were anaesthetized deeply with a ketamine (90 mg/kg)/xylazine (10 mg/kg) cocktail (Webster Veterinary Supply, Sterling, MA). Blood was collected from the left ventricle of the heart
106 before rats were decapitated and their brains extracted rapidly. Hippocampi and frontal cort ices were rapidly dissected from the left hemisphere, flash frozen and then stored at 86C until protein harvest for cytokine quantification. Although central cytokine levels in these unperfused rats could reflect circulating levels of diffusible cytokine s we neither detected immune to brain cytokine clusters nor concentrations of individual cytokines that were affected by running similarly in the blood and brain that would validate this hypothesis. Similar masses of hippocampal ( t (10) = 1.00; p = 0.34) an d cortical ( t (10) = p = 1.00) tissue were collected from controls (79.70 11.30 and 246.40 21.95 mg, respectively) and conditioned runners (65.90 8.05 and 246.60 15.69 mg, respectively). Serum supernatant was collected from blood samples afte r refrigeration cytokine quantification. The right hemisphere of the brain was post fixed overnight in freshly prepared 4% paraformaldehyde (Electron Microscopy Scienc es; Hatfield, PA) and then equilibrated in 30% sucrose ( 4 days) at 4C, before being sectioned coronally through the dentate gyrus, beginning between zing stage sledge microtome (Model 860; American Optical Corporation; IMEB Inc., San Marcos, CA). The six sets of every sixth section collected through the left side of the dentate col, 25% glycerin and 45% 0.1 M sodium phosphate buffer until processed immunohistochemically to quantify neurogenesis.
107 Protein Harvest from Brain Tissue Hippocampi and frontal cortices were thawed at 4 C in 0.1 M Tris buffered saline (TBS) containing 0.1 added just prior to use. The first protease inhibitor cocktail contained 0.5 M phenylmethylsulfonyl fluoride, 5 mg pepstatin A and 1 mg chymostatin/ml DMSO and the second contained 1 M G aminocapf roic acid, 1 M P aminobenzidine, 1 mg leupeptin and 1 mg aprotinin/mL sterile water. Tissue was mashed manually and then sonicated using a dismembrator (ThermoFisher Scientific; Pittsburgh, PA). Tissue supernatant was collected by centrifugation (12,000 rp m for 10 min at 4C) and its protein concentration quantified using a Bradford protein assay and a Bio Rad SmartSpec Plus Spectrophotometer (Hercules, CA). Similar total protein concentrations were harvested from the hippocampi ( t (10) p = 0.23) and cortices ( t (10) p = 0.94) of controls (0.93 0.19 and 1.05 0.06 mg/mL, respectively) and conditioned runners (1.20 0.09 and 1.06 0.09 mg/mL, respectively). Protein samples were stored at tions were quantified using Bio Plex technology. Immunohistochemistry Hippocampal sections were stained immunohistochemically to quantify and phenotype new (BrdU + ) cells using methods previously described (Ormerod et al., 2003, 2004; Palmer et al., 2000; Speisman et al., 2013b ) Enzyme substrate i mmunostaining. Before processing and between steps, free floating hippocampal sections were washed repeatedly in Tris buffered saline (TBS; pH 7.4). The sections were incuba ted in 0.3% H 2 O 2 in TBS for 10 min at RT to quench endogenous peroxidase, rinsed in 0.9% NaCl and then incubated in 2 N HCl for 20 min at 37 C to denature DNA. The sections were then blocked in a solution of 3%
108 normal donkey serum (NDS) and 0.1% Triton X in TBS (v/v) for 20 min and then incubated overnight in rat anti BrdU (1:500; AbD Serotec, Raleigh, NC) at 4C. The next day, the sections were incubated in biotinylated donkey anti rat IgG (Jackson ImmunoResearch, West Grove, PA; 1:500) for 4 h and then a vidin biotin horseradish peroxidase (PK 6100: Vector Laboratories, Burlingame, CA) for 2 h at RT. The diaminobenzidine tetrahydrochloride (DAB; Sigma Aldrich, St. Louis, MO) and 0 .5% H 2 O 2 in TBS. Sections were mounted on glass slides, dried overnight and dehydrated in an alcohol series prior to being cover slipped under permount (Thermo Fisher Scientific, Pittsburgh, PA). Fluorescent i mmunostaining. Sections were washed repeatedly between steps in TBS (pH 7.4). The sections were blocked in NDS solution and then incubated overnight at 4C in primary antibodies raised against the mature neuronal protein neuronal nuclei (mouse anti NeuN, 1:500; Chemicon, Temecula, CA) and the immature neuronal protein doublecortin (goat anti DCX, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) or the oligodendrocyte precursor marker chondroitin sulfate proteoglycan (rabbit anti NG2, 1:500; Chemicon, Temecula, CA) and the astrocyte/neural stem cell prot ein glial fibrillary acidic protein (chicken anti GFAP, EnCor Biotech, Alachua, FL). The following day sections were incubated with maximally cross adsorbed fluorescein isothiocyanate (FITC) conjugated anti mouse and cyanine (Cy) 5 conjugated anti goat sec ondary antibodies to reveal neurons or FITC conjugated anti rabbit and Cy5 conjugated anti chicken secondary antibodies to reveal glia for 4 h at RT (all secondary antibodies were diluted at 1:500; Jackson ImmunoResearch, West Grove, PA). Sections
109 were the n fixed with 4% paraformaldehyde, rinsed in 0.9% NaCl, incubated in 2 N HCl and then incubated in rat anti BrdU (1:500; AbD Serotec, Raleigh, NC) overnight at 4C followed by Cy3 conjugated anti rat secondary for 4 h at RT. Finally, nuclei were labeled by diamidino 2 phenylindole (DAPI; 1:10,000; Calbiochem, San Diego, CA) for 10 min. Sections were mounted on glass slides under the anti fading agent PVA DABCO (2.5% diazabicyclooctane, 10% polyvinyl alcohol and 20% glycerol in TBS; Sigma A ldrich). Cell Quantification Total new cell n umber. The total number of new (BrdU + ) cells was estimated on one 1 in through the rostral caudal extent of the dentate gyrus in the left h emisphere of each rat using stereological principles ( Boyce et al., 2010; Cameron and McKay, 1999a; Kempermann et al., 2002; Ormerod et al., 2003; West et al., 1991) We randomly selected which of the 6 collected se ts of sections to process immunohistochemically to st 6 th section taken from dentate gyrus. New cells produced in the hippocampal subgranular zone (SGZ) presumably migrate deeper into the gr anule cell layer (GCL) over the 16 21 d survival period employed. We therefore counted round or oval BrdU + cells (revealed by DAB staining) in both the SGZ and GCL on each section taken through the rostral caudal extent of the dentate gyrus in the left hem isphere of each aged rat ( 12 sections per rat) using a Zeiss Axio Observer Z1 inverted microscope under a 40X objective. Because new cells are often situated irregularly through the SGZ and GCL, we counted BrdU + cells exhaustively on each systematically uniform series of sections per rat. The mean (SEM) number of 131 12 and 191 12 BrdU + cells in the dentate gyri of control and
110 conditioned runner groups, respectively, is considered a sufficient number of events to insure precision among stereological estimates of total events ( Boyce et al., 2010 ) The total number of BrdU + cells counted in the dentate gyrus of each rat was multiplied by 6 (the section interval in each set) and by 2 (to account for the other half of the brain) to produce a stereological estimate of total number of new cells surviving in the dentate gyrus (Kempermann et al., 2002; West et al., 1991) Because age and exercise may influence vascular volumes (Fabel et al., 2003; Hattiangady and Shetty, 2008) SGZ and GCL areas (mm 2 ) on which new cells were counted were measured under a 20 objective using AxioVision software (Carl Zeiss, Thornwood, NY) and then s principle for calculating the volume of a truncated cone (Galea et al., 2000; Uylings et al., 1986) : I ( h 1 h 1 h 2 + h 2 ), where I h 1 and h 2 are the two section areas between which the volume was calculated. We also confirmed that new cell densities reflected total new cell estimates because of potential changes in vascular volumes and because we quantified neurogenesis on only of the hipp ocampus. New cell p henotypes. At least 100 BrdU + cells on quadruple fluorescent stained sections were scanned through their x, y and z planes using a Zeiss LSM 710 fully spectral laser scanning confocal microscope equipped with 405 (used to excite DAPI), 4 88 (used to excite FITC), 510, 543 (used to excite Cy3) and 633 (used to excite Cy5) laser lines under a 40 objective (with 2.3 digital zoom) to quantify the proportion that expressed neuronal or glial proteins. BrdU + cells were considered to express neu ronal + nucleus
111 clearly expressed the neuronal proteins DCX and/or NeuN, the oligodendrocyte precursor protein NG2 or the astrocyte protein GFAP. The total number of BrdU + cells was multiplied by the % of BrdU + expressing each cell phenotype to determine the total number of new neurons and glia produced in the aging brain and this number was related to water maze probe trial performance. Multiplex Quantification of Cytokines Concentr ations of immune cytokines in blood serum and neuroimmune cytokine concentrations in hippocampal and cortical protein samples were quantified using a Bio Rad Bio Plex 2000 Suspension Array system and EMD Millipore Rat Cytokine/Chemokine kits (#RCYTO 80K PM X; Billerica, MA) according to kit instructions. This kit detects the following concentrations of 24 analytes simultaneously in a single sample: IL 20,000 pg/mL), IL 20,000 pg/mL), IL 2 (3.67 20,000 pg/mL), IL 4 (2.30 20,000 pg/mL), IL 5 (2.89 20,000 pg/mL), IL 6 (9.80 20,000 pg/mL), IL 9 (12.85 20,000 pg/mL), IL 10 (5.41 20,000 pg/mL), IL 12 (4.13 20,000 pg/mL), IL 13 (23.2 20,000 pg/mL), IL 17 (1.61 20,000 pg/mL), IL 18 (4.78 20,000 pg/mL), eotaxin (3.27 20,000 pg/mL), G CSF (1.31 20,000 pg/mL), GM CSF (13.11 20,000 pg/mL), IP 10 (3.78 20,000 pg/mL), leptin (21.50 100,000 pg/mL), GRO/KC (2.06 20,000 pg/mL), IFN 20,000 pg/mL), MCP 1 (3.81 20,000 pg/mL), TNF (4.44 20,000 pg/mL), MIP 20,000 pg/mL), RANTES (54.42 20,000 pg/ mL), VEGF (4.93 20,000 pg/mL). All standards, controls and samples were prepared on ice and serum and tissue samples were run in separate plates. Seven standards (with expected concentrations of 20,000, 5,000, 1,250, 312.5, 78.13, 19.53 and 4.88 pg/mL of e ach analyte except leptin that had expected concentrations of 100,000, 25,000, 12,500, 6,250, 1,562.5, 390.63
112 and 24.41 pg/mL) were prepared by serial dilution with kit assay buffer. Serum samples were diluted 1:5 with kit assay buffer while tissue superna tant samples were kept neat supplied known control and sample were loaded in duplicate into a 96 well filter plate (EMD Millipore; Billerica, MA). Kit l and sample in the serum polystyrene beads each of 24 different color addresses were ad ded to each well and incubated for 18 h on a shaker at 4 C. Each primary antibody raised against an analyte to be quantified was adsorbed to 1 of the 24 unique sets of color addressed beads. After several washes in kit wash buffer under vacuum filtration, the beads were incubated in biotinylated secondary antibodies for 2 h at RT and then after several washes in kit wash buffer under vacuum filtration, in streptavidin phycoerythrin reporter for 30 min at RT before being resuspended in sheath fluid (Bio Rad ; Hercules, CA). Analytes were identified by color address and analyte concentrations were quantified by phycoerythrin emission intensity using a dual laser Bio Rad BioPlex 2000 system with Luminex xMAP technology (Bio Rad; Hercules, CA). Data were collect ed using BioPlex Manager Software version 4.1. A standard curve for each analyte was generated using a five parameter logistic non linear regression model on averaged duplicate observed standard concentrations. Single standard concentrations were employed in cases that its duplicate % coefficient of variation (CV) was >10% and its % recovery (observed/expected concentration) fell outside of the accepted 70 130% range. Once the positive control concentrations were
113 confirmed to fall within the expected ranges sample concentrations were compared against the standard curve. Prior to statistical analysis, duplicate sample concentrations with % CV < 10 were averaged. If the % CV for a set of duplicates was >10% and a concentration fell 2 standard deviations fro m the group mean the outlying concentration was discarded. We discarded the outlying data point of one conditioned runner rat serum leptin analysis and an outlying data point from a different conditioned runner from the serum MCP 1 analysis. Cytokine conce ntrations below the threshold of detection were set to 0 and concentrations that exceeded the maximum expected concentration were set to 20,000 pg/ml (or 100,000 pg/mL for leptin). Data were expressed in pg/mL serum or pg/mg of hippocampal or cortical tiss ue. Cytokine Cluster Analysis (Baron and Kenny, 1986; Erickson and Banks, 2011) to identify groups of cytokines with concentrations that may chang e in a coordinated fashion (i.e. in clusters) and therefore represent known or novel signaling pathways. First, cluster analyses were conducted on cytokine concentrations detected within blood, hippocampal and cortical compartments independently to both co nfirm and expand upon immune and neuroimmune cytokine signaling clusters in the aged rat. Second, cluster analyses were conducted on cytokine concentrations between blood and cortical compartments and between blood and hippocampal compartments to confirm a nd potentially reveal immune to brain signaling pathways in the aged rat. Third, we ran analyses on cytokine concentrations between cortical and hippocampal compartments to ask whether running modulates neuroimmune cytokines locally or regionally. Bonferro ni corrected alpha levels were set
114 for each analysis based upon the number of analytes exceeding the threshold of detection. Pairs of cytokines with concentrations deemed statistically related by Spearman rank correlation coefficients ( r values) after Bonf erroni corrections were ranked and plotted in descending order connected with a solid line. If one cytokine in a pair to be plotted was already plotted in a cluster, then a decision point was reached and we employed a modification to the previously reporte d procedure (Baron and Kenny, 1986; Erickson and Banks, 2011) The unplotted cytokine was added to the cluster if it correlated significantly with all of the cytokines already in the cluster. If the unplotted cytoki ne was not statistically related to one or more of the already clustered cytokines, then the cytokine pair about to be plotted was plotted as a new cluster. If a cytokine pair about to be plotted was already linked through potential mediators in the alread y plotted cluster, then the residual of its r value minus the product of the r values of the plotted pairs between the cytokines about to be plotted was compared against the Bonferroni corrected p value. If the residual r value of the cytokine pair about t o be plotted remained statistically significant, the cytokines were connected in the existing cluster with a dotted line (no mediators were detected in the current study). Statistical Analyses All statistical analyses were conducted using STATISTICA softwa re (Version 10; StatSoft; Tulsa, OK) and all data are represented in figures as the group average t tests were used to test the effect of the independent variable (conditioned r unning) on dependent measures of general health (body mass, swim speeds), strength of spatial learning and memory (probe trial discrimination index scores, number of platform
115 crossings), neurogenesis (new cell number, total new neuron number, total new gli a number) and cytokine concentration (for each of the 24 analytes). Non parametric Mann Whitney U tests were used to test the effects of the independent variable (conditioned running) on categorical percentages of BrdU + cells expressing neuronal or glial p henotypes and on inhibitory avoidance acquisition and retention latencies that were set to 900 s for animals that did not enter the shock paired side of the chamber by the end of the session. Repeated measures analyses of variance (ANOVAs) tested the effec t of the independent variable (conditioned running) on dependent measures collected repeatedly, such as spatial and non spatial acquisition of a platform location (latencies and path lengths). Newman Keuls post hoc tests were used to reveal significant dif ferences. Spearman rank correlations were run to test the relationship between the concentration of cytokine analytes modulated by running, behavioral measures and measures of neurogenesis because some analyte concentrations fell below the threshold of det level was set at 0.05. Results Aging Rats that Run Daily Locate a Visible Platform as Well as Controls but Swim Faster Since path lengths correlated positively with latencies between visible (all r values 0.69; all p values < 0.05) and hidd en (all r values 0.85; all p values < 0.01) platform trials, we report analyses on path lengths to avoid redundancy. Figure 4 2 A shows pathlengths across visible platform training blocks. An ANOVA exploring the effects of conditioned running and trainin g block on visible platform path lengths revealed that conditioned runners and controls swam similar distances to the visible platform ( F (1,9) = 1.38; p = 0.27) across training blocks ( F (4,36) = 1.56; p = 0.20 and
116 interaction effect: F (4,36) = 0.26; p = 0. 90). Because visible platform learning curves can be relatively shallow in aged rats ( Kumar et al., 2011; Speisman et al., 2013b ) planned comparisons were used to confirm that control rats and conditioned runner ra ts swam shorter path lengths on the 5 th relative to the 1 st block ( p values < 0.05, respectively). These data suggest that both conditioned runners and controls are similarly capable of learning to locate and escape to a visible water maze platform. The % of time spent swimming in the outer annulus of the pool by controls and conditioned runners was calculated as a measure of anxiety (Figure 4 2 B). An ANOVA revealed a significant effect of training block ( F (4,36) = 5.32; p < 0.01) but not conditioned running ( F (1,9) = 0.67; p = 0.44 and interaction effect: F (4,36) = 1.59; p = 0.20) on this measure. Specifically, all rats spent significantly less time in the outer annulus as training commenced (Blocks 1, 2 > 3, 5 and Block 2 > 4; all p values < 0.05), suggesting that anxiety levels dec reased in aged rats with training, regardless of exercise history. Swim speeds exhibited across blocks by controls and conditioned runners were recorded as a measure of locomotor ability (Figure 4 2 C). Although an ANOVA revealed a statistically significan t effect of training block ( F (4,36) = 5.55; p < 0.01) and a tendency for conditioned running to affect swim speed ( F (1,9) = 3.84; p = 0.08), these effects did not statistically significantly interact ( F (4,36) = 0.37; p = 0.83). Although conditioned runners tended to swim faster than controls on all blocks combined, all aged rats swam more quickly as training progressed (Blocks 1 > 3, 4 and 5 and Block 2 > 5; all p values < 0.05). These data suggest that daily exercise may potentiate the increased swimming p roficiency or reduce the floating tendencies exhibited by aging rats across visible platform training blocks. Consistent with our previous finding
117 ( Speisman et al., 2013b ) and the idea that running induced fitness r ather than mild food deprivation associated with the operant delivery of food for running affects swim speeds, the body masses of conditioned runners was similar to those of the controls at the beginning of the experiment and only tended to be smaller at t he end of the experiment (see Section 2.1). Figure 4 2. Runners and controls perform similarly on the visible platform task. Data are shown as group means ( S.E.M.). Gray circles represent control values and black squares represent conditioned runner values. (A) Conditioned runners and con trols located and escaped to a visible water maze platform with equal proficiency (interaction effect: p = 0.90). Planned comparisons confirmed decreased pathlengths on the 5th block relative to the 1 st block for control rats ( p < 0.05) and conditioned runner rats ( p < 0.05), confirming similar sensorimotor and procedural abilities between groups. (B) Regardless of exercise history (interaction effect: p = 0.20), rats decreased the amount of time in a block spent swimming arou nd the outer annulus of the water maze as training commenced (Blocks 1, 2 > 3 and 5 and Block 2 > 4; all p values < 0.05). (C) Although conditioned runners tended to swim more quickly on all visible platform training blocks combined ( p = 0.08), all rats (i nteraction effect: p = 0.83) significantly decreased their swim speeds across trials ( p < 0.01). Specifically, the aged rats swam more quickly on Block 1 versus 3, 4 and 5 and on Block 2 versus 5 (all p values < 0.05).
118 Daily Exercise Improves Spatial Ability in Aging Rats We compared pathlengths to the hidden platform across training blocks as a measure of spatial ability (Figure 4 3 A). An ANOVA revealed that pathlengths were significantly affected by conditioned running ( F (1,9) = 20.89; p < 0.01), training block ( F (4,36) = 6.55; p < 0.01) and the interaction between conditioned running and training block ( F (4,36) = 4.05; p < 0.01). All ra ts swam more directly to the hidden platform as training commenced (Blocks 1, 2 > 3, 4 and 5, p values < 0.05), but conditioned runners swam more directly across all blocks combined than controls ( p < 0.01). Conditioned runners exhibited shorter pathlength s than controls on the 1 st 4 th and 5 th training blocks ( p values < 0.05), indicating that they solved the spatial task more proficiently than the controls. However, their better performances on the 1st hidden platform training block could also indicate th at runners better learned, remembered and/or applied procedural information obtained during visible platform training conducted first (Gerlai, 2001; Ormerod and Beninger, 2002) Therefore, we confirmed that runners ( 130.22 37.53 cm/block) exhibited steeper average pathlength slopes than controls ( 5.36 35.65 cm/block) across hidden training blocks 2 5 (gray dotted lines in Figure 4 3 A; t (10) = 2.29; p < 0.05). We calculated the % of time spent in the outer annu lus of the maze on hidden platform trials by conditioned runners and controls to determine if anxiety was differentially affected by previous training (Figure 4 3 B). An ANOVA revealed significant effects of conditioned running ( F (1,9) = 6.45; p < 0.05) an d training block ( F (4,36) = 3.98; p < 0.01) but no interaction effect ( F (4,36) = 1.62; p = 0.19).
119 Figure 4 3. Conditioned runners outperformed controls on the water maze hidden platform task. Data are shown as group means ( S.E.M.). Gray circles repr esent control values and black squares represent conditioned runner values. (A) All rats combined swam more directly to the hidden water maze platform as training commenced (Block 1 > 3 and 4, all p values < 0.05), but conditioned runners swam more directl y than controls, regardless of training block ( p < 0.01), particularly on the 1 st 4 th and 5 th training blocks ( all p values < 0.05). Because runners outperformed controls on the 1st training block, we confirmed average pathlength slopes were steeper for runners 37.53 cm/block) versus controls ( 5.36 35.65 cm/block) across hidden training blocks 2 5 (g ray dotted lines; p < 0.05). A paired t test confirmed that the rats exhibited similar pathlengths on the 5th and a 6 th training block administered after the 1 h probe trial to reinforce the association between locating the platform and escape from the poo l ( p = 0.55). (B) Although conditioned runners spent significantly less time in the outer annulus than controls on all blocks combined ( p < 0.05), all rats combined reduced the % of time spent in the outer annulus of the maze (Blocks 1, 2 > 5; all p values < 0.05). (C) Conditioned runners swam significantly faster to the hidden platform than controls on all blocks combined ( p < 0.01) and while they maintained their faster swim speeds across trials, control rats swam significantly slower than conditioned run ners on Blocks 2 5 ( all p values < 0.01) and slower than they swam on the first training block (Block 1 > 2, 3, 4 and 5; a all p values < 0.01). Specifically, all rats combined spent less time in the outer annulus as training progressed (blocks 1, 2 > 5, p values < 0.05), but conditioned runners spent less time
120 than controls on all blocks combined. These data suggest that although anxiety decreases with training in all aged rats, prior training may potentiate this effect in rats that exercise daily. We calculated swim speeds on hidden platform training blocks as a measure of locomotor ability in aged conditioned runners and controls (Figure 4 3 C). Swim speeds were significantly affected by conditioned running ( F (1,9) = 13.07; p < 0.01), training block ( F (4,36) = 6.27; p < 0.01), and the interaction betw een running and training block ( F (4,36) = 3.87; p < 0.01). Conditioned runners swam significantly faster to the hidden platform than controls on all blocks combined ( p < 0.01) and maintained the swim speeds that they achieved on later visible platform tria ls across all hidden platform training blocks (see Figure 4 2 C). In all rats combined, swim speeds decreased after the first block (all p values < 0.01), but this effect was because while conditioned runners maintained their speeds across blocks, control rats swam significantly slower after the first training block (Blocks 1 > 2, 3, 4 and 5; all p values < 0.01). These data support the notion that daily exercise can potentiate the effects of water maze training on the swimming proficiency of aging rats, po tentially by improving their stamina. Aging Rats that Exercise Exhibit Better Memory for the Platform Location on Probe Trials A 60s probe trial was conducted immediately after the final hidden platform trial (Figure 4 4). An ANOVA revealed that all rats c ombined exhibited a significant quadrant preference ( F (3,27) = 24.99; p < 0.0001) and that quadrant preference significantly interacted with group ( F (3,27) = 7.54; p < 0.001) on the immediate probe. Specifically, conditioned runners spent significantly mor e time in the goal quadrant ( p = 0.0003; Figure 4 4 A) and less time in the opposite quadrant ( p = 0.045) but similar amounts of
121 time in the left ( p = 0.32) and the right quadrants ( p = 0.96) relative to controls. Similarly, conditioned runners exhibited s ignificantly better DI scores than controls ( t (9) = 4.17, p < 0.01; Figure 4 4 B) and tended to cross over the location that housed the platform on training trials significantly more frequently (4.33 0.71 crossings) than controls (2.60 0.51 crossings) did ( t (9) = 1.90; p = 0.09). A refresher block of hidden platform trials was administered after the immediate probe to minimize the probability that the association between platform localization and escape from the pool was extinguished by the immediate pr obe trial. A paired t test on the 5th and 6th hidden platform blocks confirmed that the rats exhibited similar path lengths before and after the probe trial ( t (10) = 0.29, p = 0.78; see Figure 4 3 A). A second probe trial was administered 24 h after the 5 t h hidden platform block. An ANOVA revealed that all rats combined exhibited a significant quadrant preference ( F (3,27) = 3.56; p = 0.027) and that quadrant preference interacted with group ( F (3,27) = 5.16; p = 0.006) on the 24 h probe trial (Figure 4 4 A). Specifically, conditioned runners spent significantly more time in the goal quadrant ( p = 0.026), tended to spend less time in the opposite quadrant ( p = 0.052) and spent similar amounts of time in the left ( p = 0.416) and right quadrants ( p = 0.498) rela tive to controls. Similarly, conditioned runners exhibited significantly better DI scores than controls ( t (9) = 4.39; p < 0.01; Figure 4 4 B) and crossed the location that housed the hidden platform on training trials significantly more frequently than con trols (5.17 0.40 versus 1.20 0.20 crossings, respectively; t (9) = 8.28, p < 0.01) on the delayed probe trial. These data suggest that conditioned runners both learned and remembered the hidden platform location better than controls.
122 Figure 4 4. Conditioned runners exhibit better memory in the water maze and on an inhibitory avoidance task. (A). A 60 s probe trial was conducted immediately after or 24 h after the final hidden platform trial and mean % time spent in the goal quadrant ( S.E.M. ) is depicted for the controls (gray bars) and conditioned runners (black bars). On the immediate probe ( p = 0.0003) and on the 24 h probe ( p = 0.026), conditioned runners spent significantly more time in the goal quadrant than control rats did. (B) Individual discrimination index (DI) scores were calculated for control (gray circles) and conditioned runne r rats (black squares) and then plotted. Positive scores represent better goal (versus opposite) quadrant discrimination. Conditioned runners exhibited better DI scores on both the immediate ( p < 0.01) and 24 h ( p < 0.01) water maze probe trials than con trol rats. (C) Finally, rats were trained (white bars) and then tested 1 h (light gray bars) and 24 h (dark gray bars) after training in an inhibitory avoidance task. Conditioned runners and controls entered the dark side of the inhibitory avoidance chambe r that delivered shock equally as quickly during the acquisition phase of the task ( p = 0.90). Although both control and conditioned runners exhibited similar 1 h retention latencies, conditioned runners took significantly longer than controls to re enter the dark side 24 h after training ( p < 0.05). Finally, a regressi on analysis of the distance to escape the pool on block 5 of cue discrimination training was compared to the distance to escape for block 5 of the spatial discrimination task as well as the discrimination index score obtained on the immediate probe. No association was observed indicating that acquisition of the spatial discrimination was not linked to the acquisition performance for cue discrimination.
123 Inhibitory Avoidance Scores One week after the onset of visible platform water maze training, the rats were trained and tested in an inhibitory avoidance task (Figure 4 4 C). Mann Whitney U tests confirmed that conditioned runners and controls entered the shock paired dark side of the inhibitory avoid ance chamber equally as quickly during the acquisition phase of the task ( U = 0.01; Z p = 0.92). Although 1 h latencies were similar between groups ( U = 8.00; Z p = 0.24), conditioned runners tended to have longer 24 h latencies than cont rols ( U = 5.00; Z = 1.73; p = 0.08). Spearman Rank Correlation was used to compare learning and memory on the water maze discrimination index scores and 1 and 24 h inhibitory avoidance retention latencies. The results indicated a relationship between the 2 4 h retention scores on the water maze and inhibitory avoidance ( r = 0.63, p < 0.05). Daily Exercise Increases Neurogenesis in Aged Rats by Increasing New Cell Number The total number of new cells was estimated in the dentate gyri of all rats using stereol ogical principles (Figure 4 test confirmed that the total number of BrdU + cells was higher in the dentate gyri of conditioned runners relative to controls ( t (9) = 3.44, p < 0.01; Figure 4 5 E). Although exercise could potential ly increase vascular volume within the neurogenic niche, dentate gyri volumes that new cells were estimated through were similar between controls (4.32 0.17 mm 3 ) and conditioned runners (4.53 0.30 mm 3 ; t (9) p = 0.58). As expected from these data sets, new cell densities were also higher in conditioned runners (514.77 40.95 cells/mm 3 ) versus controls (370.35 46.14 cells/mm 3 ; t (9) = 2.35, p < 0.05). Because the rats survived several weeks after Br dU was injected, these differences could reflect
124 effects on NPC division and/or the survival of new cells, but are consistent with the well known effects of physical exercise on NPC division. We confirmed that new cell differentiation was unaffected by con ditioned running by quantifying the percentage of BrdU + cells expressing immature neuronal (DCX + ), transitioning neuronal (DCX/NeuN + ), mature neuronal (NeuN + ), oligodendroglial (NG2 + ), or astroglial (GFAP + ) phenotypes (Figure 4 5 C, D and F). Mann Whitney U tests (n runner = 6 and n control = 5 in all comparisons) confirmed that the percentages of BrdU + cells expressing immature neuronal ( U = 8, Z = 1.187, p = 0.024), transitioning neuronal ( U = 14.5, Z = 0.0, p = 1.0), mature neuronal ( U = 15.0, Z = 0.0, p = 1.00), GFAP + ( U = 13.0, Z p = 0.784) and oligodendrocyte precursor ( U = 14.5, Z = 0.0, p = 1.0) phenotypes were similar between conditioned runner and control rats. Consistent with a 2.5 3 week long survival period after BrdU, most new cells ( 70%) expressed mature neuro nal phenotypes followed by astrocyte and transitioning neuronal phenotypes ( 10% each). Very few new cells expressed immature neuronal or oligodendroglial phenotypes (<3%) in the dentate gyri of all rats combined (Figure 4 5). Note that all of the BrdU/GFA P + cells were detected outside of the subgranular zone and exhibited an astrocyte rather than radial glial (or neural stem cell) like morphology. The total new cell number (Figure 4 5 E) was multiplied by the % of neurons (immature, transitioning and matur e), oligodendrocytes or astrocytes (Figure 4 5 F) for each rat to estimate total numbers of each new cell phenotype (Figure 4 5 G).
125 Figure 4 5. Conditione d running potentiated hippocampal neurogenesis in aging rats. (A) Transmitted light micrograph showing new (BrdU + ; in brown) cells located through the GCL and SGZ of aged rats under a 10X objective revealed by DAB. (B) Shows a subset of BrdU + cells depicte d in (A) under the 40 objective used for counting. (C and D) Confocal images of new cells (BrdU/DAPI + ; in white and red) expressing the neuronal proteins DCX (in blue) and NeuN (in green; [C]) or the astrocyte protein GFAP (in blue; [D]). Insets show each channel independently and scale bars represent 10 B and E) More BrdU + cells were detected in the dentate gyri of conditioned runners vs. controls ( p < 0.01). (C, D and F) Similar percentages of new cells in the dentate gyri of conditioned runners and controls expressed immature neuronal (DCX + ), transitioning neuronal (DCX/NeuN + ), mature neuronal
126 (NeuN + ), oligodendroglial (NG2 + ) or astroglial (GFAP + ) proteins. Consistent with the 2 week survival period, most new cells expressed mature neuronal phe notypes, followed by astrocyte and transitioning neuronal phenotypes. (G) The total estimated new neuron number was significantly higher in conditioned runners versus controls ( p < 0.01). Data are group means S.E.M obtained from conditioned runners (bla ck bars) and controls (gray bars). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Figure 4 6. Probe trials scores relate to measures of neurogenesis in aging rats. Spearman rank correlations were conducted on total new (BrdU + ) DCX and/or NeuN + neuron numbers and DI scores obtained from control rats (light gray squares) and conditioned runners (black squares). Total new neuron number tended to correlate with (A) DI sc ores obtained in the immediate probe trial ( p = 0.08) and (B) DI scores obtained from the 24 h probe trial ( p = 0.059). Relative to controls, conditioned runners had significantly more new neurons ( t (9) = 3.26; p < 0.01), tended to have more new astrocytes (157.41 31.27 and 227.59 22.06, respectively; t (9) p = 0.09), and had similar numbers of new oligodendrocyte precursors (26.31 16.55 and 31.43 19.89, respectively; t (9) p = 0.85). New neuron number tended to correlate positively with immediate ( p = 0.08; Figure 4 6 A) and 24 h ( p = 0.059; Figure 4 6 B) water maze probe discrimination index scores.
127 Distinct Cytokine Relationships were Detected in Serum, Hippocampal and Cortical Compartments Concentrations of 24 cytokines were quantified in blood serum and hippocampal and cortical protein samples of each behaviorally char acterized rat that neurogenesis was also quantified in (Table 4 1). Note that concentrations of eotaxin, GRO KC, IL 10, IL 13, IL 17, leptin, and RANTES were at least a magnitude higher in circulation versus the brain. I CSF, GM 2, IL 4, IL 5, IP cytokines only detected in the brain, G SCF, GM CSF, IL 10, and IP 10 were detected in the cortex but not in the hippocampus. A 2 fold higher concentration of IL MCP 1 was detected in the hippocampus versus cortex whereas a 3 fold higher concentration of IL 12 and a 2 fold higher concentration of IL 2 and IL 5 were detected in the cortex versus h ippocampus. These data suggest that in aged rats, circulating cytokine concentrations do not appear to reflect central concentrations. In addition, there appear to be regional differences in the basal expression of central cytokines and therefore, likely t heir influence. We next analyzed cytokine relationships within and between blood serum, hippocampal and cortical compartments to further explore the ideas that circulating concentrations may predict central cytokine signaling and those differences in central cytokine expression may reflect more local signal ing. Pathway analyses (see Cytokine Cluster Analysis ) revealed distinct clusters within but no clusters between compartments after Bonferroni adjustments for multiple comparisons (Table 4 2 and Figure 4 7).
128 Table 4 1. Some hippocampal (pg/mg), cortical (pg/mg) and circulating (pg/mL) cyto kines are modulated by daily exercise in aging rats. Mean (S.E.M.) values are reported. ** p < 0.01, p < 0.05, 0.05 < p < 0.10 vs. control values. Serum Hippocampus Cortex Controls Runners Controls Runners Controls Runners Eotaxin 55.06 13.76 196. 23 147.02 2.37 0.76 3.46 0.87 1.90 0.16 1.73 0.27 G CSF 0 0 2.23 2.23 0 0 0 0 0.25 0.04 0.17 0.07 GM CSF 0 0 0 0 0 0 0 0 1.09 0.11 0.99 0.33 GRO KC 1322.52 219.98 776.89 154.63 13.36 2.57 25.83 5.55 9.82 0.80 10.54 2.28 IFN 26.41 18.51 415.65 398.82 0 0 1.52 1.29 0 0 0 0 IL 0 0 233.95 233.95 2.5 5 2.55 14.81 7.03 5.60 1.06 4.96 1.58 IL 200.54 174.94 222.88 182.13 40.44 3.46 22.27 4.38 ** 13.07 2.38 13.56 1.82 IL 2 0 0 0 0 8.62 5.09 13.73 4.28 27. 50 1.75 22.47 4.18 IL 4 0 0 47.56 41.81 2.73 0.90 4.88 0.99 3.17 0.33 2.63 0.53 IL 5 0 0 18.32 13.88 1.46 1.01 1.73 0.94 4.54 0.56 3.84 0.91 IL 6 67.57 21.94 537.54 498.29 17.31 8,12 30.14 10.05 14.02 1.82 16.28 4 .19 IL 9 243.99 137.04 190.51 111.29 491.60 301.67 588.73 267.64 396.14 50.08 385.10 86.51 IL 10 59.89 59.89 59.98 59.98 0 0 0 0 4.56 0.37 4.52 1.78 IL 12 38.77 21.73 68.30 62.32 5.13 1.50 7.60 2.04 19.58 3.29 17.99 3 .55 IL 13 108.00 38.54 332.52 282.22 2.68 2.06 9.42 3.66 6.06 0.25 5.13 1.05 IL 17 20.47 8.29 29.24 19.27 0.77 0.40 0.68 0.38 0.57 0.09 0.48 0.17 IL 18 351.01 189.13 789.49 422.74 105.42 31.38 209.72 28.51 77.38 3.90 70.00 10.25 IP 10 0 0 53.56 53.56 0 0 5.72 5.00 1.57 0.11 1.41 0.19 Leptin 10001.90 850.70 5414.09 743.34 ** 17.84 5.15 19.97 6.43 10.39 0.51 9.52 1.86 MCP 1 719.92 109.32 323.09 134.99 113.92 68.04 138.22 49.40 51.23 3.85 39.93 8.78 MIP 5.67 2.25 9.42 6.61 0.61 0.25 4.05 3.09 0.34 0.02 0.43 0.09 RANTES 18364.54 1635.46 11780.8 3208.50 0 0 13.49 6.72 17.22 1.86 42.96 16.84 TNF 0 0 2.40 1.52 2.44 1.09 3.27 1.41 2.28 0.06 2.20 0.61 VEGF 21.80 21.80 23.38 23.38 3.4 6 1.52 6.46 2.68 1.91 0.25 0.98 0.33
129 In serum: (1) MCP 1 and GRO KC and (2) IL 6 and I L 13 were identified as independent clusters. In the hippocampus: (1) IL 17 and VEGF, (2) IL 5 and VEGF, (3) MCP 1, IL 2 and VEGF, (4) MCP, IL 2, TNF 5, (5) IL 2 and GRO KC, 12, (7) IL 2 and IL 4 and (8) IL 4 and IL 6 were identified as independent clusters. In the cortex: (1) IL 2 and GM CSF, (2) GM CSF and IL 18, (4) GM CSF and IL 10, and (5) IL 13 and IL 4 were identified as independent clusters. Table 4 2 shows the r values (bolded and with asterisks) of cytokine pairs that were included in these clusters because they remained statistically significant after Bonferroni corrections. While these resu lts indicate strong relationships between cytokine concentrations within each brain region and within serum, no clusters emerged between these compartments. The lack of significant relationships between serum and brain cytokine concentrations may indicate that circul ating factors neither diffuse nor are transported in detectable quantities into hippocampal and cortical regions in aging rats unchallenged by an inflammatory event (Erickson and Banks, 2011) The lack of significant relationships between hippocampal and cortical compartments suggests that basal neuroimmune signaling is a local event. Of course, the lack of signi ficant between compartments relationships could simply reflect the stringency inherent to Bonferroni adjustments, which increase the likelihood of type II errors. Measures of Behavior and Neurogenesis Relate to Concentrations of Cytokines Modulated by Runn ing To identify cytokine candidates linked to behavior and neurogenesis, we first t tests (see Table 4 1). Compared to controls, conditioned runners had significantly lower hippocampal IL t (9) = 3.14; p < 0.05), circulating MCP 1 ( t (9) = 2.28; p 0.05) and circulating leptin
130 ( t (9) = 4.06; p < 0.01) but higher hippocampal IL 18 ( t (9) p < 0.05) concentrations. Concentrations of circulating GRO KC ( t (9) = 2.08; p = 0.07) and cort ical VEGF ( t (9) = 2.16, p = 0.06) tended to be lower whereas hippocampal concentrations of GRO KC ( t (9) p = 0.09) tended to be higher in runners versus controls. Of the cytokines with concentrations that were significantly modulated by conditioned running in aged rats, several were modulated in a correlated manner (see Figure 4 7 A and Table 4 3). For example serum MCP 1, which was decreased by conditioned running, correlated positively with serum GRO KC ( p < 0.01) and both concentrations tended to correlate positively with serum leptin ( p = 0.06 and p = 0.08). Serum leptin was strongly decreased by conditioned running and correlated positively with hippocampal IL p < 0.05) but tended to correlate negatively with hippocampal IL 18 ( p = 0.06) and hippocampal GRO KC ( p = 0.08). Hippocampal IL d ecreased by conditioned running and correlated negatively with hippocampal IL 18 ( p < 0.05) and tended to correlate positively with cortical VEGF ( p = 0.07). A strong positive correlation was detected between hippocampal IL 18, which was increased with run ning, and hippocampal GRO KC ( p < 0.01). These data suggest that conditioned running modulates subsets of cytokines within and between serum, hippocampal and cortical compartments.
131 Table 4 2. Spearman rank correlation coefficients ( r s ) between cytokine pa irs detected in (A) serum, (B) hippocampal and (C) cortical compartments reveal clusters (see Figure 4 7). Note that no between compartments clusters emerged after Bonferroni corrections. After Bonferroni adjusted level: p < 0.00042 in serum, ** p < 0.00 029 in hippocampus, *** p < 0.00020 in cortex.
132 Figure 4 7. Cytokine clusters detected in the serum, hippocampal and cortical samples obtained from aging rats. To confirm and expand upon known cytokine pathways, we examined cytokines wit h concentrations that changed in a coordinated fashion. Cytokine pairs were plotted in descending order based upon Spearman r values deemed statistically significant after Bonferroni corrections. If one cytokine in a correlated pair about to be plotted was already part of a plotted cluster, and the unplotted cytokine was correlated with all cytokines in the plotted cluster, then the new pair was added to the cluster. If the unplotted cytokine of the pair about to be plotted was not significantly correlated with all cytokines in the existing cluster, the pair was plotted as a new cluster. Proteins are color coded by their known primary function and green and red lines represent positive and negative correlations, respectively. We detected two serum cytokine c lusters, eight hippocampus cytokine clusters and four cortical clusters in aging rats. Note that no between compartment clusters indicative of immune to brain signaling pathways modulated by running in aged rats were detected. Next we examined the stre ngth of relationships between variables significantly affected by conditioned running (total new neuron number, probe trial discrimination index scores and 24 h inhibitory avoidance retention latencies) using Spearman rank correlations (see Table 4 3). Int erestingly, 24 h retention latencies on the inhibitory avoidance task correlated positively with water maze 24 h probe discrimination index scores ( p < 0.05). These data suggest that these tasks are both similarly sensitive to
133 age related cognitive decline and the beneficial effects of conditioned running on spatial ability in aged rats. As mentioned previously, new neuron number tended to correlate positively with immediate ( p = 0.08) and 24 h ( p = 0.059) water maze probe discrimination index scores (see F igure 4 8 B and Table 4 3). Finally, we explored relationships between cytokine, behavioral and neurogenesis measures that were modulated by conditioned running (Table 4 3 and Figure 4 8 B). New neuron number, which was potentiated by running, correlated n egatively with serum leptin level ( p < 0.01) but positively with hippocampal IL 18 ( p < 0.001) and hippocampal GRO KC ( p < 0.01) expression and tended to correlate negatively with hippocampal IL p = 0.06). Immediate probe trial discriminati on index scores, which increased with running, correlated negatively with cortical VEGF levels ( p < 0.05) and circulating levels of leptin ( p < 0.05) MCP 1 ( p < 0.01) and GRO KC ( p < 0.01) and tended to correlate negatively with hippocampal IL p = 0.06 ). Twenty four hour discrimination index scores correlated negatively with circulating leptin levels ( p < 0.01) and hippocampal IL p < 0.05). Interestingly, serum leptin levels correlated negatively with immediate discrimination index score s ( p < 0.05), 24 h discrimination index scores ( p < 0.01) and new neuron numbers ( p < 0.01). Serum leptin level correlated positively with hippocampal IL concentrations ( p < 0.05), and hippocampal IL with 24 h water maze ( p < 0.05) and 24 inhibitory retention ( p < 0.05) performances. Cortical VE GF tended to correlate negatively with 24 h inhibitory retention latencies ( p = 0.06).
134 Table 4 3. Measures of several variables significantly modulated by daily exercise in aging rats correlate. Spearman rank correlation coefficients (r s ) were calculated test the stre ngth of the relationships between concentrations of serum (S), hippocampal (H) and cortical (C) cytokines, an d measures of spatial and hippo campal neurogenesis that were significantly modulated by conditioned running. p < 0.05, ** p < 0.01, *** p < 0.001, 0.05 < p < 0.01. Serum Hippocampus Cortex Neurogenesis and behavior MCP 1 leptin GRO KC IL IL 18 GRO KC VEGF New Neuron # Immed. DI 24hr DI 24hr IA MCP 1 (S ) 0.65 0.96 *** 0.54 0.13 ** Leptin (S) 0.65 0.56 0.76 0.52 ** ** GRO KC (S) 0.96 *** 0.56 0.42 0.07 ** IL 0.54 0.76 0.42 0.57 IL 18 (H) 0.85 ** 0.94 *** 0.51 0.54 0.16 GRO KC (H) 0.85 *** 0.79 ** 0.38 0.41 VEGF (C) 0.13 0.52 0.07 0.57 New Neuron # ** 0.94 *** 0.79 ** 0.59 0.55 0.18 Immed. DI ** 0.75 ** 0.51 0.38 0.59 0.47 0.45 24 h DI ** 0.54 0.41 0.55 0.47 0.62 24 h IA 0.16 0.18 0.45 0.62
135 Figure 4 8. Some cytokines are modulated in a coordinated fashion by conditioned running in aging rat s and relate to measures of hippocampus dependent behavior and hippocampal neurogenesis. Spearman rank correlations were run on immune and neuroimmune cytokines with concentrations that were modulated by running (see Table 4 1), water maze DI scores, inhib itory avoidance retention latencies and total new neuron number. Of the cytokines altered by daily exercise, several were modulated in a coordinated fashion. Cytokines are color coded to denote their primary, typically systemic, known function. Concentrati ons increased by running are plotted in the green circle while those that decrease are plotted in the red circle. Negatively correlated cytokines are linked with red lines while positively correlated cytokines are linked with green lines. (B) Depicts relat ionships between cytokines, behavioral measures and measures of neurogenesis that were modulated by running. Water maze discrimination index scores, inhibitory avoidance 24 h retention latencies and new neuron number were significantly affected by conditio ned running. Note that only statistically significant correlations ( p < 0.05) are shown.
136 Discussion An important goal for aging research is to identify markers of biological aging that predict cognitive decline. In the current study, we measured hippocampal neurogenesis and identified potential serum and central markers in rats after rejuvenating their cognition with a behavioral treatment. Several months of food motivated wheel running n and retain or consolidate spatial/contextual information. Hippocampal neurogenesis is a well characterized marker of brain aging that was potentiated by daily exercise in the current study along with correlated increases in hippocampal IL 18 and GRO KC. Both central and peripheral markers of inflammation have been hypothesized to contribute to age related decreases in cognitive function and we found that daily exercise decreased hippocampal IL 1 inhibitory avoidance memory scores. Due to the invasive nature of identifying markers in brain tissue, many researchers have focused upon identifying serum markers. In the aging rat, leptin emerged as a potential serum marker for age related declines in cognition and plasticity because it correlated negatively with water maze performances and new neuron number. Moreover, daily exercise decreased serum leptin along with serum MCP 1 (CCL2) leve ls and tended to decrease serum GRO KC (CXCL1) level. Interestingly, serum leptin, GRO KC and MCP 1 levels along with cortical VEGF level (which tended to decrease with daily exercise) correlated negatively with the water maze learning index. Our data sugg est that daily exercise may rejuvenate cognition and neurogenesis in aging rats by modulating immune and neuroimmune signaling pathways.
137 Although the exercise protocol employed may rejuvenate cognition and neurogenesis in aging rats by modulating immune an d neuroimmune signaling pathways, the observed benefits may also be due to a caloric restriction associated with the exercise protocol. Exercise and caloric restriction beneficially affect a number of biological processes in aged rats that include the modu lation of inflammatory signaling pathways (Chung et al., 2009) However, in the current study any caloric restriction was voluntary and very mild producing body weight changes of less than 10% (Bondolfi et al., 2004; Lee et al., 2000; Van der Borg ht et al., 2007) which would also be consistent with an exercise induced increase in fitness. Exercise is known to stimulate neurogenesis in young and aged animals (Albeck et al., 2006; Brown et al., 2003; Kobilo e t al., 2011; Parachikova et al., 2008; van Praag et al., 2005) while the effects of caloric restriction on neurogenesis may be limited to younger animals (Bondolfi et al., 2004; Lee et al., 2000; Van der Borght et a l., 2007) Regardless, the inflammation associated with obesity and a sedentary lifestyle is thought to contribute to diseases of aging and an understanding how exercise with and without caloric restriction influences inflammatory signaling cascades will be important for the development of treatments. Consistent with previous studies conducted using socially isolated animals, wheel running improved cognition (Albeck et al., 2006; Parachikova et al., 2008; van Praag e t al., 2005) and amplified basal levels of hippocampal neurogenesis without altering the percentage of new cells that acquired neuronal or glial fates (Brown et al., 2003; Farmer et al., 2004; Kannangara et al., 201 1; Kannangara et al., 2009; Kronenberg et al., 2006; Mustroph et al., 2012; Snyder et al., 2009) Although our multiple injection paradigm and 16 21 day long survival period cannot dissociate the
138 between the effects of exercise on NPC proliferation versu s the survivability of new cells, these data are consistent with those of several other studies showing that physical exercise increases NPC proliferation (Kempermann et al., 2010; van Praag et al., 1999) New neuro n number tended to correlate positively with immediate and delayed water maze probe trial discrimination index scores but not inhibitory avoidance retention latency scores. Importantly, the 900 s latency ceiling employed in the inhibitory avoidance task ma y have masked the relationship between the memory of contextual information and new neuron number. We recently reported that new neuron number strongly correlated with discrimination index scores in environmentally and socially enriched aged rats and their controls ( Speisman et al., 2013b ) While physical activity is typically considered to induce neurogenesis, environmental enrichment is typically considered to promote the survival and potentially the integration of new neurons into active hippocampal networks (Deisseroth et al., 2004; Deng et al., 2010; Gould et al., 1999a; Kobilo et al., 2011; Stephens et al., 2012 ) which may more profoundly impact hippocampal integrity ( Kempermann et al., 2010 ) Figure 4 8 illustrates a potential link between serum inflammatory markers and hippocampal cytokines associated with cognition and neurogenesis. Exercise modulated circulating leptin level (Chennaoui et al., 2008; Novelli et al., 2004) correlated negatively with maze discrimination index scores, new neuron number and the hippocampal expression of IL 18 but positively with hippocampal IL A recent work suggests that leptin can directly stimulate the proliferation of neural progenitor cells both in vitro and in vivo (Ga rza et al., 2008; Perez Gonzalez et al.,
139 2011 ) These data would suggest that serum leptin influences hippocampal neurogenesis in the aging rat through an intermediary signaling molecule or simply that the serum leptin levels detected in aged rats exceed those found in healthy young animals to the point that they become detrimental. Consistent with the latter notion, exercise decreased leptin levels in our aging rats. Indeed, leptin is emerging as a potential immune to brain signaling mediator (Hosoi et al., 2002b) because leptin levels are elevated by peripheral inflammatory stimuli (Mastronardi et al., 2005; Sarraf et al., 1997) that incidentally decrease neurogenesis (Monje et al., 2003) and leptin treatment increases brain levels of hippocampal IL (Hosoi et al., 2002b) Alternatively, serum leptin concentration in the current study may simp ly be a marker for an immune signaling cascade containing the molecule that affects neurogenesis. Indeed, serum GRO KC and MCP 1 levels were also decreased by exercise, strongly correlated with one another, and independently (along with leptin) correlated with the acquisition of a spatial search strategy in the water maze (Table 4 1 and Table 4 3 and Figure 4 8). Previous work has shown that MCP 1 levels that are elevated by high fat diet induced obesity in young mice, can be reduced by daily exercise ( Kizaki et al., 2011 ) Recently, Villeda and colleagues found that circulating CCL2, along with eotaxin (CCL11), MCP 5 (CCL12), MIP levels were related to age impaired neuroge nesis and performance in a working/reference memory radial water maze task. They then showed that circulating eotaxin levels alone could impair neurogenesis, synaptic plasticity, working/reference memory and contextual fear conditioning ( Villeda et al., 2011 ) We neither detected an effect of exercise on circulating eotaxin levels, nor relationships between circulating
140 eotaxin levels and measures of neurogenesis or water maze performance in aging rats. However, age r elated changes in circulating eotaxin may be species dependent or relate to cognition and neurogenesis by interacting with another variable that differed between studies. Nonetheless, exploring the molecular mechanisms by which circulating factors, such as leptin, GRO KC, MCP 1 and perhaps eotaxin relate to measures of cognition and plasticity are important future research avenues. Another important finding of the current study is the compartmental specificity of exercise associated changes in cytokine leve ls. After several months of daily exercise, leptin levels decreased only in serum and IL 18 increased only in the hippocampus (Table 4 1). A previous report also found that daily treadmill exercise decreases hippocampal, but not circu lating, cortical, cerebellar or pituitary levels of IL (Chennaoui et al., 2008) O ur data extends these findings to aging rats, by showing that hippocampal, but not cortical or circulating IL are reduced by exercise. Furthermore, hippocampal IL positively with serum leptin and negatively correlated with both water maze and inhibitory avoidance memory sco res (Table 4 3 and Figure 4 8). Leptin is actively transported across the blood brain barrier (Morrison, 2009) and previous work has demonstrated that leptin treatment increases IL (Hosoi et al., 2002b) providing one possible mechanism for the observed relationship. Moreover, the inverse relationship b etween IL work indicating that elevated hippocampal IL plasticity in young and aged rats (Barrientos et al., 2009; Barrientos et al., 2006; Barrientos et al., 2003; Chennaoui et al., 2008; Hein et al., 2010; O'Callaghan et al.,
141 2009) Together these data suggest that hippocampal IL reliable biomarker of mnemonic decline and, along with circulating lept in, a target for nootropic drug development. In contrast to IL 18 and GRO KC levels were increased in the hippocampus of the exercise group. Hippocampal IL 18 and GRO KC concentrations correlated positively with one another and independently with ne w neuron number, while hippocampal IL 18 concentration correlated negatively with hippocampal IL concentration (Figure 4 8). Daily exercise has been shown previously to potentiate ress human amyloid protein (Parachikova et al., 2008) Interestingly, these mice also exhibited improved water radial arm maze performance and decreased hippocampal IL (Nichol et al., 2008; Parachikova et al., 2008) GRO KC stimulates adult rat spinal cord oligodendrocyte (Robinson et al., 1998) and fetal ventral midbrain precursor (Edma n et al., 2008) proliferation and IL 18 may attenuate neuronal differentiation in cultured fetal rat derived neural progenitor cells (Liu et al., 2005) but this is the first report of a relationship between either factor and adult NPC behavior, to our knowledge. Conflicting reports suggest that IL 18 promotes neuroprotection and spatial ability ( Ryu et al., 2010; Yaguchi et al., 2010 ) but also age related cognitive decline (Blalock et al., 2003; Mawhinney et al., 2011 ) Our data showing that exercise increased hippocampal IL 18 levels correlate positively with new neuron number (which correlate positively with spatial ability in aging rats; Table 4 2 and Speisman et al., 2012b) are consistent with the former notion. The question remains as to how IL 18 could be linked to improved hippocampal function. One possibility is that exercise may improve hippocampal health
142 and stimulate neurogen esis in the aging brain by improving vascular health (Palmer et al., 2000) GRO KC and IL 18 exhibit pro angiogenic properties and are linked through ras raf ERK MAPK signaling (Park et al., 2001; Zhong et al., 2008) Although we did not observe an exercise induced shift in hippocampal VEGF levels, previous research indicates that running potentiates hippocampal neurogenesis through VEGF activity in young mice (Fabel et al., 2003; Tang et al., 2010) and increases hippocampal VEGF levels in middle aged mice ( Latimer et al., 2011 ) However, VEGF levels are known to decline in the aged brain (Shetty et al., 2005) and may require rejuvenation before exercise can potentiate neurogenesis beyond a basal level (Figure 4 6 versus Speisman et al., 2012a). Note that although every analyte measured was detected in the h ippocampus and/or cortex (from which similar amounts of protein were harvested), the volume of protein obtained from of the hippocampus could have been too small to quantify changes in the concentration of low level analytes, such as VEGF. Our cluster an alyses (see Figure 4 7) further suggest that cytokine signaling in aging rat runners and their controls is compartmentalized. In serum, we found strong positive correlations between GRO KC and MCP 1 and between IL 6 and IL 13 concentrations. Coordinated mo nocyte/macrophage derived serum MCP 1 and GRO KC concentrations have been reported in models of wound repair induced angiogenesis and in the serum of LPS treated mice (Barcelos et al., 2004; Erickson and Banks, 2011) and coordinated circulating IL 13 and IL 6 concentrations may be consistent with the heightened Th2 response hypothesized to occur with age ( Grolleau Julius et al., 2010 ) Although IL 13 was detected in cortical c lusters and GRO KC and MCP 1 were detected hippocampal clusters we did not detect between compartments correlations
143 that would indicate direct diffusion or transport across the blood brain barrier to either location. In the brain, we detected correlated co ncentrations of cytokines that were distinct in the cortex and hippocampus. In the cortex, concentrations of structurally and functionally homologous IL 13 and IL 4, which are expressed by microglia and typically associated with anti inflammatory and neuro protective effects in the brain were correlated (Opal and DePalo, 2000; Ponomarev et al., 2007; Shin et al., 2004) Cortical GM CSF was detected in separate clusters with IL 2, IL 10 and IL 18. Astrocytic GM CSF act s on its receptors expressed by microglia and oligodendrocytes (Kimura et al., 2000) IL 2 and its receptor protein is thought to be expressed by neurons, glia and microglia while IL 18 mRNA is expressed by microgli a with its receptors being expressed by neurons, astrocytes and microglia throughout the brain (Hanisch and Quirion, 1995; Quirion et al., 1995; Tambuyzer et al., 2009) While the relationship between brain IL 2 and GM CSF is unclear, IL 10 decreases but IL 18 increases GM CSF production by peripheral immune cells and IL 10 may suppress microglial inflammatory responses ( Lee et al., 2010 ) In the hippocampus, VEGF correlated i ndependently with IL 2 and MCP 1, IL 5, and finally IL 17. Endothelial VEGF production can be stimulated by IL 2, and microglial, endothelial cell or smooth muscle cell MCP 1 expression in response to vascular injury (Parenti et al., 2004) IL 5 and IL 17, often associated with allergic reactions, can also stimulate VEGF production. Interestingly, the injury induced expression of MIP MCP 1, GM CSF, and TNF and induce phagocytosis (Ousman and David, 2001) The coordinated concentration of
144 IL 2 with this cluster is interesting because IL 2 is often associated with self recognition (Kolls and Linden, 2004) IL 2 and IL 4 are co regulated by exhaustive acute exercise in muscle and in serum presumably to stimulate repair processes ( Rosa Ne to et al., 2011 ) Relationships between IL 2 and IL 4, IL 2 and GRO KC, and between IL4 and IL 6 that we detected in the hippocampus have not yet been reported in the brain, to our knowledge. Future work that confirms and expands these regionally distinct neuroimmune signaling pathways and tests their effects in the aging brain will be critical for understanding their impact on cognition and plasticity. Certainly, regional changes in other signaling systems are under exploration in the aging brain [for exa mple, see ( McQuail et al., 2012 ) ]. Implications Daily exercise improved spatial/contextual ability, perhaps by stimulating hippocampal plasticity in the form of neurogenesis and by modulating immune and neuroimmune signaling. Daily exercise was associated with the decreased expression of factors that correlated negatively with learning, memory and neurogenesis measures but the increased expression of factors that correlated positively with our neurogenesis measure. T he picture of how immune and neuroimmune signaling impacts cognition and plasticity is growing. We add to this picture by showing that exercise modulates factors distinctly in serum and in the brain, suggesting that immune factors do not appreciably diffus e or are transported into the brains of aging rats that exercise and their controls. We also found that exercise modulated neuroimmune factors distinctly in the cortex and hippocampus, which supports the notion that in the brain, neuroimmune signaling is r egion specific. Serum leptin emerged as a biomarker for both brain and cognitive aging. Along with serum leptin, serum MCP 1 and GRO KC levels may predict
145 spatial ability. We confirmed that hippocampal IL mnemonic ability and w e discovered that hippocampal IL 18 and GRO KC levels correlated positively with neurogenesis. In summary, our work suggests that engaging in physical activity may reverse some aspects of age related cognitive decline, perhaps by stimulating neurogenesis a nd by modulating beneficial and detrimental aspects of immune and neuroimmune signaling. Our correlation data begin to provide a framework for systematically manipulating these immune and neuroimmune signaling molecules to test their effects on cognition a nd neurogenesis across lifespan in future experiments.
146 CHAPTER 5 CONCLUSION Major Experimental Findings While the elderly population grows and the cost of care for those individuals with cognitive impairments increases, we must develop strategies to pre dict and treat non pathogenic dementia. Here I revealed a novel relationship between measures of hippocampal neurogenesis and hippocampus dependent behavior in experiments that introduced variability in both measures with differential experience. Both wee ks long physical activity and exposure to environmental enrichment improved performance in a rapid acquisition water m aze task and increased ongoing basal levels of neurogenesis measured weeks after behavioral testing to minimize potential confounds. These data show that age related changes in both neurogenesis and performance in the rapid acquisition water maze task can be rescued by simple lifestyle changes. These data also suggested that developing an assay that could predict age related cognitive declin e could be beneficial for encouraging a lifestyle c hange as a preventative measure To this end, I assayed circulating and central inflammatory and stress hormone levels that were found to correlate with age related cognitive decline and measures of neurog enesis. Interestingly, physical exercise rescued neurogenesis and cognitive ability by possibly modulating these inflammatory biomarkers that were altered with age. In fact, some of the inflammatory biomarkers that were modulated with running correlated wi th each other along with the rescued measures of neurogenesis and cognitive ability allowing us to draw out potential pathways for treating and/or preventing age related cognitive decline. A biomarker assay of circulating inflammatory and stress molecules in the blood may one day be a beneficial tool to not only diagnose but also
147 predict cognitive impairment in the elderly. The results are discussed in the context of how they and the analyses I pioneered to obtain them have moved the fields of age related c ognitive decline and biomarker development forward. A Novel Relationship Between Hippocampal N eurogenesis and Hippocampus dependent Task Performance in Aged R ats some advancement s in the field of behavioral neuroscience, particularly in detecting a novel relationship between hippocampus dependent task performance and hippocampal neurogenesis in aged rats. In all of our experiments we utilized a rapid acquisition version of the Mor ris water maze task developed by the Foster laboratory, which is sensitive to age related cognitive decline (Carter et al., 2009; Foster and Kumar, 2007; Foster et al., 2003) The rapid acquisition task may be more sensitive to this age specific relationship than distributed training water maze protocols (Bizon and Gallagher, 2003; Bizon et al., 2004; Merrill et al., 2003) since it likely requires faster and more flexible proc essing application of spatial clues by the hippocampus (Foster, 2012) The increased task difficulty also allowed us to characteri ze rats as either impaired or unimpaired in order to examine differences in potential prognostic and diagnostic biomarkers of age related cognitive decline in Chapters 2 and 3. This task may also detect the beneficial effects of differential experience on cognition in aged rats (Kumar et al., 2012). It is important to note that exposure to environmental enrichment and physical exercise in Chapter 2 and 4 respectively may have also introduced the necessary variability to tease out the relationship between ne urogenesis and cognition. My data add to those of other groups suggesting that we should further explore how neurogenesis impacts cognition with age and whether methods modulating
148 neurogenesis could be used as possible treatments for reversing or preventin g age related cognitive decline. Identification of Prognostic and Diagnostic Biomarkers of Age related Cognitive D ecline Individual biomarkers of inflammation (Blalock et al., 2003; De Martinis et al., 2005; Gimeno et al., 2008; Krabbe et al., 2004; Magaki et al., 2007; Rafnsson et al., 2007; Solfrizzi et al., 2006; Villeda et al., 2011) and stress (Issa et al., 1990; Lupien et al., 1998; McEwen, 1998) have been previously s hown to change with age Here, I used Bio Plex technology to map changes in 27 different analytes in the blood and brain of aging rats. Utilizing behavioral scores from the water maze task and measures of hippocampal neurogenesis I was able to identify whi ch of these proteins correlated with changes in age related cognitive decline. I hypothesized that serum levels that were modulated in middle aged rats could be used as potential prognostic biomarkers and in aged rats as diagnostic biomarkers. Hippocampal proteins that were modulated with age could also be used as diagnostic biomarkers or could elucidate possible mechanisms for better understanding how these inflammatory molecules potentiate their effects on hippocampal neurogenesis and cognition. While ex amining blood serum for potential biomarkers, I uncovered novel age induced i ncreases in circulating levels of GRO KC, IFN IL 2, IL 5, IL 13, IL 17, leptin and MIP confirmed known analytes that increase with age in humans and rodents such as corticosterone, eotaxin, IL 6, IL 10, IL 18, IP 10, MCP 1 and R ANTES (Gangemi et al., 2003; Gerli et al., 2000; Krabbe et al., 2009; Mariani et al., 2006; Palmeri et al., 2011; Seidler et al., 2010; Villeda et al., 2011; Zhao et al., 2010) To elucidate possible mechanisms behi nd age related cognitive decline, I
149 examined analyte levels in the hippocampus and found that IL 5 and IL 12 dec reased while IL 9, IL 18, MIP related changes in the inflammatory molecules adds to the knowledge of aging and inflammation research while opening up avenues for future experiments to assess prevention, treatment and role of individual analytes in the aging process. Importantly, our data revealed region specific age induced changes which may be important when crafting a targeted molecular intervention since modulating systemic levels of a particular ana lyte may do more harm than good Through the correlation analyses in Chapter 4 I have identified MCP 1, leptin and GRO KC as potential immune and GRO KC and IL 18 as potential neuroimmune signaling molecules that may play an imperative role in adult hippo campal neurogenesis and/or cognitive decline Since I found that GRO KC and IL 18 were both elevated in the hippocampi of rats with increased neurogenesis and GRO KC has been reported to induce proliferation in other cell types (Edman et al., 2008; Robinson et al., 1998) while IL 18 reduces neuronal differentiation (Liu et al., 2005) it would be interesting to examine the effects of GRO KC and IL 18 on a NPC culture. Central d elivery to the hippocampus of GRO KC, IL 18 or adsorbing antibodies to explore the effect in vivo will require a creative methodology since an injection may damage the blood brain barrier and elicit an inflammatory response masking the effects of the molec ule(s) in question. However, GRO KC, leptin and MCP 1 which were downregulated in the serum related to immediate water maze probe scores and could more easily be injected systemically before behaviorally testing rats to examine these relationships further.
150 Hypothesis driven Pathway A nalysis Reveals I nflammatory Prognostic and Diagnostic B iomarker s of Hippocampal Neurogenesis and I ntegrity Using our innovative hypothesis drive correlation cluster analysis I found that many cytokines, chemokine s growth fa ctors and stress hormones that are modulated with age change in a synergistic fashion. Again, I confirmed known relationships such as the one between serum IL 6 and IL 13 (de Waal Malefyt et al., 1993) and revealed a few novel pathways like that between h ippocampal IL 4, IL 12 and TNF which was noted in the experiments in both Chapter 3 and 4. Since IL 4 has previously been shown to inhibit production of TNF 12, possibly through the transcription factor S TAT 6 pathway in peritoneal exudate macrophage cells (Levings and Schrader, 1999) experiments examining this relationship in a hippocampal co culture could reveal a new method for attenuating neuroinflammation. Or the neuroimmunomodulatory target eotaxin, which I found also linked to both TNF 12, may be targeted for modulation as it has recently been linked to neurogenesis and cognitive ability in aged rodents (Villeda et al., 2011) In Chapter 3, I employed a mu ltiple regression approach to determin e aged and aged rats based on their behavioral performance and biomarker profile s This analysis identified candidate biomarkers that could separate out memory impaired from memory unimpaired rats. Both the methodology and data are translational and could be used to test for similar biomarkers in the aging human population. KC. Interestingly, GRO KC serum levels in aged rats were decreased with running and correlated with measure of learning while hippocampal levels of GRO KC were
151 increased and correlated positively with total new neuron number in Chapter 4 This builds upon existing reports of exercise in duced hippocampal expression mice (Parachikova et al., 2008) and GRO (Barcelos et al., 2004; Zhong et al., 2008) I also found that running decreased s erum levels of MCP 1 and leptin. Since serum levels of leptin correlated with measures of cognition and neurogenesis, plays a role in response to inflammatory assault (Mastronardi et al., 2005; Sarraf et al., 1997) and has the ability to influence inflammatory molecules in the brain (Hosoi et al., 2002a, b) it may be a promising molecule to test in future experiments to modulate both the inna te and adaptive immune systems. Lif estyle Changes can Restore Hippocampal Neurogenesis and Hippocampal Integrity, Possibly through Modulation of Inflammatory Mediators age related cognitive decline. Differenti al experience through environmental enrichment physical activity and/or social interaction has been shown to promote neuroplasticity among other health benefits in both the young and aging brain in rodents and humans alike. In Chapter 2 and 4, I present d ata supporting long term environmental enrichment and physical activity as possible interventions for curbing age related cognitive decline, even when this intervention is begun later in life. Specifically, in Chapter 2, I expanded upon work showing t hat e nvironmental enrichment could rescue age related learning impairments potentially by improving new neuron production and survival (Kempermann et al., 2002; Kempermann et al., 1998; Leal Galicia et al., 2008; Segovia et al., 2006) This study suggests that simple lifestyle changes including environmental enrichment and social interaction can restore age
152 related impairments to levels seen in young rats, even when introduced later in life. Since the beneficial effects o f environmental enrichment may be due to improved electrophysiology (Kumar et al., 2011) increases in vascular volumes (Palmer et al., 2000) or the induced expression of neurogenic factors such as brain derived neurotrophic factor fibroblast growth factor 2, vascular endothelial growth factor, and insulin growth factor 1 (Lee et al., 2002; Obiang et al., 2011; Shetty et al., 2005) future experiments should examine such measures in conjunction with neurogenesis. In Chapter 4 I demonstrated that daily exercise improved spatial /contextual ability, stimulated hippocampal neurogenesis and modulated immune and neuroimmune signaling. While the benefits of exercise on memory and neurogenesis are well documented (van Praag et al., 1999a; van Praag et al., 1999b; van Praag et al., 2005) we are the first to show a comprehensive examination of immune and neuroimmune molecules that are modulated in aged rats by running and their relationship to neuroplasticity and cognition. Future experiments will assess the identified candidate biomarkers for their predictive potential and effects of modulation for intervention. Specifically, I found that a ged rats given access to a running wheel had decreased expression of circul ating GRO KC, MCP 1 and leptin and correlated negatively with learning while leptin was also linked to measures o f memory and neurogenesis. As I found that GRO chronological age, with increased levels found in aged rats with poor cognitive ability in Chapter 3 it is promising to not e that daily exercise may be able to all a y this possible deleterious increase with age. I also identified leptin as a candidate for modulation due the blood brain barrier (Morrison et al., 2009) and influence
153 hippocampal expression of inflammatory cytokines like IL (Chennaoui et al., 2008; Hosoi et al., 2002b) which has been linked to memory (Barrientos et al., 2009; Barrientos et al., 2006; Barrientos et al., 2003; Chennaoui et al ., 2008; Hein et al., 2009) Levels of GRO KC and IL 18 were both elevated in the hippocampi of exercised aged rats and correlated positively with total new neuron number and therefore should be tested for their effects on proliferation, differentiation a nd survival of NPCs in future in vitro experiments. Overall, I showed that physical activity might reverse some aspects of age related cognitive decline, perhaps by stimulating adult hippocampal neurogenesis and by modulating immune and neuroimmune signali ng. This data is truly translational as l ifelong or even later in life, physical and mental exercise may also contribute to preserved cognition in aged humans as well (Christensen and Mackinnon, 1993; Churchill et al., 2002; Erickson et al., 2010; Kramer et al., 2004) Furthermore, this data suggests that modulation of inflammatory biomarkers may also improve cognition through enhanced hippocampal neurogenesis. However, whether cognition is related to neurogenesis in humans is a question that will require the advance ment of biomedical technology that can measure neurogenesis in vivo Applications and Implications Why some elite agers remain sharp with an intact memory while others suffer from cognitive impairments without any identifiable pathology is unknown. This phenomenon is plagued with unanswered questions making it a particularly attractive field for research. Methods for early detection of individuals that will go on to have serious cognitive deterioration are critical for prevention, treatment and understanding the mechanisms of dementia of non pathological origin. The experiments presented in
154 this dissertation further the understanding of the relationship between cognition, neurogenesis and inflammation wh ile also identifying potential biomarkers to diagnose and/or predict age related cognitive decline in rats are particularly innovative. The question, approach, method and analyses I used are unique and allow for this research to be truly translational. De velopment of a Biomarker Assay for the Detection of Age related Cognitive impairments The identification and utilization of biomarkers to diagnose and/or predict a physiological or disease process is rather progressive and attractive concept. Our proposed approach of using molecular concentrations of inflammatory cytokines and stress hormone as biomarkers to assess cognitive impairment with age is particularly an innovative model. Certain aspects of these relationships have been proposed but not integra ted as I did here in the current experiments. Inflammation (Blalock et al., 2003; Chung et al., 2009; Gimeno et al., 2008; Krabbe et al., 2009) as well as stress hormone (He et al ., 2008; Issa et al., 1990; Lupien et al., 1998) have been shown to increase with age while the number of new neurons (Kuhn et al. 1996) and cognitive ability are diminished (Gage et al., 1984b) However, no experiment looks at all these factors collectively across age in rats, until now. While these variables have all b een dependently linked to age I proposed that potential biomarker expression in middle age could predict the cognitive outcome in old age. However, I found that cytokine and stress hormone measurements along with neurogenesis were more appropriately used as diagnostic biomarkers of age related cognitive impairment. Perhaps the most advanced aspect of our methodology is the use of multiplex t echnology.
155 growth in interest in this technology. The Bio Rad Bio Plex system allows for the simultan eous analysis of up to 100 different analytes in a single a 96 well micro plate platfor m. Here I tested 27 different inflammatory cytokines, chemokines, growth factors and stress hormones in blood serum and brain tissue of aging ra ts. However, there is a large assortment of pre made panels to test for other molecules in various species including humans. Furthermore, the prospect of manufacturing a panel of distinctive molecules specific to your research question adds another aspect of novelty to this experiment. For example, our cluster analyses have identified GRO KC, MCP 1, RANT ES, TNF 4 and melatonin in various combinations as biomarkers on future blood serum panels. Now that I have identified potential biomarkers of age related cognitive decline I can create an age related cognitive decline panel to continue our research in rats or cross over form lab bench to bedside and examine the senescent human population. Throughout our experiments I used Bio Plex technology to analyze both circulating and central concentrations of our suspected biomarkers, which allowed us to answe r many questions. Analyte concentrations in the blood serum offered an evaluation of the peripheral status of the rats. In previous experiments in our lab intraperitoneal injection of LPS results in an acute systemic inflammatory response with a cascade of increasing pro inflammatory cytokines such as IL 6 (Asokan, 2010) However, when examining changi ng in basal levels across age I noted a robust but much more subtle elevation in both pro and anti inflammatory cytokines,
156 chemokines, growth factors and s tress hormones. In the brain, I meas ured analyte concentrations from supernatant collected from hippocampal and cortical tissue allowing us to analyze specific regional differences in the brain. Interestingly, while numerous analytes with varying function were elevated in the cortex, the hip pocampus showed a more reserved expression pattern change with age. Perhaps, these regional differences create a pattern of neurodegeneration that starts in the cortex (Hof and Morrison, 2004; Lu et al., 2004; Sowell et al., 2003) and progresses with age. The molecular contrast between blood serum, hippocampus and cortex helped to identify prospective mechanisms for how inflammation and stress are related to mnemonic ability and overall cognition. Since I measured bo th blood serum and bra in levels in the same animals I was also able to assess how the brain and periph ery communicate. For example, I found that aged rats given access to a running wheel had decreased serum levels of the metabolic hormone leptin, which cor related positively with decreased levels of hippoc ampal IL (Hosoi et al., 2002b) as the relationship between Leptin and IL (Morrison, 2009) Interestingly both leptin and IL our memory task. Th us, I have demonstrated how analyte levels in the blood, which are easy to attain, may provide a readout of brain biology, which is additionally linked to cognitive decline. Data Compilation, Pathway Analysis and Machine Learning The approach I used to id entify our biomarkers is also quite innovative, as I utilized behavioral measures to uncover the biology behind age related cognitive decline. In our experiments, I use performance on a rapid acquisition water maze task to categorize middle age and age d ra ts as either memory unimpaired (MU) or memory
157 impaired (MI). I then assess between group differences within each age to identify relationships with neurogenesis and potential biomarker concentration. This inventive approach classifies the rats based on the ir behavior allowing for the assessment of both categorical data (MU vs. MI) and quantitative data (water maze scores) while lessening the effect of variability that is typical for behavior in aged rats (Gage et al., 1984b) Before I could draw conclusions on the relationships between learning and memory, neurogenesis and in flamma tion across age I had to first tackle our large multivariate data sets. Water maze testing with many measures (latency, pathlength and speed) collected for multiple trials across blocks for different tasks and the quantification of neurogenesis (new cell n umber, dentate volume, and the percent of new cells expr essing each of the phenotypes I examined) are notorious for creating larg e sets of data. Additionally, I examined the concentration of 27 different analytes in blood serum along with hippocampal and c ortical tissue. With the collection of all this data, it was imperative to create an efficient technique for analysis, a meticulous means of interpretation and finally an artistic approach to presenting the findings. In addition to standard statistical an alyses and the presentation of our Bio Plex da ta in easy to read heat maps, I created a novel method to identify analyte clusters that are modulated synergistically and multiple regression analyses to predict the middle ag ed or aged rats. I began our correlation cluster analyses by adapting existing pathway analyses (Baron and Kenny, 1986; Erickson and Banks, 2011) to answer the questions: 1) Do analytes that change with age do so in a correlated fashion? 2) Do circulating analyte concentrations directly influence central analyte concentration? And 3) Do analytes that change with age
158 correlate with measures of learning and memory or neurogenesis? In order to answer these questions I p lotted Spearman Rank correlation pairs that remained significant after Bonferroni correction for multiple comparisons and used residual analyses to determine whether relationships between analytes that were already plotted e xisted through mediating pairs. Using this approach I identified relationships between analytes within the same compartment (blood, hippocampus or cortex) or across compartments. Our multiple regression analyses highlighted predictive analytes that based on behavioral characterization a nd analyte concentrations could approximate the biological age rather than chronological age of each rat. This is also a novel approach to look at system biology as I ask the question: can blood serum analyte levels be used to estimate analyte levels in br ain tissue and vice versa. Furthermore, as our data set grows I can apply a machine learning approach to verify our findings. The application of these techniques to this type of data set is completely innovative and allows for the data to be tested for tru e prognostic capability. Chronological age cannot solely be used to assess cognitive decline but rather biological age, a measure of vital factors or biomarkers in relation to ones peers (Karasik et al., 2005) may be critical in assessment of successful aging. The research presented in this dissertation is clinically translational as the findings can be easily applied to a human clinical trial and then to the burgeoning aging population. A biomarker detectable in an easily obtained and non invasive blood serum sample followed over time may help to diagnose and/or predict cognitive decline and even identify patients at higher risk for impairments so they can take preventative measures.
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185 BIOGRAPHICAL SKETCH Rachel Speisman was born and raised in Gainesville, Florida. She received her Bachelor of Science in Agricultural and Biological Engineering from the University of Florida in 2008. Later that year, she joined the J. Crayton Pruitt Family Department of Biomedical Engineering at the University of Florida and began her doctoral research under the guidance of Dr. Brandi K. Ormerod. developing a biomarker assay to predict/diagnose age related cogni tive decline. She aspires to become a leading investigator and professor in the field of neuroregeneration with a focus on immune signaling and neurological disorders