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Investigation of Retinal Pigment Epithelium Regeneration in Wound Healing Mice

Permanent Link: http://ufdc.ufl.edu/UFE0044038/00001

Material Information

Title: Investigation of Retinal Pigment Epithelium Regeneration in Wound Healing Mice
Physical Description: 1 online resource (97 p.)
Language: english
Creator: Xia, Huiming
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: amd -- regeneration -- rpe -- woundhealing
Genetics (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Regenerative medicine holds the promise of restoring cells and tissues that are destroyed in human disease, including degenerative eye disorders. However, development of this approach in the eye has been limited by a lack of animal models that show robust regeneration of ocular tissue. Here, we test whether MRL/MpJ mice, which exhibit enhanced wound healing, can efficiently regenerate the retinal pigment epithelium (RPE) after an injury that mimics the loss of this tissue in age-related macular degeneration. The RPE of MRL/MpJ and control AKR/J mice was injured by retro-orbital injection of sodium iodate at 20 mg/kg body weight, which titration studies indicated was optimal for highlighting strain differences in the response to injury. Five days after sodium iodate injection at this dose, electroretinography of both strains revealed equivalent retinal responses that were significantly reduced compared to untreated mice. At one and two months post-injection, retinal responses were restored in MRL/MpJ but not AKR/J mice. Brightfield and fluorescence microscopy of eyecup cryosections indicated an initial central loss of RPE cells and RPE65 immunostaining in MRL/MpJ and AKR/J mice, with preservation of peripheral RPE. Phalloidin staining of posterior eye wholemounts confirmed this pattern of RPE loss, and revealed a transition region characterized by RPE cell shedding and restructuring in both strains, suggesting a similar initial response to injury. At one month post-injection, central RPE cells, RPE65 immunostaining and phalloidin staining were restored in MRL/MpJ but not AKR/J mice. BrdU incorporation was observed throughout the RPE of MRL/MpJ but not AKR/J mice after one month of administration following sodium iodate treatment, consistent with RPE proliferation. These findings provide evidence for a dramatic regeneration of the RPE after injury in MRL/MpJ mice that supports full recovery of retinal function, which has not been observed previously in mammalian eyes. This model should prove useful for understanding molecular mechanisms that underlie regeneration, and for identifying factors that promote RPE regeneration in age-related macular degeneration and related diseases.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Huiming Xia.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Scott, Edward W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044038:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044038/00001

Material Information

Title: Investigation of Retinal Pigment Epithelium Regeneration in Wound Healing Mice
Physical Description: 1 online resource (97 p.)
Language: english
Creator: Xia, Huiming
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: amd -- regeneration -- rpe -- woundhealing
Genetics (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Regenerative medicine holds the promise of restoring cells and tissues that are destroyed in human disease, including degenerative eye disorders. However, development of this approach in the eye has been limited by a lack of animal models that show robust regeneration of ocular tissue. Here, we test whether MRL/MpJ mice, which exhibit enhanced wound healing, can efficiently regenerate the retinal pigment epithelium (RPE) after an injury that mimics the loss of this tissue in age-related macular degeneration. The RPE of MRL/MpJ and control AKR/J mice was injured by retro-orbital injection of sodium iodate at 20 mg/kg body weight, which titration studies indicated was optimal for highlighting strain differences in the response to injury. Five days after sodium iodate injection at this dose, electroretinography of both strains revealed equivalent retinal responses that were significantly reduced compared to untreated mice. At one and two months post-injection, retinal responses were restored in MRL/MpJ but not AKR/J mice. Brightfield and fluorescence microscopy of eyecup cryosections indicated an initial central loss of RPE cells and RPE65 immunostaining in MRL/MpJ and AKR/J mice, with preservation of peripheral RPE. Phalloidin staining of posterior eye wholemounts confirmed this pattern of RPE loss, and revealed a transition region characterized by RPE cell shedding and restructuring in both strains, suggesting a similar initial response to injury. At one month post-injection, central RPE cells, RPE65 immunostaining and phalloidin staining were restored in MRL/MpJ but not AKR/J mice. BrdU incorporation was observed throughout the RPE of MRL/MpJ but not AKR/J mice after one month of administration following sodium iodate treatment, consistent with RPE proliferation. These findings provide evidence for a dramatic regeneration of the RPE after injury in MRL/MpJ mice that supports full recovery of retinal function, which has not been observed previously in mammalian eyes. This model should prove useful for understanding molecular mechanisms that underlie regeneration, and for identifying factors that promote RPE regeneration in age-related macular degeneration and related diseases.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Huiming Xia.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Scott, Edward W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044038:00001


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1 INVESTIGATION OF RETINAL PIGMENT EPIT HELIUM REGENERATION IN W OUND H EALING MICE By HUIMING XIA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 201 2

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2 20 1 2 Huiming X i a

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3 To my beloved wife Luning Zhuang

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4 ACKNOWLEDGMENTS I would first like to thank my mentor, Dr. Edward Scott, for the excellent training and opportunities I received in his lab. He taught me patiently in all the aspect of both life and science. Due to his mentorship I have gained invaluable knowledge on how to organize my research. I would also like to thank the all the members of my committee, Dr. Alfred Lewin, Dr. Edward Chan and Dr. Maria Grant, for their time, energy, effort and guidance in my research for the past several years. Your invaluable suggestions are critical for my project. I am deeply grateful to my partner Dr. M ark Krebs for his unselfish guidance and help on the project. His rigorousness, passion on science let me realized what qualities a scientific researcher should have. Without his help, my project cannot proceed so confluent ly I would also like to thank Dr Liya Pi for the encouragement, for the discussion and suggestions on my research. In addition, I want to thank Gary Brown for his mouse expertise, computer and network support. I must thank Li Lin and Dustin Hart for all the support on the reagents and f acilities. My deepest thanks also are extended to my fellow graduate students, Seung Bum Kim, Anitha Shenoy and David Lopez. The time I spend in the lab with you I will forever cherish. I would like to thank Hong Li for her help on the plastic sectioning. I would also like to thank the staffs of the core facilities at UF, Marda Jorgensen, Neal Benson and Doug Smith for their support and expertise. Finally, I would like to thank my family members, my father in the heaven, my mother my father and mother i n law and my wife Luning. Your unconditioned love is my motivation and strength.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 10 CHAPTER 1 BACKGROUND AND SIGNIFICANCE ................................................................... 12 AMD and RPE ........................................................................................................ 12 Introduction to AMD .......................................................................................... 12 Pathobiology of AMD ........................................................................................ 12 RPE Structure and Function ............................................................................. 14 Current Strategies for AMD Treatment ................................................................... 15 Antioxidant Therapy ......................................................................................... 15 Suppressing Inflammation ................................................................................ 15 Anti neovascularization .................................................................................... 16 Surgical Approaches ........................................................................................ 16 Regenerative Medicine and RPE Restoration in AMD ............................................ 17 Regenerative Medicine ..................................................................................... 17 Treatment of RPE Loss in AMD ....................................................................... 17 Wound Healing and Regeneration .......................................................................... 19 Wound Healin g ................................................................................................. 19 MRL/MpJ Mice ................................................................................................. 20 p21 Knockout Mice and Wound Healing .......................................................... 21 Stem Cell and Regeneration ................................................................................... 22 The Definition and Function of Stem Cell ......................................................... 22 Wnt Signal Pathway ......................................................................................... 23 Notch Signaling Pathway .................................................................................. 23 Smad Signaling Pathway .................................................................................. 24 PLK1 with Cancer and Cell Cycle ..................................................................... 25 2 MATERIALS AND METHODS ................................................................................ 35 Animals ................................................................................................................... 35 Sodium Iodate Administration ................................................................................. 36 Irradiation and Bone Marrow Transplantation ......................................................... 36 Bone M arrow C ell C ollection ............................................................................ 36 Irradiation and Bone M arrow Cell I njection ....................................................... 37 Electroretinography (ERG) ...................................................................................... 38 M ouse Eye Preparation .......................................................................................... 38

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6 Cardiac Perfusion ............................................................................................. 3 8 Mouse Eye Analysis ......................................................................................... 39 Flatmount Staining and RPE imaging ..................................................................... 40 OCT Sectioning ................................................................................................ 40 Immunohistochemistry ...................................................................................... 41 Plastic Sectioning ............................................................................................. 41 Flatmount ......................................................................................................... 41 Brdu Staining for Proliferation ................................................................................. 42 Microinjections ........................................................................................................ 42 Intra Vitreal Injection ........................................................................................ 42 Subretinal Injection ........................................................................................... 43 FluorescenceActivated Cell Sorting (FACS) .......................................................... 44 Realtime PCR ......................................................................................................... 45 3 SODIUM IODATE DAMAGE TO RPE AND OPTIMIZING DOSAGE FOR REGENERATION ................................................................................................... 48 Validation of R egeneration P henotype by E ar P unch ............................................. 48 Comparision the R egeneration U nder 40 mg/kg Dose ........................................... 49 Optimizing Sodium Iodate Dose ............................................................................. 50 4 ENHANCED RETINAL PIGMENT EPITHELIUM REGENERATION AFTER INJURY IN MRL/MPJ MICE .................................................................................... 56 Time Course of ERG Response in MRL/MpJ Mice after Chemical Ablation of RPE ..................................................................................................................... 56 Loss and Restoration of RPE65 Expression in MRL Mice Posterior Cup after Injury .................................................................................................................... 57 RPE Restructuring and Loss within Days of Sodium Iodate Injury .......................... 58 Enhanced Restoration of RPE Morphology at One Month ...................................... 59 Confirmation of Morphological Changes in Plastic Embedded Sections ................. 60 Cell Proliferation ..................................................................................................... 60 5 INVESTIGATION OF REGENERATION MECHANISM .......................................... 70 Introduction of p21 K.O. Mice as Another Regeneration Strain .............................. 70 Compari son of Gene Expression in the Posterior Cup ............................................ 71 Comparison of Circulating Stem Cell and Bone Marrow Cell Difference ................ 71 Whole Bone Marrow Cross Transplantation ........................................................... 72 6 DISCUSSION AND CONCLUSION ........................................................................ 79 LIST OF REFERENCES ............................................................................................... 86 BIOGRAPHICAL SKETCH ............................................................................................ 97

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7 LIST OF FIGURES Figure page 1 1 Schematic diagram of healthy retina and illustration of layers of the neural retina. ................................................................................................................ 26 1 2 Layers of neural retina. ....................................................................................... 27 1 3 Retina structure under age related macular degeneration .................................. 28 1 4 RPE cell layer as an indispensable part for vision function ............................... 29 1 5 Strategies of AMD treatment. ............................................................................. 30 1 6 Illustration of limb regeneration ........................................................................... 31 1 7 HSC fate decision ............................................................................................... 32 1 8 Notch, Wnt and Smad signaling pathways ......................................................... 33 1 9 PLK1 and cell cycle regression ........................................................................... 34 2 1 Overview of the experiment design. ................................................................... 46 2 2 Schematic diagram showing different routes of ocular delivery .......................... 47 3 1 Confirmation of the MRLGFP Black wound healing phenotype. ........................ 51 3 2 Comparison of ERG signaling between C57/BL6 and MRL/MpJ after 40mg/kg of sodium iodate ................................................................................. 52 3 3 Comparison of ONL thickness between MRL/MpJ and C57/BL6 30 day s P.I. of 40 mg/kg sodium iodate ................................................................................. 53 3 4 Comparison of bwave amplitudes of MRL/MpJ and AKR/J, MRLBL/GFP and C57BL/6 at various dos e ................................................................................... 54 3 5 RPE cells that were considered to differentiated fr o m donor CD133+ cells. ....... 55 4 1 Single and average ERG traces at 5dB intensity. .............................................. 62 4 2 Analysis of ERG signal ....................................................................................... 63 4 3 Immunostaining of RPE65 in cryosections of MRL/MpJ and AKR/J 30 days post injection ....................................................................................................... 64 4 4 Mosaic image of posterior eyecup whole mounts stained with rhodamine phalloidin. ........................................................................................................... 65

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8 4 5 Whole mounts of MRL/MpJ and AKR/J stained with rhodaminephalloidin ........ 66 4 6 Measurement of outer nuclear thickness (ONL) after 20mg/kg sodium iodate injury ................................................................................................................... 67 4 7 Comparison of RPE integrity in plastic embedding sectioning of MRL/MpJ and AKR/J after sodium iodate injury ................................................................. 68 4 8 Observation of cell proliferation by BrdU staining ............................................... 69 5 1 Reat time P CR analysis of gene expression profile difference between different strains in the posterior cup 5 days post injection. ................................. 74 5 2 FACS analysis of peripheral cell difference in MRL/MpJ and AKR/J 5 days post sodium iodate injection. .............................................................................. 75 5 3 ERG analysis of C57BL/6, p21 K.O. and MRL/MpJ mice post sodium iodate injection recovery. ............................................................................................... 76 5 4 ERG analysis of cross bone marrow transplanted chimeric mice before and after sodium iodate treatment ............................................................................. 77 5 5 Flatmount of bone marrow transplanted cohorts stained with FITC phalloidin .. 78 6 1 A schematic diagram that suggests the stem cell, micro enviroment and the interactions that may contribute to RPE regeneration. ....................................... 85

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9 LIST OF ABBREVI ATIONS AMD Age Related Macular Degeneration CDK Cyclin dependent kinase CNV C horoidal neovasculariztion Brdu Bromodeoxyuridine ECM E xtracellular matrix ERG Electroretinography FACS Fluorescence Activated Cell Sorting GFP Green Fluorescent Protein HSC He matopoietic stem cells K.O. Knockout FBS Fetal Bovine Serum TG F Transforming growth factor MMP M atrix metalloproteinase MRL/MpJ Murphy Roths Large derived by the Murphy (Mp) group of the Jackson Laboratory ONL Outer nuclear layer ROS R eactive oxygen species RPE Retinal Pigment Epithelium sFRP2 Secreted frizzledrel ated protein 2 PBS Phosphate Buffered Saline PFA P araformaldehyde P.I. Post Injection PLK1 Polo like kinase 1

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10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirement s for the Degree of Doctor of Philosophy INVESTIGATION OF RET INAL PIGMENT EPITHEL IUM REGENERATION IN W OUND H EALING MICE By Huiming Xia M ay 201 2 Chair: Edward William Scott Major: Medical Sciences Molecular Genetics and Microbiology Regenerative medi cine holds the promise of restoring cells and tissues that are destroyed in human disease. However, development of this approach in the eye has been limited by a lack of mammalian models that show robust regeneration of ocular tissue. Here, the potential of retinal pigment epithelium (RPE) regeneration ability in wound healing strains and possible mechanisms were investigated. W e test whether MRL/MpJ mice, which exhibit enhanced wound healing, can efficiently regenerate the RPE after an injury that mimics t he loss of this tissue in agerelated macular degeneration. The RPE of MRL/MpJ and control AKR/J mice was injured by injection of sodium iodate at 20 mg/kg body weight, which titration studies indicated was optimal for highlighting strain differences in the response to injury. Five days after injection, electroretinography (ERG) of both strains revealed equivalent and reduced retinal responses. At one and two months post injection, retinal responses were restored in MRL/MpJ but not AKR/J mice. Brightfield a nd fluorescence microscopy of eyecup cryosections indicated an initial central loss of RPE cells in MRL/MpJ and AKR/J mice, with preservation of peripheral RPE. Phalloidin staining of posterior eye wholemounts

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11 confirmed this pattern of RPE loss, and reveal ed a transition region characterized by RPE cell shedding and restructuring in both strains, suggesting a similar initial response to injury. At one month post injection, central RPE cells, RPE65 immunostaining and phalloidin staining were restored in MRL/ MpJ but not AKR/J mice. BrdU incorporation was observed throughout the RPE of MRL/MpJ but not AKR/J mice one month following sodium iodate treatment, cons istent with RPE proliferation. These findings provide evidence for a dramatic regeneration of the RPE after injury in MRL/MpJ mice that supports full recovery of retinal function, which has not been observed previously in mamm alian eyes. In order to elude the genetic background uncertainty of MRL/MpJ mice, B6.129S6(Cg) Cdkn1atm1Led/J mice ( p21 K.O.), which has a single gene knockout and has similar wound healing ability, was introduced in later study. To investigate possible underlying regeneration mechanism, cross bone marrow transplantation was conducted among wound healing and nonwound healing strains. After sodium iodate injury, C57BL/6 transplanted with p21 K.O. bone marrow cells displayed regeneration ability similar to the donor, while p21 K.O. host transplanted with C57BL/6 chimera shown limited regeneration ability. This result indicated that the b one marrow derived cells may contribut e more than the microenvironment. Our study should prove useful for understanding molecular mechanisms that underl y regeneration, and for identifying factors that promote RPE regeneration in agerelated macular degeneration and related diseases.

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12 CHAPTER 1 BACKGROUND AND SIGNI FICANCE AMD and RPE Introduction to AMD The Age relatedmacular degeneration (AMD) is an incurable eye dis order. It is the leading cause of the blindness of the western community including Austr alia and a ffect s more than 10 million patients in U.S. alone, causing devastating impact on quality of life in a significant fraction of the elderly population worldwide ( 1 ) The number of affected population is forecasted to reach 14.6 million in 2050 ( 2 ) Study the potential treatment of this disease will benefit dramatically to the community. In general, AMD is the multifactorial disease of aging Many risk factors, including environment al, life habit s, like smoking, and genetic factors will alter an individual s suscepti bility A ging changes in the RPE Bruch membrane choriocapillaris complex are highly associated with AMD. The prevalence of this disease increases significantly with age ( 3 5 ) Pathobiology of AMD AMD is characterized by the degenerative changes in the macula, the central region of the retina bearing the highest concentration of cones and responsible for central vision and visual acuity ( 6 ) Clinically and histological ly, it can be divided into atrophic AMD ( dry AMD ) and exudative AMD ( wet AMD ). Dry AMD is featured by outer RPE atrophy and subjacent choriocapillaris degeneration representing an early form of AMD. Wet AM D is characterized by choroidal neovasculariztion (CNV) followed by subsequent clinical phenotype including hemorrhage, exudative retinal detachment,

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13 disciform scarring, and retinal atrophy ( 7 ) .T h e normal and pathological features of retina are illustrated in Figure 1 1, Figure 12 and Figure 13 ( 8 ) AMD is a progressive disease with exudative AMD a more advanced and more damaging type. The pathological process is initiated by the aging changes in the RPE and choroicapillary which lead to the chronic inflammation Extensive damage to RPE, choroid and the extra damage introduced by infl ammation results in the abnormality of extracellular matrix (ECM) change of composition and permeability of Bruch s membrane The diffusion of product wastes, hormones and nutrients through Bruch s membrane may stimulate the secretion of VEGF by RPE. The change of RPE choriocapillaris behavior will ultimately leading to atrophy of the retina, RPE and CNV ( 7 ) In the process, the accumulation of extracellular debris changes the and lead to the diffusion of product wastes, hormones and nutrients to RPE layer. In response to this metabolic distress, the RPE probably produce VEGF and basic fibroblast growth fact o rs and stimulate CNV. Drusen, the extracellular deposit of protein and lipid that accumulate beneath the RPE, is one of the distinct disease characteristics in AMD. Drusen composition and origin have been analyzed extensively in the hope of searching for the pathogenesis of AMD. It is believed that most of the drusen components are from RPE, neural retina, or choroidal cells but some are from extraocular sources. Many different types of molecules have been identified. Among them are apolipoprotein E, as well as inflammatory mediator s and complement components This findings have led to the suggestion the association of AMD with atherosclerosis

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14 (vitronectin, apolipoproteins B and E, complement and lipid), with inflammation (amyloid P component, C5 and 1 antitrypsin; vitronectin, apolipoprotien E and C5; C5, C5b9 and C3) and with c holesterol metabolism (c holesterol and its transporter, apoE ) ( 7 9 10) In this pathological process of AMD, dysfunction and eventual loss of the RPE is considered as critical st ep in AMD pathology The abnormal changes in the RPE and choriocapillaris will finally lead to the atrophy of the retina, and change of RPE choriocapillaris These pathological changes will progressively lead to the CNV and severe damage to photoreceptor c ells ( 7 ) R PE Structure and F unction RPE layer is a monolayer of cuboidal cells which separates the p hotoreceptors and the choroid forming a part of the blood/retina barrier ( 11) With its specific apical tight junctions and asymmetrical distributed vectorial transport proteins, the RPE nourishes retinal visual cells by taking up nutrients such as glucose, retinol, and fatty acids from the blood and deliver to photoreceptors and by exchang ing ions, water, and metabolic end products with the circulating system RPE cells also participate in vision function by absorbing light energy and working as reisomeriz e enzyme in vision cycle. As a result, the RPE layer is essential for maintaining retinal health and vision function (Fig u re 1 4 ) ( 12) It is commonly believed that the RPE cells are terminally differentiated and will not proliferate or at most at very low rate ( 13) As a result, there is no restorative treatment for AMD, unless replenish source of RPE can be explored and utilized in clinical trials, to replace the damaged or loss of RPE cells in AMD patients.

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15 Current S trategies for AMD T reatme nt So far, m any strategies and methods targeting different pathological procedure have been developed (Fig ure 1 5). Most of them are design to slow the pathological progression or alleviate the symptom As a result, they cannot eventually reverse the pathological damage and recovery of normal physiological function of the retina. Regenerative approach is promising for the recovery of damaged or loss of unrenewable tissues. Antioxidant T herapy The macula is a location with high metabolic activity and RPE i s therefore exposed to high levels of reactive oxygen species (ROS) Polymorphism of genes involved in controlling oxidative stress is found to be one of the risk factors of AMD ( 14) Accumulating evidence indicate that oxidative damage contributes to the pathogenesis of AMD by directly damage RPE, interfere impairs local complement inhibition and accelerate neovascularization ( 1517 ) Age R elated Eye Disease Study (AREDS) results indicate that supplementing of high dose antioxidants plus zinc significantly reduce the risk of advanced AMD and its associated vision loss ( http://www.nei.nih.gov/amd/ ) T his nutrient therapy is highly cost effective ( 18) A ntioxidant gene therapy may also provide an option for the long term protection against damage and degeneration caused by oxidative stress ( 19, 20 ) Suppressing Inflammation P roteomic studies of the composition of drusen, as well as genetic association studies have revealed a connection between complement system and AMD ( 21) POT 4 and eculizumab are two drugs that work to interfere with the complement system and inhibit the activation of downstream cascade. POT 4 is an analogue of the small cyclic

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16 synthetic peptide compstatin. By blocking the amplification of the complement response at central stage complement cascade, POT 4 act to inhibit downstream effector functions ( 22) E culizumab is an anti compl ement protein C5 mono antibody. By specific binding by eculizumab, the complement protein C5 evade the cleavage by C5 convertase and prevent the generation of C5b9 b [ www.clinicaltrials.gov/ct2/show/NCT00935883 ]. Both drugs have been entered into the phase II clinical trials to test the effect on reduction on drusen volume and area of geographic atrophy. Anti neovascularization The more debilit at ing and rapid progressive form of AMD is the exudative form, which is characterized by the choroidal neovascularization (CNV). Since 1970, laser was adopted to ablate CNV in the exudative AMD. Macular Photocoagulation Study reported that l aser photocoagulation of CNV in exudative AMD reduced the risk of severe visual loss ( 23, 24) Administration of ant i angiogenesis agents, such as a nti VEGF drugs is usually utilized cooperatively with the laser surgery, aiming to inactivate v ascul ar endothelial growth factor and inhibit CNV ( 25, 26 ) Two anti VEGF agents, pegaptanib sodium and ranibizumab, have been approved in clinical trials and proved to be effective in preventing vision loss ( 27) Besides, genetic tools are investigated to be used as novel strategies for the silencing of VEGF gene expression ( 2830 ) Surgical Approaches One surgical approach is to remove hemorrhagic choroidal neovascular ization. It is reported that the patient group whose hemorrhage was surgically removed will have decreased chance of losing visual acuity (VA) compared to untreated group ( 31) This approach was later abandoned due to the low efficiency and replaced by the subretinal

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17 in jection of recombinant tissue plasminogen activator and 20% SF6 gas. The possible mechanism is to liquefy and displace the hemorrhage clot. Complete displacement of submacular hemorrhage was achieved in as many as 50% of treated patients ( 32) The second surgical approach is called Macular Translocation Surgery The goal is to lift the macula away from underlying blood vessels and move it to a new, healthier l ocation to restore central vision in patients who are losing the last of their central vision to AMD The macular function after RPE choroid graft transplantation can be maintained up to 7 years with low rate of recurrence ( 33) Regenerative M edicine and RPE R estoration in AMD Regenerative Medicine The motivating concept of regenerative medicine is that physically or functionally damaged cells, tissues, and org ans might be restored in patients with severe injuries or chronic diseases ( 34 ) Inspiring examples include the regeneration of amputated limb (Fig ure 1 6) lesioned spinal cord, lens, jaws and tails in amphibian ( 3538) and truncated heart in zebrafish ( 39) Regeneration is not limited to amphibians, regeneration of blood cells ( 40) antler ( 41) liver ( 42, 43) and digital tip ( 44) are common phenomena in high vertebrate including human. Using regenerative medicine as a therapy can be traced back to 1960 when whole bone marrow transplantation is used to treat leukemia It is working towards treating lots of s tubborn diseases, like hear t disease ( 4547 ) Treatment of RPE Loss in AMD AMD is o ne important disease target due to low efficacy of current treatment strategies and the fact that mammalia n RPE is essentially nonrenewable by itself ( 13 48) Due to the critical functions RPE carrying during v isual activities, without replenish

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18 RPE, no ne of these treatments can fully restore the vision health. As a result, it is critical to develop a therapeutic approach, which can provide replacement source for the damage or loss of RPE in AMD to fully restore the normal vision function. Several regenerative approaches have been investigated, as discussed below. An ex vivo way is to introduce cultured RPE cells deriv ing from embryonic or induced pluripotent stem cells into the subretinal space This approach s how some efficacy but the application is limited by the immune rejection, a limited source of donor cells, and surgical complications as well as the lack of effect on halting disease progression ( 49, 50) A second approach is to deliver bone marrow derived stem cells into the intravitreous or circulating system anticipated to working as a regeneration source for RPE replacement. Intriguing results have been reporting by other lab and ours identifying RPE cells transdifferentiated from bone marrow derived cells in rodent models Shortcoming of this approach is the efficiency which is currently too low to be consid ered as a robust thera py ( 5156) Another untested strategy is to trigger reprogram ming of resident stem cells or undamaged RPE cells to repopulate the damaged t issue, by local introducing regeneration chemically to wound site, as proposed in other organs ( 57) The investigation of organisms with high regenerative capacity like MRL/MpJ mice is a nother promising direction for regenerative medicine T h e rationale is to identify regenerative factors and then make modifications on the damaged locus so that it can recapitulate the regeneration in regenerative orga nisms

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19 Wound H ealing and Regeneration Wound H ealing Tissue repair or wound healing is an essential ability for survival allowing animals to escape danger and recover from injury. There are two kinds of tissue repair categories : s carring and scar free heal ing. Fibrosis and scarring is the normal outcome of tissue repair seen in adult advanced vertebrates including mammals. Wound healing is composed of a series of sequential events. It begins from fibrin clot deposition, platelets aggregation, followed by inflammation, keratinocytes reestablishing, granulation tissue formation, remodeling and scar forming. Fetal wound repair, on the other hand, represents another category of tissue repair which was characterized by absent of scarring and fibrosis and by complete restoration of normal structure and function. This kind of regeneration only exists during the first third of human development ( 58, 59) It will be clinically attractive if non scar wound healing be extended throughout life. Wound healing is a complex process involving many factors. Among them, molecular signaling plays critical roles. The close associati on between T GF family member T GF 1, T GF 2, and T GF 3 expression profile and the pattern of tissue repairing indicates that they are important mediators in wound signaling. It is indicated that low level of T GF 1, T GF 2 and high level of T GF 3 will result in scar free tissue repair, as seen in embryonic wounds healing. Inhibition of T GF 1, T GF 2 ( 60) and exogenous a ddition of T GF 3 ( 61) were able to induce scar free tissue repair. Similarly, TGF family members were also found involved in amphibian limb, tail regeneration ( 62, 63 ) As a result, there see ms to have some strong connection between TGF

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20 family and proliferation and regeneration. Study or modification the expression profile of T GF 1,2, and 3 may provide some clues on investigation of RPE regeneration. MRL/MpJ M ice The ability to regenerate damaged tissue and/or organs is seen in many nonmammalian species. This ability however, disappears in most of advanced vertebrates which end up with scaring as mentioned above. A laboratory inbreeding strain, MRL/MpJ mice is among few examples of adult m ammal regeneration, which has the ability to completely close 2 mm ear punch, a classic way of numbering mice colony. Upon ear punch injury, MRL mice regrow cartilage, skin, and hair follicles, which reminiscent of regeneration in amphibians ( 64 ) The regeneration is not limited to the ear but also observed in other organs as well including heart ( 65) cornea ( 66 ) articular cartilage ( 67) and axon ( 68 ) The MRL mice strain was generated through the interbreeding among LG mouse, the AKR mouse, the C3H mouse and the C57BL/6 mouse. Crossbreeding experiments indicated that the regeneration process is a complex mutagenic process involving 20 loci ( 69) It is also found that the regeneration is sexually dimorphic with female mice heal faster than the male. Studying of regeneration mechanism revealed an upregulation of Ki 67 expression in the early stage of injury and BrdU incorporation suggesting the involvement of cell division or differentiation ( 64) Recent evidence indicate t he MRL mice have superior m esenchymal stem cell resulted from the inhibition of Wnt signaling by sFRP2 ( 70) Biochemistry study data discovered that a number of features of embryonic metabolism were found to be retained in adult MRL mice. Populations of cells that express the markers of embryonic stem cells are also found to be retained. Both of them are rare in mammals ( 71) Proteomic study by using tissue pro filing matrix -

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21 assisted laser desorption ionization (MALDI) mass spectrometry (MS) implicated the involvement of calcium binding proteins calgranulin A and B, calgizzarin, and calmodulin in the wound healing procedure ( 72 ) Another key event during the regeneration is the destruction of the basal membrane, a process associated with matrix metalloproteinase (MMPs) and its inhibitor including TIMP. It is found that upon injury, the level of MMP 2 and MMP 9 was up regulated in the healing situation, indicative of creating a permissive environment for regeneration ( 63) These molecules are mostly expressed by inf lammatory cells and brought to the site soon after wounding ( 64) Suggested by this as well as other evidence, the involvement of immune system in the response to regeneration is now becom e a hot crossover topic. p21 Knockout Mice and W ound H ealing MRL/MpJ m ouse is a hybrid strain of many ancestor strains. The difficulty of study the regeneration process in MRL/MpJ mice is that there so many regeneration loci, it would be impossible to make genetic modification on normal situation to make it regeneration like MRL/MpJ. This problem is bypassed by studying the regeneration of another strain, B6.129S6(Cg) Cdkn1atm1Led/J a strain that contains a mutation in a single gene. In this strain, a p21 ( Cdkn1a), cyclin dependent kinase inhibitor 1A is knocked out, and interestingly, the regeneration is enhanced ( 2 ) The regeneration is p53 independent indicating is it not involved in the p21/p53 pathway. Detailed mechanism still unclear but the single gene mutant regeneration strain is extremely useful to study and in turn to apply the regeneration mechanism in clinical trials in the future ( 69)

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22 Stem Cell and Regeneration The D efinition and F unction of S tem C ell Stem cell is an indispensable component in the regeneration process. It is defined as a cell population capable of both self renew and differentiation into at least one specified cell type. Stem cells are essential for the maintenance of normal function of tissue with high rate of cellular turnover and for the repair of injury in adults. There are two kinds of self renewal pattern: symmetr ic and asymmetric self renewal. In the symmetric self renewal, both daughter cells retain stem cell property while in the asymmetric self renewal, on daughter cell remain stem cell property and the other differentiate. The stem cell fate are tightly regulated by a combination of factors including cytokines, growth factors, transcription factors, chromatin modifiers and cell cycle regulators (Fig ure 1 7) ( 73) Hematopoietic stem cell (HSC) is the most common type of adult stem cel ls responsible for the regeneration of give rise to all the blood cell types It has been used for treatment of leukemia for at least 40 years and recently been reported to be able to differentiate into a variety of specified cell types ( 74) For example, our lab proved that bone marrow derived cells are able to differentiate into retinal pigm ented cells ( 51, 54 ) As a result, the stem cells are considered to be a potential source for the restoration of tissue function and a lot of clinical trials have been taken aiming to develop alternative treatment strategy for refractory disease( 75) However, most of the stem cell treatments are passive in that there is no artificial regulation of the stem cell differentiation to certain direction. Understanding of the signal governing stem cell fate is extremely importance for the utilizing of stem cell as a medical treatment tool. There are mainly three signal transduction pathways that involve in the regulatio n of stem cell differentiation, the Wnt, Notch and Smad.

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23 Wnt S ignal P athway Wnt signaling plays an important role in the regulation of cell proliferation and stem cell differentiation (Fig ure 1 8) caternin is the main actor. In the unstimulated situation, the free caternin is phosphorylated by the and degraded. When Wnt biding to its Fz receptor, complex is inhibited and caternin stab ilized Transcriptional activity of caternin /Tcf thus initiates the expression of series of genes. Wnt signaling may regulate self renewal of hematopoietic stem cells. Activation of wnt signaling pathway by soluble Wnt protein or over expression of ca ternin will promote proliferation and inhibit differentiation resulting in sustained self renewal of HSC. Because of its function on stem cell proliferation and self renewal, Wnt and their signaling mediators are attractive therapeutic agents ( 76 ) It is, however, paradox to f i nd that the inhibition of Wnt signaling by sFRP2 will promote the restoration of the myocardial function by mesenchymal stem cell ( 70) Not ch S ignaling P athway Notch signaling pathway plays pivotal role in the regulation of fundamental cellular process including stem cell maintenance and proliferation in adult and during development (Fig ure 1 8) After ligand binding, the intracellular part is cleaved off and enters the cell nucleus where it binding to RBP J, to activates transcription of genes containing RBP J binding sites In the absent of ligand, the RBP J works as a repressor for the Notch target gene through recruiting of corepressor. W hen the Notch signaling is activated, the released Notch intracellular domain will replace the corepressor and result in the derepression of genes containing RBP J binding site. Consequently, the recruiting of co activator will lead to the expression of Notch targeting genes.

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24 Notch signaling pathway is a critical regulator in the stem cell maintenance. For example, Notch signaling inhibits neural differentiation through repressing the expression of proneural genes Failure to activate Notch signaling wil l result in the formation of more neuronal clusters while constitutive activation of Notch signaling will suppresses neural differentiation in Drosophila In vertebrates, Notch signaling is associated with stem cell maintenance. For example, activation of Notch signal will inhibit differentiation of crypt progenitors while post natal gut specific inactivation of RBP J results in the complete loss of proliferating transient amplifying cells i n the gut ( 77) Smad S ignaling P athway The Smad signaling is implicated in the maintenance of pluripotency and self renewal of stem cells (Fig ure 1 8) The signaling ligands include the TGF family proteins, bone morphogenetic proteins (BMPs) and activins. The signaling cascade begins when the ligands bind to the type II receptor which will recruit and activate by phosphorylate the type I receptor. Type I receptor is a single pass serine/threonine kinase receptors which can then phosphorylates receptor regulated SMADs (R SMADs) R SMADs can bind the coSMAD and the R SMAD/coSMAD complexes accumulate in the nucleus and regulate the transcription of target gene expression ( 78) Smad signaling pathway plays important role in ESC self renewal, maintenance of pluripotency and regulation of differentiation by exert ing multiple and s ometimes opposite effects depending on cellular context It is currently believed t he self renewal promoting activity of animal serum is attributing to BMP2 and BMP4 It is believe the relative quiescence of HSC is due to the strong growth inhibition by TG F However,

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25 lower concentrations of BMP 4 induced proliferation and differentiation of primitive human hematopoietic progenitors. Because of its redundant function of Smad pathway which causes the embryonic lethality of knockout mice, the precise functio n on stem cells remains unclear. However, overall, the Smad signaling plays pivotal role in regulation of stem cell fate ( 73 79) PLK1 with C ancer and C ell C ycle PLK1 is expressed primarily in proliferating cells due to its crucial role in numerous mitotic events (Fig ure 1 9) ( 80) Overexpression of PLK1 is an early event of cancer cells and inhibition of PLK1 activity results in a potent antitumor effects both in vitro and in vivo suggesti ng that PLK1 can be act as oncology targets ( 81) A recent study on zebrafish heart regeneration suggests that PLK1 is an ess ential component of regeneration ( 82) U pon the observation that p21 and PLK1 are involved in regeneration and the fact that both play critical role in cell cycle regression, we hypothesized that cell cycle regulation genes are essential for regeneration and the modification of the expression level to mimic that of wound healing mice strains may reproduce the regeneration in normal mice.

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26 Figure 11. Schematic diagram of healthy retina and illustration of layers of the neural retina. Cone cells are in red, green or blue ( 8 )

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27 Figure 12. Layers of n eural retina illustrated by H.E. staining of cryosection of posterior cup and plastic embedding sectioning stained with toluidene blue.

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28 Figure 13 Retina structure under age related macular degeneration. A) Diagram of the posterior cup subretinal regi on with early sign of AMD. B) Diagram of the posterior cup subretinal region with wet AMD ( 8 )

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29 Figure 14 RPE cell layer as an indispensable part for vision function ( 12)

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30 Figure 15 Strategies of AMD treatment.

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31 Figure 16 Illustration of limb regeneration ( 59 )

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32 Figure 17 HSC fate decision ( 73)

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33 Figure 18 Notch, Wnt and Smad signaling pathways ( 73)

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34 Figure 19 PLK1 and cell cycle regression ( 80)

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35 CHAPTER 2 MATERIALS AND METHOD S In this chapter, the main method utilized for accomplishing this thesis is described. The materials and methods described in this section are optimized over a number of years from multiple previous reports and own observations. All animal procedures were reviewed and approved by the University of Florida Animal Care and Use Committee and performed in an Association for Assessment of Laboratory Animal Care approved facility according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. A general flowchart of the experiment is given in Fig ure 2 1. T h e details of each procedur e are described in following sections. Animals MRL/MpJ and B6.129S6(Cg) Cdkn1atm1Led/J mice were obtained commercially (Jackson Laboratory, Bar Harbor, ME). Two ancestral strains of MRL/MpJ, AKR/J (Jackson Laboratory) and C57BL/6 (Charles River Laboratori es, Wilmington, MA) were chosen as albino and pigmented control strains, respectively. For studies of pigmented animals, MRLBL/GFP, a laboratory derivative of MRL/MpJ mice was used. This strain was constructed by crossing MRL/MpJ and Tg (CAG EGFP)B5Nagy/ J mice (Jackson Laboratory) to obtain black mice expressing green fluorescent protein (GFP), which were then backcrossed against MRL/MpJ for >5 generations with retention of these phenotypes. Ear punch closure analysis confirmed the regeneration phenotype. Mice were raised on cage racks with unrestricted access to food and water under fluorescent lighting with a 12hour light/12hour dark cycle.

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36 Sodium Iodate Administration Sterile s odium iodate (SigmaAldrich, St. Louis, MO) in 1x Dulbeccos phosphate bu ffered saline (PBS Gibco. Carlsbad, California, 14190136) at dose of 20 40mg/kg was injected into adult mice (weighting ~35 grams for MRL/MpJ and MRLBL/GF P ~20 grams for other strains) through retro orbital sinus The sodium iodate stock solution was prepared by dissolving 20 mg of sodium iodate into 2 mL of sterile 1x PBS. The stock solution was then filtered through 0.22 m syringe f ilter ( Fisher Scientific ). This led to a sterile solution at concentration of 1 0mg/ ml. Further seri al dilution by adding appropriate volume of sterile 1x PBS yielded the solutions at the concentration of 7.5 mg/ml and 5 mg/ml. All the mice were weighted and injected proportionally with x l of sodium iodate solution where x=body w eight (in gram)*4. For mice treated with 20mg/kg, 30mg/kg and 40mg/kg dose were injected with the 5 mg/ml, 7.5 mg/ml and 10 mg/ml solutions respectively. Untreated animals were injected w ith a comparable volume of PBS. RPE injury was monitored at 5 days, 30 day s and 60 days period by RPE/sclera flatmounts and retinal cross sections (refer to corresponding sections for more details). For the sodium iodate treatment of mice with whole bone marrow transplantation, sodium iodate was injected 4 days ahead of the irradiation. Irradiation and Bone Marrow Transplantation Bone M arrow C ell C ollection Adult donor mice were euthanized and the long bones in the legs (femurs and tibias) were immediately excised. All muscle, tendon, and ligature were peeled to the end of j oint by hand and cut by the blade discarded. The long bones were immediately immersed in ice cold 1x PBS supplemented with 2% Fetal Bovine Serum (FBS; Gibco) and was cut on each end to expose the marrow cavity trabecular region. The bone

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37 marrow was was flu shed out into a tissue culture treated plate by inserting a 26 gauge needle into one end of the bone and washing ~3 mL of PBS with 2% FBS through the hollow bone core by quickly push the cartridge of 3ml syringe. By p i p e t t ing using a 1 ml p i p e tte and th rough a 18gauge needle, the bone marrow was manipulated into single cell suspension. The cell suspension was then filtered through the BD RoundBottom t ube with cell strainer cap to get rid of debris ( BD 352235). The filtered cell suspension was then pell eted by centrifugation at 1000 x gravity performed at 4 Celsius ( C ) The pellet was then counted and resuspended in 1x PBS with 2% FBS with appropriate volume. More than 10^5 donor cells were injected to each recipient mouse. Irradiation and Bone M arro w Cell I njection The mice received 950 Rads of whole body irradiation. The bone marrow transplantation was conducted by retroorbital injection ( 83 ) Before injection, the mice were anaesthetized br iefly with isofluorane (Baxter; Deerfield, Illinois). T h e anesthetized mice was positioned on its side and restrained with thumb and middle finger of nondominant hand. By gently press ing the surrounding tissue, the eyeball was pushed out and needle was inserted at approximately 45 degree beneath the globe of the eye, pierc ing through the conjunctiva and directly into the retroorbital sinus. No resistance, the feeling of bump up by the nondominant finger, and a little bit blooding is a sign of correct inj ection. A sharp cutting needle is preferred in order to minimize tissue distortion and damage. The volume of injection is up to 200 l. The bone marrow cell suspension (~10^5) was administrated as described. The irradiated and bone marrow transplanted mic e was then watered with 2.5 mL antibiotics supplement Baytril 1 00 ( 100

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38 mg/ml, enrofloxacin, Bayer HealthCare; Shawnee Mission, Kansas) into 500 mL water bottle, for minimum of two weeks Electroretinography (ERG) Mice were dark adapted overnight before analysis. Pupils were dilated with 0.5% proparacaine hydrochloride (Akorn, IL) and 0.5% phenylephrine hydrochloride (Bausch and Lomb, FL). Mice were anesthetized with avertin at 0.5 mg/g body weight The mice should be anesthetized completely (otherwise will generate noise) and placed on a temperaturecontrolled working platform at 37C. Gold rimmed contact electrodes were placed on the corneal surface and visual responses were recorded with a UBA 4204 visual electrodiagnostic ERG system (LKC Technologies, Gaithersburg, MD) using white light stimuli at intensities of 45, 35, 25, 15, 5 and 5 dB with LED light. After the measurement, the mice eyes were applied with vetropolycin and put on the 37 C slide warmer The ERG traces were recorded and expor t ed as CSV files which were used for analysis by using O ffice Excel software. M ouse Eye Preparation Cardiac Perfusion To get better morphology of the eye in histological analysis, the mice were fixed by myocardial perfusion with 4% paraformaldehyde (PFA). Prior to perfusion operation, the mice were anesthetized with an intraperitoneal (I.P.) injection of 240mg/kg avertin (2,2,2 Tribromoethanol, SigmaAldrich). Deep anesthetized mice were s preadeagle on a dissecting board with 20gauge needles. The lower limbs we re spread very far apart whereas the upper limbs should be secured closer to the body of the animal to relieve tension on the thorax 70% alcohol was s pread on the mice body M a d e a shallow cut on the fur of the mice abdomen by using force ps and a scissor The fur on the abdomen

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39 was t o r n apart along the cut in the vertical direction to spinal. Ma d e a cut on the upper part of the fur and using hand to tear the furs along the midline of the body. The abdomen was then opened without cutting on the v isceral or blood vessels. T horacic cavity was then opened up by making side cuts and with an 18guage needle to fix the surrounding tissue to the board. In the hood, by using a switch valve, which has one side installed 3ml syringe of 1x PBS and the other side with 5ml syringe of 4% PFA, perfuse the mice first with PBS and then with 4% PFA. The perfusion was operated as follows: slowly insert the 26 gauge needle into the left ventricle, and mad e a punch on right atrium with another 26 gauge needle, slowly press e d the cartilage to perfuse the 1x PBS through the circulating system. After washing the system with 1x PBS, switch ed the valve and continue the perfusion with 4% PFA. With successful perfusion, we c ould see the leaking of blood from the right atrium and t urning from red to whit of the lungs as well as hardening of the body. M ouse Eye Analysis The perfused mice from last step was blew with air for 30 second to make the eye dry and mark ed at the 12 o'clock point of the eye to make the orientation of the eye in following steps easier. E yes were c arefully e nucleated by diggin g into the eye with sharp blade and with gentle to to preserve the morphology as much as possible. In 1x PBS, extraocular tissue were carefully removed the by using bonn forceps ( World pre cision instruments 555055F T). The processed eye ball s were put the in 4% P FA for 5 minutes and remove d the anterior part (cornea and lens) of the eye by using bonn forceps and w estcott stitch scissors ( Katena products K4 4100). Cutting around lens to dis connect the lens from iris will make the enuleation of lens easier. Mad e a minor single cut on the marked 12 o'clock point With one of the posterior cup, ma d e 56

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40 radical cuts extending to optic nerve to flatmount the eye. Depending on different purpose, either separate the retina from the RPE chroidsclera or leave it as a whole mount ( 84 ) T he second eye was transfer red it to a 24 well plate well with 4% PFA and fi x for overnight. After fixation the eye cup wa s transferred to 20% sucrose in PBS for overnight at 4C and to 30% sucrose in PBS for another overnight incubat ion at 4C and f inally to 30% sucrose supplemented with 20% tissue freezing medium OCT ( Optimal Cu tting Temperature compound, VWR 25608 930) and incubat ed overnight at 4C By using forceps, the eye cups were slowly put into the cryomold ( Tissue Tek Biopsy 6253410) with OCT and oriented under dissecting microscope carefully to prevent bubbles 2 met hyl butane (Fisher 035514) was poured into a container with dry ice. The tissue (in cassette and OCT) was then slowly placed in dry ice/ 2 methylbutane mixture making sure that the exposed OCT never touches the liquid until it is frozen solid to prevent bubbling of OCT. When the tissue in OCT was frozen, the block was marked and placed in 80C freezer. Flatmount S taining and RPE imaging OCT S ectioning The OCT blocks were transported on dry ice to sectioning machine and let it sit for 5 minutes for equilibr ate to cryostat temperature (~ 20 C). After m ark ing the 12 point the block was attached to the holding knob of the sectioning machine with OCT for at least 5 min B lock s were orient ed and serial cut at 14 m thickness and sections collect ed onto the posi tive charged slides ( Superfrost/Plus, Fisher Scientific, Pittsburgh, PA). Every four sections were collected per slides. All the slides were dr ied under room temperature overnight and placed in 80C freezer Sections with optic nerve were selected for fur ther imaging.

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41 Immunohis to chemistry For immunohistochemistry, sections were treated with target retrieval solution (DAKO, Carpinteria, CA), protein blocking buffer (DAKO), and biotin and avidin blocking kit (Vector Laboratories, Burlingame, CA). Sections w ere stained with mice anti RPE65 antibody ( 401.8B11.3D9, NOVUS, Littleton, CO) at 1:150 dilution as primary and donkey anti m ouse IgG AlexaFluor 488 (Invitrogen, Carlsbad, CA) at 1:500 dilution as secondary by using ARK kit (DAKO). After extensive washes in Tris buffered saline, the slides were counterstained and mounted in antifade medium (Vectashield; Vector Laboratories) with 4 6 diamidino2 phenylindole (DAPI). Microscopy was performed with a spinning disk confocal microscope (BX61WI DSU; Olympus, Cen ter Valley, PA). Green and blue channels were acquired with a 20X objective and merged with Volocity (Perkin Elmer) and tiled with the MosaicJ plugin ( 85) in ImageJ (Wayne S. Rasband, U. S. National Institutes of Healt h, Bethesda, Maryland, USA; available at http://rsb.info.nih.gov/ij/download.html ) Plastic Sectioning To check for morphological changes after injury, harvested eyes were fixed overnight in 2% glutar aldehyde and 4% paraformaldehyde in PBS. The eye cups were washed in Cacodylate (pH 7.4) for 3x 10 minutes and then transferred to 1% osmium for 4 hours at 4C and embedded in epoxy resin. Sections were cut at 1 m thickness and stained with toluidine blue ( 86) Flatmount For whole mounts, left posterior eyecups were stained at room temperature with DAPI and rhodamine phalloidin (Invitrogen, Carlsbad, CA) in PBS contai ning 1% Triton X Eyecups were cut radially, mounted with Vectashield and imaged

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42 with a spinning disk confocal microscope (IX81DSU or BX61WI DSU; Olympus, Center Valley, PA) with acquisition software (Slidebook, Olympus; or Volocity, res pectively). Brdu S taining for P roliferation Bromodeoxyuridine ( BrdU Sigma Aldrich, St. Louis, MO ) incorporation was used to observe cell proliferation after injury Prior to Brdu injection, the mice ( MRL/MpJ and AKR/J mice ), w ere treated with sodium iodat e at the dose of 20mg/kg body weight The 100 mg Brdu powder was dissolved in 10ml 1x PBS to make it a 10mg/ml solution. The sodium iodate treated mice were administered daily with 5 fold body weight (g) volume ( ) of the Brdu solution by intraperitoneal injection to the dose of 50 mg/kg body weight. Eyes were harvested 30 days post sodium iodate injection. Eye cups were embedded in OCT. The OCT blocks were sectioned, antigen retrieved with 0.1% Trypsin for 7 mi nutes at 37 C and stain ed with the BrdU staining Kit (Invitrogen, Carlsbad, CA 93 3943). Pictures were taken under bright field. By creating and subtracting a blank background will improve the quality of the image. Microinjections There are v arious ad ministration options for the delivery of therapeutic reagents to the eye. Fig ure 2 2 gives an illustration of mice eye and the injection delivery sites. Injection of compound precisely and with minimum of trauma is desired to maximize treatment effect. Int ra V itreal I njection I ntravitreal administration of drugs is a popular method used to treat many retinal diseases, including AMD. Prior injection, sterile injection reagents should be prepared and placed on ice. Hamilton 901 N 10 L Syringe was washed firs t with water and later with 70% alcohol and dry in the hood. After anesthetize animals with avertin, the

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43 treatment reagents (stem cells, drugs et al.) was drawn into tuberculin syringe from a sterile bottle with t hirty two gauge needles (blunt). Gonak (Ako rn, IL) was applied to prevent hemorrhage during the injection. At the site of the infero temporal quadrant a shallow punch was made with the Insulin syringe 28G1/2 The blunt needle of Hamilton 901 N 10uL Syringe was slowly inserted through the punch int o the intravitreal space. The cartilage of the syringe is pushed by assistant slowly to avoid jet for mation or Using a single smooth continuous maneuver, the treatment agent was injected into the eye. Gently and slowly remove the needle from the eye. Multiantimicrobial drug was applied to avoid infection and placed o n the slide warmer for better recovery. Sometimes the f luorescein isothiocyanate (FITC, Sigma Aldrich, St. Louis, MO ) was used as an indicator to track the treatment drug. A successful injection will lighten up the eye with minor fluorescein flow out. The maximum volume of intra v itreal injection is up to 2 l for adult mice ( 59 ) Subretinal I njection The subretinal space is an excellent target site for RPE loss treatment. It is commonly used clinically and has been used in many animal models including mice. Like intravitreal injection, sterile injection reagents should be prepared and placed on ice Hamilton 901 N 10uL Syringe w as washed first with water and later with 70% alcohol and dry in the hood before injection. Prior anesthetizing, the mice pupil was dilated with first with atrophine sulfate (Bausch&Lomb) twice every half hour and then p henylephrine (Bausch& Lomb ) once Under deep anesthetization, the mouse was positioned with its nose pointing away from the surgeon. A drop of gonak was applied to prevent hemorrhage By gently tighten ing the surrounding tissue, the eye was pushed

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44 little bit out. A sharp 26 gauge needle was used as a lance to make a punch on the cornea. The thirty two gauge blunt needle was advanced to the retina opposite to the puncture site until it was but a few tip diameters away from the optic nerve head. The view of needle tip was magn ified by the lens By a gentle pressure was applied to penetrate the neural retina into the subretinal space. Too much pressure will penetrate or damaged the RPE sheet. Appropriate extent of press can only be judged according the experience and feeling. By slowly press the cartilage of the syringe, the treatment agent was slowly delivered to the subretinal space. Slowly pull out the needle and apply vetropolycin (Nada # 065016) T h e maximum injection volume of subretinal injection is up to 2 l ( 73) Fluorescence A ctivated C ell S orting (FACS) Peripheral blood was collected th r ough the saphenous vein of the cheek using a 5.0 mm GoldenRod animal lancet (MEDIpoint, Inc. Mineola, New York) by dropping 58 blood drops in to a 5mL f alcon tube (Fisher Scientific) containing 0.5 mL of 1X PBS and 5 mM EDTA which act as an anticoagulant. P eripheral blood mononuclear cells (PBMC) were collected apart from t he erythrocytes and granulocytes by using FicollPaque PLUS (Amersham Biosciences. Uppsala, Sweden) purification. 1.5mL of Ficoll Paque PLUS was conducted to the bottom of the blood/PBS mix ture by using a Pasteur pipette This solution was centrifuged at 1000rpm at 4 C for 40 minutes. PBMC will be located in the cloudy layer of the stratified blood sample. By using Pasteur pipette, the PBMC were collected into new 5mL falcon tubes where the cells were washed in 5x volumes of 1x PBS and then blocked in 10% normal rat serum for 30 minutes at 4C Antibodies ( ies to CD3e (T cells), CD11b (macrophages), B220 (B cells), CD117, Scar1 and IgG control antibodies ( for exclude

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45 nonspecific binding) conjugated to FITC (BD Pharmingen) were used to label collected PBMC cell. After staining, the cells suspensions were wa shed in 5x volumes of 1x PBS and resuspend in the left over solution. FACS was performed by using FACScaliber (BD Biosciences). Realtime Reverse transcription PCR Mice tis sue was collected and put in RNAlater (Qiagen, 76104) and homogenized with the molar. RNeasy mini kit (Qiagen, 74104) was used to extract RNA. cDNA was synthesized by using SuperScript TM III FirstStrand Synthesis System (Invitrogen, 18080051 ). Realtime PCR primers are obtained from the following website: http://pga.mgh.harvard.edu/primerbank/ http://mouseprimerdepot.nci.nih.gov/ QuantiTect SYBR Green PCR Kit (Qiagen, 204141) was used to compare the mRNA expression level of different genes. Betaactin mRNA expression level was used as an internal standard. Each sample w as triplicated in the assay carrying out in the 96 well plate.

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46 Figure 21 Overview of the experiment design.

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47 Fig ure 22 Schematic diagram showing different routes of ocular delivery, including intravitreal and subretinaled after cell division ( 87)

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48 CHAPTER 3 SODIUM IODATE DAMAGE TO RPE AND OPTIMIZING DOSAGE FOR REGENERATION Sodium iodate has been used for several decades as a specific tox in of RPE. Though t he complete mechanism is not well understood, sodium iodate has been shown to inhibit lysosomal enzyme activities within RPE cells, especially acid phosphatase activity, which is essential for RPE cell survival and normal function ( 88) The area and the extent of damage is sodium iodate dose dependent. Low dose (15, 20mg/kg) of sodium iodate cause specific damage on RPE and around the central retina, while high dose of sodium iodate cause a more dramatic damage affecting a vaster area including the peripheral region and damage not only RPE but also photorecept or cells ( 60, 61) To develop a model for RPE regeneration, we sought to induce damage and compare functional and morphological recovery of the RPE in MRL/MpJ with other strains. An immediate issue was the choice of control strains. MRL/MpJ is an inbred albino strain initially generated from a series of crosses among several laboratory strains, making congenic comp arison impossible. Albino strains have also been reported to be more sensitive to sodium iodate induced RPE damage ( 89 ) We chose control strains with normal woundhealing capability, including C57BL/6 and AKR/J, which were used as stock in the generation of MRL/MpJ mice. Furthermore, AKR/J serves as an albino control for the pigmented C57BL/6 strain. We also have crossed the MRL healer phenotype onto a pigmented GFP positive background to generate MRLBLGFP as a pigmented counterpart of albino MRL/MpJ for our study. Validation of R egeneration P henotype by E ar P unch T o begin with we have to validate that the strains we were using doe s have the regeneration ability, especially MRLBL GFP for the reason that it has been cross

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49 breeded with nonregeneration strain. Ear punch wa s adopted since it is simple, straightforward and visible. Adult C57BL/6, MRL/MpJ and MRLBL GFP at the age of approximately 45 weeks old were throughand through ear punched, the closure of the ear punch was photographed at week 1, 2, 3 and 5 (Fig ure 3 1). We found that the ear hole was recovery and almost closed on the third week after the punch both on MRL/MpJ and MRLBL GFP but not in C57BL/ 6 indicating that MRL/MpJ and MRLBL GFP have enhanced wound healing ability Comparis on the R egeneration U nder 40 mg/kg Dose To avoid possible secondary effects that might hinder regeneration ( 51) our first objective was to identify conditions in which recovery of injured RPE in MRL/MpJ mice could be easily distinguished from that of control mice. In many mouse studies of RPE injury ( 51, 54) sodium iodate was used at a dose of 40 mg/kg body weight or higher. This dose resulted in substantial RPE damage with minimal r ecovery in preliminary experiments. In our initial trials, we used the 40mg/kg body weight to compare the regeneration between the C57BL/6 and MRL/MpJ, the wound healing strain. The regeneration was observed to be more robust in MRL/MpJ than in C57BL/6 by ERG analysis, by morphology and by the outer nuclear (ONL) thickness measurement (Fig ure 3 2 Fig ure 3 3 ). Moreover, intravitreal injection of CD133+ cells were conducted at the dosage of 40mg/kg with the same protocol as described previously ( 54) .Though donor derived cells were observed to differentiate into RPE like cells (Fig ure 3 5), the occurrence and efficiency is not stable. In all, the regeneration under 40 mg/kg sodium iodate treatment wa s not stable and robust as we expected, p ossibly due to the reason that the regeneration environment was destroyed at 40 mg/kg dose.

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50 The regeneration observed in MRL/MpJ mice under this dose was far from being significant for future investigation and for clinical trials. Optimizing S odium I odate D ose We hypothesized that highdose sodium iodate may cause excessive damage to the tissue microenvironment, leading to the loss or alteration of repair signals and environmental guidance cues that are required for regeneration. Massive damage would most likely lead to scar formation regardless of strain background. Four mice strains ( C57BL/6 vs MRLBL/GFP AKR/J vs MRL/MpJ) were injected retroorbitally with sodium iodate at various doses (20, 30 and 40 mg/kg body weight) and examined one month later by ERG. One month is a time point when a plateau stage has been reached in C57BL/6 mice injected with low dose of sodium iodate ( 60) Significantly greater bwave amplitudes were observed in MRL/MpJ and MRLBL/GFP mice compared with all other strains tested at all sodium iodate doses The differences in ERG amplitudes between MRL/MpJ, MRLBL/GFP and control cohorts were greatest at a dose of 20 mg/kg body weight. The 20 mg/kg dose also resulted in a near flat line ERG response in the control strain that was not significantly different from the damage observed with higher doses of sodium iodate. Moreover, regardless of pigment phenotype, MRL strains showed a more robust recovery of the ERG signal than control strains (Fig ure 3 4 ), suggesting that the previously reported influence of pigment on sodium iodate susceptibility ( 89) does not apply to recovery. In all, s odium iodate will cause selective toxic effect on RPE cell layer. MRL/MpJ mice were able to recover f ro m sodium i odate damage at low rate. High dose of sodium iodate can cause detrimental effect on regeneration. 20 mg/kg is a dose where the recovery difference can be amplified. Based on these results, we chose a sodium iodate dose of 20 mg/kg for further studies.

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51 Figure 31 Confirmation of the MRL GFP Black wound healing phenotype. Bothe MRL/MpJ and MRLGFP Black mice were able to completely close the ear punch.

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52 Figure 32 Comparison of ERG signaling be tween C57/BL6 and MRL/MpJ after 40mg/kg of sodium iodate (Note: y axis change) There is difference on the response to the sodium iodate injury. MRL/MpJ strain was able to recovery at low rate after sodium iodate damage.

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53 A B C D 0 10 20 30 40 40 0 40 0ONL thickness (pixels) C57BL/6 NaIO3 (mg/kg) MRL/MpJ Figure 33 Comparison of ONL thickness between MRL/MpJ and C57/ BL6 30 days post 40 mg/kg sodium iodate injection. A ) C57/BL6 treated at 40mg/kg sodium iodate. B ) MRL/MpJ treated at 40mg/kg sodium iodate C ) C57BL / 6 treated with PBS D ) MRL/MpJ treated with PBS. E) Comparison of ONL thickness between the two strains aft er treatment (a sterisk indicate P value less than 0.05 n 3 for each group) E

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54 Figure 34 Comparison of bwave amplitudes of MRL/MpJ and AKR/J, MRLBL/GFP and C57BL/6, at a dosage of 20, 30 and 40 mg sodium iodate/kg body weight 30 days post injection. 3 for each group. Significant level at P<0.05 is indicated by asterisks.

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55 Figure 35 RPE cells that were considered to differentiated f r o m donor CD133+ cells. Pigmented, RPE like cells (arrow) were observed on the flatmount of MRL/MpJ mice intravitrea l injected CD133+ from MRL GFP Black.

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56 CHAPTER 4 ENHANCED RETINAL PIGMENT EPITHELIUM REGENERATION AFTER INJURY IN MRL/MPJ MICE It is not a simple task to prove regeneration The one month single time point ERG difference after treatment is not sufficient to prove that MRL/MpJ mice have the ability to regenerate RPE regeneration. The difference can be explained as the resistance or due to the less damage at the first beginning. The difference will not be convinc ing until we provide evidence indicate that the RPE cells were damaged and lost after sodium iodate treatment, and after a period of regeneration, restored both structurally and functionally Time C ourse of ERG R esponse in MRL/MpJ M ice after C hemical A blation of RPE As established in the previous chapter MRL/MpJ showed higher ERG responses than control strains at post injection day 30. To follow the kinetics of the recovery process and to exclude the possibility that MRL/MpJ mice are more resistant to the initial injury, we monitored the ERG r esponse in MRL/MpJ and AKR/J mice as a function of time after sodium iodate injection. Prior to treatment, both strains exhibited typical scotopic ERG responses characterized by aand bwaves with superimposed oscillatory potentials (Fig ure 4 1 ). At early times after injection, both strains showed attenuated responses. At later times, the ERG response of MRL/MpJ mice was substantially restored, while that of AKR/J remained low (Fig ure 4 1 ). To account for strain differences in the pretreatment ERG response, we performed the analysis with ERG amplitudes normalized to the pretreatment values for the bwave at 5 dB flash intensity. This analysis revealed nearly identical initial damage in both strains followed by recovery in MRL/MpJ but not AKR/J mice (Fig ure 4 2 A ) Similar damage and recovery trends were observed with intensity response data collected at flash intensities

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57 from 45 dB to +5 dB (Figure 4 2 B ). Statistical analysis of the 15 dB, 5 dB and +5 dB b wave amplitudes at 5, 30 and 60 days post injec tion with a mixed model statistical analysis indicated no significant difference in initial damage between the two strains (P=0.1214). Analysis at these stimulus intensities also indicated significant recovery in MRL/MpJ mice at 30 and 60 days compared to 5 days post injection (P<0.0001 and <0.0001, respectively but not in AKR/J mice (P=0.4910 and 0.6258, respectively). These results suggest ed that MRL/MpJ and AKR/J mice are equally susceptible to sodium iodate injury, but only MRL/MpJ mice recover signi ficantly. Thus, the increased ERG response in MRL/MpJ mice after sodium iodate treatment is not due to a greater resistance to the initial injury but rather to enhanced regeneration. Loss and R estoration of RPE65 E xpression in MRL M ice P osterior C up after I njury We conducted anti RPE65 immunostaining to correlate the observed functional recovery with structural changes to the RPE in cryosections of posterior eyecups before and after sodium iodate injury. Prior to injury, immunohistochemical analysis with antibody against RPE65, an RPE specific protein ( 90) revealed a thin, positively stained band at the expected location between the neurosensory retina and the choroid (Fig ure 4 3 A and D ). At higher magnification, polygonal RPE65positive cells with large central nuclei characteristic of the RPE were clearly identifiable (Fig ure 4 3 G L ). RPE65 staining was dramatically reduced in both strains at 5 days post inje ction (Fig ure 4 3 B and E ). Strong central RPE65 staining reappeared at 30 days post injection in MRL/MpJ but not AKR/J mice (Fig ure 4 3 C and E ), consistent with the recovery of ERG responses (Fig ure 4 1 and Fig ure 4 2 ). The decrease in RPE65 staining f ollowing injury may be due to a reduction in the cellular expression of RPE65, a loss of RPE cells or

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58 both. Measure and comparison of the ONL thickness of the stained cryosections of the eyes revealed a gradual thinning of ONL in the AKR/J strain but not obvious in MRL/MpJ mice (Fig ure 4 6). This may due to apoptosis of photoreceptor cells due to the lack of nourishing and supporting from the RPE cells. These results suggest that RPE cellular integrity was similarly disrupted by sodium iodate in both strains, but recovered more effectively in MRL/MpJ mice. RPE R estructuring and L oss within D ays of S odium I odate I njury To further confirm the loss of RPE cellular integrity, we analyzed posterior eyecup preparations stained with rhodamine phalloidin, which detects filamentous actin (F actin) on the apical border of RPE cells. Since conventional RPE/choroid/sclera flat mounts are subject to potential artifacts that arise when the adherent retina is removed, we examined eyecup whole mounts, in which the retina is retained. At two days following sodium iodate injection, both strains showed a similar extent of damage (Fig ure 4 4 A and B ). Polygonal RPE cells were detected in the eyecup periphery, but were absent from the center, consistent with RPE cell atrophy an d loss. The transition zone between healthy and atrophic tissue was characterized in both strains by irregularly shaped RPE cells whose border curvature and area increased towards the eyecup center (Fig ure 4 4 A and B ). Visualization of the RPE in whole m ount image stacks was challenging because of the abundance of F actin and unevenness of tissue. Maximum merge z projection of image stacks encompassing the RPE layer was typically uninterpretable (Fig ure 4 4 C ). We therefore developed a flattening macr o in ImageJ to extract the phalloidinstained RPE cell borders (Fig ure 4 4 D ) and generate an image containing the RPE apical surface in a single plane (Fig ure 4 4 E ). These images clearly revealed structures

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59 reminiscent of RPE cellular shedding events wi thin the transition zone of sodium iodateinjured mice in both AKR/J and MRL/MpJ (Fig ure 4 4 E asterisks). Taken together, these results establish that sodium iodate treatment at 20 mg/kg body weight produces a gradient of RPE loss and cellular tissue restructuring that is similar in both strains. Enhanced R estoration of RPE M orphology at O ne M onth In order to better characterize the loss and regeneration of RPE in the MRL and control animals we examined phalloidinstained posterior eyecup whole mounts at one month post injection (Fig ure 4 5 ). Mosaic images of AKR/J and MRL/MpJ mice were generated starting at the superior, periphery and ending at the optic nerve head. AKR/J mice at one month post injection retained extensive areas toward the optic nerve head that were completely devoid of polygonal RPE cells as detected by phalloidin staining (Fig ure 4 5 A and B ). By comparison, and in contrast to the appearance at two days post injection, phalloidin staining of MRL/MpJ eyecups at one month post injection showed the polygonal RPE cells throughout the posterior eye (Fig ure 4 5 C and D ) a striking improvement to the disrupted morphology observed at two days post injection. The large, irregularly shaped cells observed at early times after injury (Fig ure 4 4 A ) were mostly absent. Polygonal cells were observed mainly in the periphery of AKR/J eyes, which might contribute to the small amount of ERG recovery in the AKR/J strain (Fig ure 3 4 Fig ure 4 1, Fig ure 4 2 ) Phalloidin stained F actin structures reminiscent of those in fibroblasts were observed at the transition between the normal and atrophic RPE, suggestive of scar formation. These results demonstrate that the RPE region damaged by sodium iodate can be efficiently regenerated in MRL/MpJ but not AKR/J mi ce.

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60 Confirmation of Morphological C hanges in P lastic E mbedded S ections To confirm the post injury morphological changes, ul tra thin plastic embedded sections were prepared at day 0, 5 and 30 post injection. The images revealed severe central RPE damag e at 5 days post injection in both MRL/MpJ and AKR/J mice (Fig ure 4 7 arrowheads) At 30 days post injection, the typical structure of the RPE layer w as restored in MRL/MpJ mice but not in AKR/J mice (Fig ure 4 7 arrows ). At 30 days, the outer nuclear lay er and photoreceptor inner and outer segment were largely preserved in MRL/MpJ mice consistent with a restoration of RPE function. Th ese result s further confirm ed RPE65 and phalloidin staining result suggesting that the MRL/MpJ mice were able to regenerate RPE efficiently after sodium iodate injury. Cell P roliferation To test if cell proliferation contributes to the observed RPE regeneration, BrdU w as injected daily following sodium iodate treatment. At 30 days post injection, many BrdU positive cells were observed in the subretinal space of sodium iodate treated MRL/MpJ mice (Fig ure 4 8 D and H ) but only sporadically in AKR/J mice (Fig ure 4 8 B and F ). Some of these cells have large nuclei and located in a monolayer above Bruch's membrane suggesting they are RPE cells. BrdU incorporation w as not observed in uninjured mice (Fig ure 4 8 A, B E and F ) indicating t hat incorporation reflects a post injury proliferation. These results suggest that after injury, there are proliferating cells in the subretinal space in MRL/MpJ mice but not AKR/J mice that may contribute to the regeneration of the RPE. In this chapter, we investigated RPE recovery in MRL/MpJ mice after sodium iodate treatment. Electroretinography and BrdU labeling and histology was performed to as sess RPE damage and restoration in MRL/MpJ and control strains. We found that

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61 MRL/MpJ mice show more robust structural and functional regeneration than control mice after injury with a full recovery of ERG responsiveness. Our result provides the first demonstration of enhanced RPE regeneration in rodents and provides a significant new tool for future studies of RPE regenerative therapy.

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62 Figure 41 Single and average ERG traces at 5dB intensity. ERG traces in MRL/MpJ and AKR/J mice at an intensity of 5 dB were recorded at 0, 2, 5, 30 and 60 days after sodium iodate injection. Thick line : mean ERG response. Thin line:individual traces. n 3 for each group.

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63 Figure 4 2 Analysis of ERG a and bwave amplitude under different flash intensity A) Respective time based aand bwave amplitude and percentage analysis at 5 db B ) F lash dose dependent response of AKR/J and MRL/MpJ at different

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64 Figure 43 Immunostaining of RPE65 in cryosections of MRL/MpJ and AKR/J 30 days post injection. A C) Fluorescence micrographs of posterior cup cyosections of AKR/J D F) Fluorescence micrographs of posterior cup cyosections of MRL/MpJ. G L) High magnification view of A F. Scale bar A F G L: nerve head. Green: AlexaFluor 488; Blue: 4 6 diamidino2 phenylindole (DAPI).

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65 Figure 4 4 Mosaic image of posterior eyecup whole mounts stained with rhodamine phalloidin A) AKR/J B) MRL/MpJ C E ) The transitional zone examined at higher magnification A sterisks, RPE apical surface and possible shedding events within the transitional zone. ONH: optic nerve head.

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66 Figure 4 5 W hole mounts of MRL/MpJ and AKR/J stained with rhodaminephalloidin. A B) Superior and central whole mounts of AKR/J. C D) Superior and central whole mounts of MRL/MpJ E H) Higher magnitude view of box area in A D. ONH: optic nerve head. Scale bars: A

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67 Figure 46 Measurement of outer nuclear thickness (ONL) after 20mg/kg sodium iodate injury. The thickness of ONL in the immunostained cryosections of posterior cup was measured and compared at different time point after 20 mg/kg sodium iodate injection. ANOVA analysis and s imultaneous comparison between the tissues collected different time point and different strains indicate the ONL become thinner sug gesting some degeneration process taking place.

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68 Figure 4 7 Comparison of RPE integrity in plastic embedding sectioning of MRL/MpJ and AKR/J after sodium iodate injury A) B right field images of plastic embedded eyecups B) Higher magnitude view of p anel A Scale bars: upper pa ONH: optic nerve head. Arrows: healthy RPE layer. Arrow heads: damaged RPE layer. B A

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69 Figure 4 8 Observation of cell proliferation by BrdU staining. A) Bright field pictures of eye sections from AKR/J mice treated with PBS B) Bright field pictures of eye sections from AKR/J mice treated with 20 mg/kg sodium iodate. C) Bright field pictures of eye sections from MR L/MpJ mice treated with PBS. D) Bright field pictures of eye sections from MRL/MpJ mi ce treated with 20 mg/kg. E H) Higher magnitude picture of the boxed area of panel A D. BrdU positive cells (arrows), Scale bars: A

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70 CHAPTER 5 INVESTIGATION OF REGENERATION MECHANISM We have established the RPE regeneration ability in the MRL/MpJ mice in the pre vious chapter. In this chapter, possible regeneration mechanisms were investigated. Modif ications by whole bone marrow transplantation were conducted aiming to reproduce the regeneration in the normal genetic background. Introduction of p21 K.O. M ice as A nother R egeneration S train Due to the genetic complex of the natur al mutant strain MRL/MpJ investigation of the regeneration mechanism will be continued in this chapter with the single gene knockout strain B6.129S6(Cg) Cdkn1atm1Led/J ( p21 K.O). Compared to the MRL/MpJ mice, the p21 K.O. strain has at least three advantages: 1) it is a single gene knock out strain of B6 mice so the genetic background is well documented; 2) unlike MRL/MpJ, the p21 K.O. strain will not develop any autoimmune disease; 3) it has k halplotype, which is the same as C57BL/6 so that the bone marrow transplantation is compatible with C57BL/6 and most of the other strains. The first question we have to ask is does p21 K.O. mouse able to regenerate tissues other than the ear and heart, especially RPE ? Due to the fact that the regeneration is highly tissuespecific, we hav e to reexamine the RPE regeneration ability of p21 K.O. mice. We treat the C57BL/6, MRL/MpJ and p21 K.O. mice with 20mg/kg sodium iodate. We found that the p21 K.O. mice can restore the ERG response more like a MRL/MpJ mice and clearly different from its parent strain C57BL/6 (Fig ure 5 1).

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71 Comparison of G ene E xpression in the P osterior C up The fundamental question in this chapter is to ask what are the factors that may contribute to the RPE regeneration. Generally speaking, the factors can be classified as local environmental factors and circulating stem cells We want to compare the differences in each aspect. To compare the local gene expression difference, we examined the gene expression profile in the posterior cups of different strains at five d ays post injection, a time point we believe the regeneration start. By the reported candidate genes involved in the regeneration of other tissues in MRL/MpJ mice, Axolotl and zebrafish ( 63, 82 91) we choose MM P 2, MMP 9, TGF 1, TGF 2, TGF 3 and Plk 1 as candidates. Five days post sodium iodate injectio n, the posterior eye cups were collected, homogenized and RNA extracted. The cDNA were synthesized by reverse transcriptase. Real time reverse transcript PCR was conducted in triplicate with the primers recognizing murine MM 2, MMP 9, TGF 1, TGF 2, TGF 3 and Plk 1 cDNAs with beta actin as an internal standard. T h e expression levels were compared as shown in Fig ure 5 2. We find that the relative expression level of TGF 3, MMP 2, MMP 9 and Plk 1 were significantly higher in MRL/MpJ mice after sodium iodat e injection. This result may indicate that the reduced scarring (TGF 3,), increased permeability and enhanced cell proliferation that contribute to the regeneration Comparison of C irculating S tem C ell and B one M arrow C ell D ifference Accumulating evidence suggested that the bone marrow derived cells can contribute to the replenishing of RPE cell loss ( 51, 92 ) As a result, we ask ed is there any difference with the circulating hematopoietic stem cells or the bone marrow cells

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72 between the regeneration strains and nonregeneration strains. The hypothesis is that a more immature bone marrow or circulating stem cell may be account for the enhanced regeneration ability, immature in terms of there are more stem/progenitor cells within the circulating system and the bone marrow. By using FACS system, we check ed the percentage of stem cell/lineage marker positive cells. W e found that in MRL/MpJ mice, the fraction of CD117+ cells, one of stem cell markers, were larger when compared to the control group (Fig ure 5 2 ). This discovery is consistent with the fact that p opulations of cells express ing the embryonic stem cells mark ers of are retained as well as number of embryonic metabolism features ( 71) Whole B one M arrow C ross T ransplantation As from the previous experiment, it is sugges ted that that circulating/bone marrow cells were different between the regenerative and nonregenerative strains. It will be not so convincing until we can provide the evidence that the change of the circulating/bone marrow cells will also change the regenerative ability. To achieve that, after sodium iodate treatment, p21 K.O. and C57BL/6 mice were irradiated and under whole bone marrow transplantation with bone marrow cells from either the p21 K.O. or the C57BL/6 mice forming four cohorts of chimeric mice. After one month period of recovery, the ERG response of each cohort was measured and compared (Fig ure 5 4). We find that the ERG response was higher in the cohorts transplanted with the p21 K.O. bone marrow cells than those transplanted with C57BL/6 bone marrow cells. Moreover, p21 K.O. host transplanted with C57BL/6 donor showed no significant advantage over the cohorts with C57BL/6 host and C57BL/6 donor. However, cohorts with p21 K.O. host transplanted with p21 K.O. donor did show better regeneration a bility over the cohorts with C57BL/6 host and p21 K.O. donor indicating the contribution by the host

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73 background. Analysis of the flatmount of posterior cup of the chimera mice (host/donor) also yield similar conclusion: ( p21 K.O.)/( p21 K.O.) chimera has the healthiest look RPE layers and (C57BL/6)/(C57BL/6) has the worst; (C57BL/6)/( p21 K.O.) has better RPE integrity than the ( p21 K.O.)/(C57BL6) (Fig ure 5 5). It might be reasonable to predict that there might be some synergetic effect between the circulating stem cell and the micro environment.

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74 Figure 5 1. Rea l time reverse transcript PCR analysis of gene expression profile difference between different strains in the posterior cup 5 days post injection The expression level of each gene is normalized to that of actin. The expression level in MRL/MpJ mice is then normalized by the average of the expression level in the control conhorts.

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75 Figure 5 2 FACS analysis of peripheral cell difference in MRL/MpJ and AKR/J 5 days post sodium iodate injection. Th e collected peripheral blood samples from MRL/MpJ and AKR/J were stained with various FITC conjugated antibodies and analyzed by FACS. T he percentage of each cell population in MRL/MpJ mice were normalized by the average of the percentage in AKR/J mice. n 3.

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76 Figure 53. ERG analysis of C57BL/6, p21 K.O. and MRL/MpJ mice post sodium iodate injection recovery C57BL/6, p21 K.O. and MRL/MpJ mice were treated with 20mg/kg body weight. The ERG responses were recorded before and 30 days post injection.

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77 Figure 5 4 ERG analysis of cross bone marrow transplanted chimeric mice before and 30 days after treated with 20 mg/kg sodium iodate A) Comparison of C57BL/6 and p21 K.O. before bone marrow transplantation. B) C57BL/6 and p21 K.O. mice treated with 20 mg/kg sodium iodate and self MBT. C) C57BL/6 and p21 K.O. mice treated with 20 mg/kg sodium iodate and crossMBT D) a wave analysis of A), B) and C). E) bwave analysis of A), B) and C). A B C D E

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78 Figure 5 5 Flatmount of bone marrow transplanted cohorts stained with FITC phalloidin. We will annoted the chimera mice as (Host)/(Donor). T h e chimeras were treated with 20 mg/kg sodium iodate prior bone marrow transplantation. The posterior cups were harvested 30 days post injection. A) Flatmount ( p21 K.O.)/( p21 K.O.) B) (p21 K.O.)/(C57BL6/J). C) (C57BL6/J)/( p21 K.O.). D) (C57BL6/J)/ (C57BL6/J)

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79 CHAPTER 6 DISCUSSION AND CONCL USION Our group has previously showed the bone marrow derived progenitor cells can be differentiated into RPE cells ( 51, 54) with low efficiency. The goal of the current work is to identify a mouse model with robust regeneration of RPE in response to inj ury that can be used in future studies to elucidate the underlying pathways. Our analysis indicates that RPE regeneration after sodium iodate injury is significantly enhanced in MRL/M pJ mice compared to AKR/J mice. We identified a sodium iodate dose of 20 mg/kg under which a clear difference in recovery was observed between these strains. At this dose, the ERG response at 60 days after damage in MRL/MpJ recovered beyond pretreatment amplitudes, consistent with a more than sufficient restoration of RPE funct ion, which is essential for photoreceptor viability and activity ( 12 ) By monitoring tissue histology and morphology as a function of time after sodium iodate injury, we demonstrated that the RPE of both strains undergoes similar cellular damage after injury, but only MRL/MpJ recovers significantly. BrdU incorporation studies indicate that MRL/MpJ recovery correlates with an increased labeling of subretinal nuclei, consistent with a contribution of cell division t o regeneration in this MRL/MpJ tissue. The loss and subsequent recovery of RPE function and structure in MRL/MpJ mice satisfies the criteria of a regenerative process ( 34) A modest level of sodium iodateinduced injury appears to be critical for detecti ng RPE regeneration in rodents. Although published studies are difficult to compare because of differences in the sodium iodate dose, mode of adm inistration, age of animals and time of analysis after injection, major trends can be identified. Complete RPE ablation in mice is often achieved with sodium iodate at 40 100 mg/kg body

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80 weight injected intravenously or intraperitoneally. Under these conditions, little ( 93) to no ( 89, 94 95 ) regeneration has been found. We observed a similarly low level of regenerati on in MRL/MpJ and control mice, including pigmented strains, with retroorbital sodium iodate at 40 mg/kg body weight, in agreement with our previous work ( 51, 54) By contrast, significant regeneration has been sug gested to occur at lower doses. For example, intravenous sodium iodate in C57BL/6 mice at 15 and 25 mg/kg body weight caused a dip and subsequent part ial recovery of visual function as measured by a sensitive optomotor kinetic reflex assay, possibly indicating RPE regeneration ( 60) Consistent with these studies, we found robust regeneration of MRL/MpJ RPE with retro orbital sodium i odate at 20 mg/kg body weight. The importance of a lower dose may reflect a need to preserve adult RPE cells or tissueresident stem cells that repopulate the damaged tissue. Alternatively, high doses may damage circulating stem cells required for regeneration, as suggested previously ( 51) ; alter the tissue microenvironment so that it no longer supports RPE regeneration; or induce secondary damage due to inflammatory responses, as suggested as an explanation for RPE cell loss in a genetic ablation model ( 96) The observed dependence of regeneration on the severity of sodium iodate injury fits with the variable outcomes of tissue regeneration in MRL/MpJ mice ( 65 67 97106 ) Unlike reports of patchy RPE loss in response to a low sodium i odate dose ( 60 94) our results indicate contiguous damage of the central posterior RPE with preservation of t he periphery. We did not observe patches of RPE cell loss in the strains we examined, possibly because we avoided damage to the RPE by examining whole eyecup preparations in whi ch the retina remained intact. In earlier attempts where the

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81 retina was removed, we found that adherence of the RPE and retina introduced artifacts that made it difficult to interpret changes in RPE morphology, especially in sodium iodateinjured eyes. The loss of central RPE with peripheral preservation with low sodium iodate doses has also been reported in C57BL/6 mice ( 95) and rabbits ( 107 ) Greater central damage has also been noted at higher sodium iodate doses in mice ( 89 93) and rats ( 93) and is supported by fluorescein ang iography in rabbits and monkeys, which reveal a sodium iodateinduced breakdown of RPE barrier function in the central eye early after injury ( 108) Since major blood vessels of the choroidal and retinal circulation enter and exit the central eye at the optic nerve and have their largest diameter there, we speculate that delivery of and damage from sodium iodat e is greatest in this region. However, it is also possible that damage is uniform throughout the eye, but peripheral regions are repaired more rapidly due to the presence of progenitor or stem cells in these regions ( 109) Features of the transition zone between normal and atrophic RPE in MRL/MpJ and AKR/J mice reveal a similar initial response to sodium iodate injury in both strains. Flower like cell shedding structures in the transition zone have previously been observed in chick embryonic RPE ( 110) in transgenic mice following genetic ablation of the RPE ( 96) and in cell culture models of epithelial shedding ( 111 112 ) RPE sh edding structures are associated with apical ejection of a central dead or dying RPE cell ( 110) Enlarged cells with irregular borders at the central edge of the transition zone may be similar to those reported at the boundary of RPE atrophy in retinal cross sections and in RP E/choroid/sclera flat mounts of sodium iodateinjured mouse and rabbit eyes ( 113 114) These observations l ead us to propose an initial injury response

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82 in which dead or dying RPE cells are shed by a purse string closure process characteristic of epithelial monolayers ( 115) Neighboring cells rearrange to cover the area occupied by the shed cells, as suggested in a genetic RPE ablation model ( 96 ) resulting in enlarged cells with irregular boundaries. In the face of continued atrophy, RPE cells at the edge of the transition zone dedifferentiate, losing their characteristic polygonal shape and RPE65 expression. Further studies may reveal whether the RPE is repopulated in sodium iodateinjured MRL/MpJ mice by proliferation of cells at the central edge of the transition zone. The establishment of enhanced RPE regeneration in MRL/MpJ mice is an important first step towards identifying factors that may improve regenerat ive approaches for agerelated macular deg eneration and related diseases. MRL/MpJ mice are not universal healer mice as they were originally nicknamed ( 101 106 ) and even the original ear punch regeneration phenotype has been observed at much lower levels in other strains, including C57BL/6 ( 116 117) Nevertheless, there is general agreement that tissue regeneration is most robust in MRL/MpJ strain, making it the most experimentally tractable for studying this process. In many ways the fact that MRL/MpJ mice are near ly normal makes them a potentially more relevant model for regenerative studies, as long as the tissue of interest is repaired in this background. Our results clearly show full restoration of ERG function in MRL/MpJ mice following acute sodium iodate injur y of the RPE. The association between low expression level of p21 and elevated level of plk1 suggested a possible connection of cell cycle regulation to regeneration. It is proposed that the lack of check points during cell cycle due to the p21 knockout, the percentage

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83 of G2/M population will increase. A high G2/M cell percentage is also a feature of regenerative tissues like liver. Unscheduled entering into S phase will cause the phenotype of enhanced proliferating ( 118) Overexpression of pololike kinase 1 may create similar situation including the unscheduled entering into S phase and accumulation of G2/M phase cells. The distinct expression profile of dif ferent TGF family members provides an interesting index of types of tissue repairing. It is not easy to explain our result that TGF 1, TGF 2 and TGF3 were all found to be expressed in higher level in regenerative strain. One explanation might be that the high level of TGF 3 may dominate the regenerative like situation. Possible experiment can be carried out by inhibiting the TGF 1, TGF 2 and/or over expressing TGF 3 and check the RPE regeneration. A promising therapeutic approach can be developed if this works. T h e investigation of the possible regeneration mechanisms on chapter five provide general hint of the role circulating stem cell and microenvironment played during the regeneration process. Though circulating stem cell may play a pivotal im portant role in the regeneration, as suggested by others ( 70 ) synergetic effect may exist between the circulating stem cell and microenvironment during regeneration process by the result that the cohort with both p21 K.O. bone marrow cell and host regenerated better than the cohorts with either one factor It is not surprising since the regeneration is a complex process. The motivation and differentiation requires series of signals and cytokines ( 73) Recent studies provide additional information on how enhanc ed RPE regeneration can occur. Similar conclusion were draw in a study of a mouse myocar dial injury and recovery model which indicate c irculating stem cells m ay be superior in MRL/MpJ

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84 mice ( 70) Alternatively, the peripheral region of the posterior eye, which has been suggested to contain stem or progenitor cells ( 109) may be more proliferative in MR L/MpJ mice than in AKR/J mice. Finally, a less destructive inflammatory response in MRL/MpJ mice ( 119 120 ) may promote the engraftment and regeneration of RPE cells or the proliferation of other tissue derived cells. Studies in MRL/MpJ mice and other animal models have indicated that the status of systemic inflammatory factors and the local balance of proand anti inflammatory cytokines profile are critical in determining whether a wound heals with or without a scar ( 120123 ) Whicheve r hypothesis proves correct, if the mechanism of enhanced RPE regeneration in the MRL/MpJ mice can be clarified, it will be useful for developing therapies for clinical recovery from RPE damage and loss. The simple fact that RPE regeneration occurs robustl y in MRL/MpJ mice may help efforts to identify the cell sources that effect RPE repair in a mammalian system. Studies from our own and other laboratories have shown that HSC derived donor cells can form cells within the RPE layer that are morphologically i ndistinguishable from native RPE ( 5156 ) However, only low levels of donor cell incorporation were observed and it was challenging to confirm that these cells we re fully functional. The extensive RPE regeneration in our wound healing mouse model may permit more robust incorporation of donor cells and facilitate functional testing of these populations within the RPE layer.

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85 Figure 6 1. A schematic diagram that s uggest s the stem cell, micro enviroment and the interactions that may contribute to RPE regeneration.

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86 LIST OF REFERENCES 1. Kanda, A., Abecasis, G., and Swaroop, A. 2008. Inflammation in the pathogenesis of agerelated macular degener ation. Br J Ophthalmol 92:448450. 2. Rein, D.B., Wittenborn, J.S., Zhang, X., Honeycutt, A.A., Lesesne, S.B., and Saaddine, J. 2009. Forecasting agerelated macular degeneration through the year 2050: the potential impact of new treatments. Arch Ophthalmo l 127:533540. 3. Klein, R., Klein, B.E., Jensen, S.C., and Meuer, S.M. 1997. The fiveyear incidence and progression of agerelated maculopathy: the Beaver Dam Eye Study. Ophthalmology 104:721. 4. Mitchell, P., Wang, J.J., Foran, S., and Smith, W. 2002. Five year incidence of age related maculopathy lesions: the Blue Mountains Eye Study. Ophthalmology 109:10921097. 5. Mukesh, B.N., Dimitrov, P.N., Leikin, S., Wang, J.J., Mitchell, P., McCarty, C.A., and Taylor, H.R. 2004. Fiveyear incidence of agerelat ed maculopathy: the Visual Impairment Project. Ophthalmology 111:11761182. 6. Patel, M., and Chan, C.C. 2008. Immunopathological aspects of age related macular degeneration. Semin Immunopathol 30:97110. 7. Zarbin, M.A. 2004. Current concepts in the pathogenesis of agerelated macular degeneration. Arch Ophthalmol 122:598614. 8. Swaroop, A., Chew, E.Y., Rickman, C.B., and Abecasis, G.R. 2009. Unraveling a multifactorial lateonset disease: from genetic susceptibility to disease mechanisms for agerelated macular degeneration. Annu Rev Genomics Hum Genet 10:19 43. 9. Isas, J.M., Luibl, V., Johnson, L.V., Kayed, R., Wetzel, R., Glabe, C.G., Langen, R., and Chen, J. 2009. Drusen deposits contain soluble and mature amyloid fibrils. Invest Ophthalmol Vis Sci 1 0. Malek, G., Johnson, L.V., Mace, B.E., Saloupis, P., Schmechel, D.E., Rickman, D.W., Toth, C.A., Sullivan, P.M., and Bowes Rickman, C. 2005. Apolipoprotein E allele dependent pathogenesis: a model for agerelated retinal degeneration. Proc Natl Acad Sci U S A 102:1190011905. 11. Rizzolo, L.J. 2007. Development and role of tight junctions in the retinal pigment epithelium. Int Rev Cytol 258:195234. 12. Strauss, O. 2005. The retinal pigment epithelium in visual function. Physiol Rev 85:845881.

PAGE 87

87 13. Al Hus saini, H., Kam, J.H., Vugler, A., Semo, M., and Jeffery, G. 2008. Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo. Mol Vis 14:17841791. 14. Zanke, B., Hawken, S., Carter, R., and Chow, D. 2010. A genetic appr oach to stratification of risk for agerelated macular degeneration. Can J Ophthalmol 45:2227. 15. Yildirim, Z., Ucgun, N.I., and Yildirim, F. 2011. The role of oxidative stress and antioxidants in the pathogenesis of agerelated macular degeneration. Clinics (Sao Paulo) 66:743 746. 16. Beatty, S., Koh, H., Phil, M., Henson, D., and Boulton, M. 2000. The role of oxidative stress in the pathogenesis of agerelated macular degeneration. Surv Ophthalmol 45:115134. 17. Dong, A., Xie, B., Shen, J., Yoshida, T., Yokoi, K., Hackett, S.F., and Campochiaro, P.A. 2009. Oxidative stress promotes ocular neovascularization. J Cell Physiol 219:544552. 18. Trevithick, J., Massel, D., Robertson, J.M., Tomany, S., and Wall, R. 2004. Model study of AREDS antioxidant supplementation of AMD compared to Visudyne: a dominant strategy? Ophthalmic Epidemiol 11:337346. 19. Qi, X., Sun, L., Lewin, A.S., Hauswirth, W.W., and Guy, J. 2007. Long term suppression of neurodegeneration in chronic experimental optic neuritis: antioxidant gene therapy. Invest Ophthalmol Vis Sci 48:53605370. 20. Chen, B., Caballero, S., Seo, S., Grant, M.B., and Lewin, A.S. 2009. Delivery of antioxidant enzyme genes to protect against ischemia/reperfusioninduced injury to retinal microvasculature. Invest Ophthalmol Vis Sci 50:55875595. 21. Sparrow, J.R. 2010. Bisretinoids of RPE lipofuscin: trigger for complement activation in agerelated macular degeneration. Adv Exp Med Biol 703:63 74. 22. 2009. Deal watch: Alcon licenses complement pathway inhibitor for macular degeneration. Nat Rev Drug Discov 8:922. 23. 1991. Laser photocoagulation of subfoveal recurrent neovascular lesions in agerelated macular degeneration. Results of a randomized clinical trial. Macular Photocoagulation Study Group. Arch Ophthalmol 109:12321241. 24. 1991. Subfoveal neovascular lesions in agerelated macular degeneration. Guidelines for evaluation and treatment in the macular photocoagulation study. Macular Photocoagulation Study Group. Arch Ophthalmol 109:12421257.

PAGE 88

88 25. Shah, A.M., Bressler, N.M., and Jampol, L.M. 2011. Does laser still have a role in the management of retinal vascular and neovascular diseases? Am J Ophthalmol 152:332339 e331. 26. Zampros, I., Praidou, A., Brazitikos, P., Ekonomidis, P., and Androudi, S. 2012. Ant ivascular endothelial growth factor agents for neovascular agerelated macular degeneration. J Ophthalmol 2012:319728. 27. Ciulla, T.A., and Rosenfeld, P.J. 2009. Antivascular endothelial growth factor therapy for neovascular agerelated macular degenerati on. Curr Opin Ophthalmol 20:158165. 28. Singerman, L. 2009. Combination therapy using the small interfering RNA bevasiranib. Retina 29:S4950. 29. Elbarbary, R.A., Takaku, H., Tamura, M., and Nashimoto, M. 2009. Inhibition of vascular endothelial growth f actor expression by TRUE gene silencing. Biochem Biophys Res Commun 379:924927. 30. Pechan, P., Rubin, H., Lukason, M., Ardinger, J., DuFresne, E., Hauswirth, W.W., Wadsworth, S.C., and Scaria, A. 2009. Novel anti VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization. Gene Ther 16:1016. 31. Bressler, N.M., Bressler, S.B., Childs, A.L., Haller, J.A., Hawkins, B.S., Lewis, H., MacCumber, M.W., Marsh, M.J., Redford, M., Sternberg, P., Jr., et al. 2004. Surgery for hemorrhagic choroidal neovascular lesions of agerelated macular degeneration: ophthalmic findings: SST report no. 13. Ophthalmology 111:19932006. 32. Hillenkamp, J., Surguch, V., Framme, C., Gabel, V.P., and Sachs, H.G. 2010. Management of submacular hemorrhage with intravitreal versus subretinal injection of recombinant tissue plasminogen activator. Graefes Arch Clin Exp Ophthalmol 248:511. 33. Hillenkamp, J., Surguch, V., Framme, C., Gabel, V.P., and Sachs, H.G. 2012. Management of submacular hemorrhage wi th intravitreal versus subretinal injection of recombinant tissue plasminogen activator. Graefes Arch Clin Exp Ophthalmol 248:511. 34. Gurtner, G.C., Callaghan, M.J., and Longaker, M.T. 2007. Progress and potential for regenerative medicine. Annu Rev Med 58:299312. 35. Nacu, E., and Tanaka, E.M. 2011. Limb regeneration: a new development? Annu Rev Cell Dev Biol 27:409440. 36. Chernoff, E.A., Stocum, D.L., Nye, H.L., and Cameron, J.A. 2003. Urodele spinal cord regeneration and related processes. Dev Dyn 2 26:295 307.

PAGE 89

89 37. Del Rio Tsonis, K., and Tsonis, P.A. 2003. Eye regeneration at the molecular age. Dev Dyn 226:211224. 38. Dawley, E.M., S, O.S., Woodard, K.T., and Matthias, K.A. 2012. Spinal cord regeneration in a tail autotomizing urodele. J Morphol 273 :211225. 39. Poss, K.D., Wilson, L.G., and Keating, M.T. 2002. Heart regeneration in zebrafish. Science 298:21882190. 40. Rossi, L., Challen, G.A., Sirin, O., Lin, K.K., and Goodell, M.A. 2011. Hematopoietic stem cell characterization and isolation. Meth ods Mol Biol 750:47 59. 41. Kierdorf, U., and Kierdorf, H. 2012. Antler regrowth as a form of epimorphic regeneration in vertebrates a comparative view. Front Biosci (Elite Ed) 4:16061624. 42. Michalopoulos, G.K. 2007. Liver regeneration. J Cell Physiol 213:286300. 43. Hongbo, S., Yu, C., Ming, K., Honglin, S., Yuanping, H., and Zhongping, D. 2012. Augmenter of Liver Regeneration may be a Candidate for Prognosis of HBV Related AcuteonChronic Liver Failure as a Regenerative Marker. Hepatogastroenterolo gy 59. 44. Fernando, W.A., Leininger, E., Simkin, J., Li, N., Malcom, C.A., Sathyamoorthi, S., Han, M., and Muneoka, K. 2011. Wound healing and blastema formation in regenerating digit tips of adult mice. Dev Biol 350:301310. 45. Okano, H. 2011. Strategic approaches to regeneration of a damaged central nervous system. Cornea 30 Suppl 1:S1518. 46. Penn, M.S., Dong, F., Klein, S., and Mayorga, M.E. 2011. Stem cells for myocardial regeneration. Clin Pharmacol Ther 90:499501. 47. Guan, X., Furth, M.E., and C hilders, M.K. 2011. Stem cell use in musculoskeletal disorders. Pm R 3:S95 99. 48. Seagle, B.L., Gasyna, E.M., Mieler, W.F., and Norris, J.R., Jr. 2006. Photoprotection of human retinal pigment epithelium cells against blue light induced apoptosis by melanin free radicals from Sepia officinalis. Proc Natl Acad Sci U S A 103:1664416648. 49. Boulton, M., Roanowska, M., and Wess, T. 2004. Ageing of the retinal pigment epithelium: implications for transplantation. Graefes Arch Clin Exp Ophthalmol 242:7684. 50. Lee, E., and Maclaren, R.E. 2010. Sources of RPE for replacement therapy. Br J Ophthalmol

PAGE 90

90 51. Harris, J.R., Brown, G.A., Jorgensen, M., Kaushal, S., Ellis, E.A., Grant, M.B., and Scott, E.W. 2006. Bone marrow derived cells home to and regenerate retinal pigment epithelium after injury. Invest Ophthalmol Vis Sci 47:21082113. 52. Vossmerbaeumer, U., Kuehl, S., Kern, S., Kluter, H., Jonas, J.B., and Bieback, K. 2008. Induction of retinal pigment epithelium properties in ciliary margin progenitor cells. Clin Experiment Ophthalmol 36:358366. 53. Carr, A.J., Vugler, A.A., Hikita, S.T., Lawrence, J.M., Gias, C., Chen, L.L., Buchholz, D.E., Ahmado, A., Semo, M., Smart, M.J., et al. 2009. Protective effects of human iPS derived retinal pigment epithelium cell tr ansplantation in the retinal dystrophic rat. PLoS One 4:e8152. 54. Harris, J.R., Fisher, R., Jorgensen, M., Kaushal, S., and Scott, E.W. 2009. CD133 progenitor cells from the bone marrow contribute to retinal pigment epithelium repair. Stem Cells 27:45746 6. 55. Idelson, M., Alper, R., Obolensky, A., Ben Shushan, E., Hemo, I., YachimovichCohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., et al. 2009. Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cell s. Cell Stem Cell 5:396408. 56. Uygun, B.E., Sharma, N., and Yarmush, M. 2009. Retinal pigment epithelium differentiation of stem cells: current status and challenges. Crit Rev Biomed Eng 37:355375. 57. Xu, Y., Shi, Y., and Ding, S. 2008. A chemical appr oach to stem cell biology and regenerative medicine. Nature 453:338344. 58. Metcalfe, A.D., and Ferguson, M.W. 2007. Tissue engineering of replacement skin: the crossroads of biomaterials, wound healing, embryonic development, stem cells and regeneration. J R Soc Interface 4:413437. 59. Wicker, J., and Kamler, K. 2009. Current concepts in limb regeneration: a hand surgeon's perspective. Ann N Y Acad Sci 1172:95109. 60. Shah, M., Foreman, D.M., and Ferguson, M.W. 1994. Neutralising antibody to TGF beta 1, 2 reduces cutaneous scarring in adult rodents. J Cell Sci 107 ( Pt 5):11371157. 61. Shah, M., Foreman, D.M., and Ferguson, M.W. 1995. Neutralisation of TGF beta 1 and TGF beta 2 or exogenous addition of TGF beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 108 ( Pt 3):9851002. 62. Ho, D.M., and Whitman, M. 2008. TGF beta signaling is required for multiple processes during Xenopus tail regeneration. Dev Biol 315:203216.

PAGE 91

91 63. Levesque, M., Gatien, S., Finnson, K., Desmeules, S., Villiard, E., Pilote, M., Philip, A., and Roy, S. 2007. Transforming growth factor: beta signaling is essential for limb regeneration in axolotls. PLoS One 2:e1227. 64. Heber Katz, E., Leferovich, J., Bedelbaeva, K., Gourevitch, D., and Clark, L. 2004. The scarless heart an d the MRL mouse. Philos Trans R Soc Lond B Biol Sci 359:785793. 65. Leferovich, J.M., Bedelbaeva, K., Samulewicz, S., Zhang, X.M., Zwas, D., Lankford, E.B., and Heber Katz, E. 2001. Heart regeneration in adult MRL mice. Proc Natl Acad Sci U S A 98:9830 9835. 66. Ueno, M., Lyons, B.L., Burzenski, L.M., Gott, B., Shaffer, D.J., Roopenian, D.C., and Shultz, L.D. 2005. Accelerated wound healing of alkali burned corneas in MRL mice is associated with a reduced inflammatory signature. Invest Ophthalmol Vis Sci 4 6:40974106. 67. Fitzgerald, J., Rich, C., Burkhardt, D., Allen, J., Herzka, A.S., and Little, C.B. 2008. Evidence for articular cartilage regeneration in MRL/MpJ mice. Osteoarthritis Cartilage 16:13191326. 68. Kostyk, S.K., Popovich, P.G., Stokes, B.T., Wei, P., and Jakeman, L.B. 2008. Robust axonal growth and a blunted macrophage response are associated with impaired functional recovery after spinal cord injury in the MRL/MpJ mouse. Neuroscience 156:498 514. 69. Masinde, G.L., Li, X., Gu, W., Davidson, H ., Mohan, S., and Baylink, D.J. 2001. Identification of wound healing/regeneration quantitative trait loci (QTL) at multiple time points that explain seventy percent of variance in (MRL/MpJ and SJL/J) mice F2 population. Genome Res 11:20272033. 70. Alfaro M.P., Pagni, M., Vincent, A., Atkinson, J., Hill, M.F., Cates, J., Davidson, J.M., Rottman, J., Lee, E., and Young, P.P. 2008. The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair. Proc Na tl Acad Sci U S A 105:1836618371. 71. Naviaux, R.K., Le, T.P., Bedelbaeva, K., Leferovich, J., Gourevitch, D., Sachadyn, P., Zhang, X.M., Clark, L., and Heber Katz, E. 2009. Retained features of embryonic metabolism in the adult MRL mouse. Mol Genet Metab 96:133 144. 72. Caldwell, R.L., Opalenik, S.R., Davidson, J.M., Caprioli, R.M., and Nanney, L.B. 2008. Tissue profiling MALDI mass spectrometry reveals prominent calcium binding proteins in the proteome of regenerative MRL mouse wounds. Wound Repair Regen 16:442449. 73. Blank, U., Karlsson, G., and Karlsson, S. 2008. Signaling pathways governing stem cell fate. Blood 111:492503.

PAGE 92

92 74. Grove, J.E., Bruscia, E., and Krause, D.S. 2004. Plasticity of bone marrow derived stem cells. Stem Cells 22:487500. 75. T uch, B.E. 2006. Stem cells --a clinical update. Aust Fam Physician 35:719 721. 76. Luo, J., Chen, J., Deng, Z.L., Luo, X., Song, W.X., Sharff, K.A., Tang, N., Haydon, R.C., Luu, H.H., and He, T.C. 2007. Wnt signaling and human diseases: what are the therapeutic implications? Lab Invest 87:97 103. 77. Borggrefe, T., and Oswald, F. 2009. The Notch signaling pathway: transcriptional regulation at Notch target genes. Cell Mol Life Sci 66:16311646. 78. Watabe, T., and Miyazono, K. 2009. Roles of TGF beta family signaling in stem cell renewal and differentiation. Cell Res 19:103115. 79. Seuntjens, E., Umans, L., Zwijsen, A., Sampaolesi, M., Verfaillie, C.M., and Huylebroeck, D. 2009. Transforming Growth Factor type beta and Smad family signaling in stem cell function. Cytokine Growth Factor Rev 80. Barr, F.A., Sillje, H.H., and Nigg, E.A. 2004. Polo like kinases and the orchestration of cell division. Nat Rev Mol Cell Biol 5:429440. 81. McInnes, C., and Wyatt, M.D. 2011. PLK1 as an oncology target: current stat us and future potential. Drug Discov Today 16:619625. 82. Jopling, C., Sleep, E., Raya, M., Marti, M., Raya, A., and Izpisua Belmonte, J.C. 2010. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 464:606609. 83. Yardeni, T., Eckhaus, M., Morris, H.D., Huizing, M., and Hoogstraten Miller, S. 2011. Retroorbital injections in mice. Lab Anim (NY) 40:155160. 84. Xia, H., Krebs, M.P., Kaushal, S., and Scott, E.W. 2011. Enhanced retinal pigment epithelium regeneration after injury in MRL/MpJ mice. Exp Eye Res 93:862872. 85. Thevenaz, P., and Unser, M. 2007. User friendly semiautomated assembly of accurate image mosaics in microscopy. Microsc Res Tech 70:135 146. 86. Li, Q., Timmers, A.M., Hunter, K., Gonzalez Pola C., Lewin, A.S., Reitze, D.H., and Hauswirth, W.W. 2001. Noninvasive imaging by optical coherence tomography to monitor retinal degeneration in the mouse. Invest Ophthalmol Vis Sci 42:29812989. 87. Ralph, G.S., Binley, K., Wong, L.F., Azzouz, M., and Ma zarakis, N.D. 2006. Gene therapy for neurodegenerative and ocular diseases using lentiviral vectors. Clin Sci (Lond) 110:3746.

PAGE 93

93 88. Menger, B., Vogt, P.M., Kuhbier, J.W., and Reimers, K. 2010. Applying amphibian limb regeneration to human wound healing: a review. Ann Plast Surg 65:504510. 89. Redfern, W.S., Storey, S., Tse, K., Hussain, Q., Maung, K.P., Valentin, J.P., Ahmed, G., Bigley, A., Heathcote, D., and McKay, J.S. 2011. Evaluation of a convenient method of assessing rodent visual function in safety pharmacology studies: effects of sodium iodate on visual acuity and retinal morphology in albino and pigmented rats and mice. J Pharmacol Toxicol Methods 63:102 114. 90. Hamel, C.P., Tsilou, E., Pfeffer, B.A., Hooks, J.J., Detrick, B., and Redmond, T.M. 1 993. Molecular cloning and expression of RPE65, a novel retinal pigment epithelium specific microsomal protein that is post transcriptionally regulated in vitro. J Biol Chem 268:1575115757. 91. Douglas, H.E. 2010. TGF ss in wound healing: a review. J Woun d Care 19:403406. 92. Enzmann, V., Yolcu, E., Kaplan, H.J., and Ildstad, S.T. 2009. Stem cells as tools in regenerative therapy for retinal degeneration. Arch Ophthalmol 127:563571. 93. Mizota, A., and Adachi Usami, E. 1997. Functional recovery of retina after sodium iodate injection in mice. Vision Res 37:1859 1865. 94. Enzmann, V., Row, B.W., Yamauchi, Y., Kheirandish, L., Gozal, D., Kaplan, H.J., and McCall, M.A. 2006. Behavioral and anatomical abnormalities in a sodium iodateinduced model of retinal pigment epithelium degeneration. Exp Eye Res 82:441448. 95. Machalinska, A., Lubinski, W., Klos, P., Kawa, M., Baumert, B., Penkala, K., Grzegrzolka, R., Karczewicz, D., Wiszniewska, B., and Machalinski, B. 2011. Sodium iodate selectively injuries the pos terior pole of the retina in a dosedependent manner: morphological and electrophysiological study. Neurochem Res 35:18191827. 96. Longbottom, R., Fruttiger, M., Douglas, R.H., Martinez Barbera, J.P., Greenwood, J., and Moss, S.E. 2009. Genetic ablation o f retinal pigment epithelial cells reveals the adaptive response of the epithelium and impact on photoreceptors. Proc Natl Acad Sci U S A 106:1872818733. 97. Chadwick, R.B., Bu, L., Yu, H., Hu, Y., Wergedal, J.E., Mohan, S., and Baylink, D.J. 2007. Digit tip regrowth and differential gene expression in MRL/Mpj, DBA/2, and C57BL/6 mice. Wound Repair Regen 15:275 284. 98. Gourevitch, D.L., Clark, L., Bedelbaeva, K., Leferovich, J., and Heber Katz, E. 2009. Dynamic changes after murine digit amputation: the M RL mouse digit shows waves of tissue remodeling, growth, and apoptosis. Wound Repair Regen 17:447455.

PAGE 94

94 99. Tolba, R.H., Schildberg, F.A., Decker, D., Abdullah, Z., Buttner, R., Minor, T., and von Ruecker, A. 2010. Mechanisms of improved wound healing in Murphy Roths Large (MRL) mice after skin transplantation. Wound Repair Regen 18:662670. 100. Naseem, R.H., Meeson, A.P., Michael Dimaio, J., White, M.D., Kallhoff, J., Humphries, C., Goetsch, S.C., De Windt, L.J., Williams, M.A., Garry, M.G., et al. 2007. R eparative myocardial mechanisms in adult C57BL/6 and MRL mice following injury. Physiol Genomics 30:4452. 101. Cimini, M., Fazel, S., Fujii, H., Zhou, S., Tang, G., Weisel, R.D., and Li, R.K. 2008. The MRL mouse heart does not recover ventricular function after a myocardial infarction. Cardiovasc Pathol 17:3239. 102. Grisel, P., Meinhardt, A., Lehr, H.A., Kappenberger, L., Barrandon, Y., and Vassalli, G. 2008. The MRL mouse repairs both cryogenic and ischemic myocardial infarcts with scar. Cardiovasc Pathol 17:1422. 103. Robey, T.E., and Murry, C.E. 2008. Absence of regeneration in the MRL/MpJ mouse heart following infarction or cryoinjury. Cardiovasc Pathol 17:6 13. 104. Davis, T.A., Amare, M., Naik, S., Kovalchuk, A.L., and Tadaki, D. 2007. Differential cutaneous wound healing in thermally injured MRL/MPJ mice. Wound Repair Regen 15:577588. 105. Abdullah, I., Lepore, J.J., Epstein, J.A., Parmacek, M.S., and Gruber, P.J. 2005. MRL mice fail to heal the heart in response to ischemiareperfusion injury. Wo und Repair Regen 13:205208. 106. Oh, Y.S., Thomson, L.E., Fishbein, M.C., Berman, D.S., Sharifi, B., and Chen, P.S. 2004. Scar formation after ischemic myocardial injury in MRL mice. Cardiovasc Pathol 13:203206. 107. Korte, G.E., Perlman, J.I., and Polla ck, A. 1994. Regeneration of mammalian retinal pigment epithelium. Int Rev Cytol 152:223263. 108. Ringvold, A., Olsen, E.G., and Flage, T. 1981. Transient breakdown of the retinal pigment epithelium diffusion barrier after sodium iodate: a fluorescein ang iographic and morphological study in the rabbit. Experimental Eye Research 33:361369. 109. von Leithner, P.L., Ciurtin, C., and Jeffery, G. 2010. Microscopic mammalian retinal pigment epithelium lesions induce widespread proliferation with differences in magnitude between center and periphery. Mol Vis 16:570581. 110. Nagai, H., and Kalnins, V.I. 1996. Normally occurring loss of single cells and repair of resulting defects in retinal pigment epithelium in situ. Exp Eye Res 62:5561.

PAGE 95

95 111. Florian, P., Schoneberg, T., Schulzke, J.D., Fromm, M., and Gitter, A.H. 2002. Single cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions. J Physiol 545:485499. 112. Rosenblatt, J., Raff, M.C., and Cramer, L.P. 20 01. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actinand myosindependent mechanism. Curr Biol 11:18471857. 113. Kiuchi, K., Yoshizawa, K., Shikata, N., Moriguchi, K., and Tsubura, A. 2002. Morphologic characteris tics of retinal degeneration induced by sodium iodate in mice. Curr Eye Res 25:373379. 114. Korte, G.E., Mrowiec, E., Landzberg, K.S., and Youssri, A. 1995. Reorganization of actin microfilaments and microtubules in regenerating retinal pigment epithelium Exp Eye Res 61:189 203. 115. Garcia Fernandez, B., Campos, I., Geiger, J., Santos, A.C., and Jacinto, A. 2009. Epithelial resealing. Int J Dev Biol 53:15491556. 116. Reines, B., Cheng, L.I., and Matzinger, P. 2009. Unexpected regeneration in middle aged mice. Rejuvenation Res 12:45 52. 117. Costa, R.A., Ruiz deSouza, V., Azevedo Jr, G.M., Vaz, N.M., and Carvalho, C.R. 2009. Effects of strain and age on ear wound healing and regeneration in mice. Braz J Med Biol Res 42:11431149. 118. Bedelbaeva, K., Sny der, A., Gourevitch, D., Clark, L., Zhang, X.M., Leferovich, J., Cheverud, J.M., Lieberman, P., and Heber Katz, E. 2010. Lack of p21 expression links cell cycle control and appendage regeneration in mice. Proc Natl Acad Sci U S A 107:58455850. 119. Heber Katz, E., and Gourevitch, D. 2009. The relationship between inflammation and regeneration in the MRL mouse: potential relevance for putative human regenerative(scarless wound healing) capacities? Ann N Y Acad Sci 1172:110114. 120. Li, X., Mohan, S., Gu, W ., and Baylink, D.J. 2001. Analysis of gene expression in the wound repair/regeneration process. Mamm Genome 12:5259. 121. Anam, K., Amare, M., Naik, S., Szabo, K.A., and Davis, T.A. 2009. Severe tissue trauma triggers the autoimmune state systemic lupus erythematosus in the MRL/++ lupus prone mouse. Lupus 18:318331. 122. Eming, S.A., Hammerschmidt, M., Krieg, T., and Roers, A. 2009. Interrelation of immunity and tissue repair or regeneration. Semin Cell Dev Biol 20:517527.

PAGE 96

96 123. Zins, S.R., Amare, M.F., Anam, K., Elster, E.A., and Davis, T.A. 2010. Wound trauma mediated inflammatory signaling attenuates a tissue regenerative response in MRL/MpJ mice. J Inflamm (Lond) 7:25.

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97 BIOGRAPHICAL SKETCH Huiming Xia was born on 19 81 i n Lanxi, Zhejiang Province, China He attended Nankai University on 2000 where he graduated first with bachelor s degree on life science and later with master s degree on microbiology Huiming enrolled into the Interdisciplinary Program in Biomedical Sciences at the University of Florida in August of 200 7 where he began his doctoral study under the guidance of Dr. Edward Scott in the Department of Molecular Genetics and Microbiology supported by Alumni Fellowship After joining the laboratory of Dr Edward Scott, Huiming began his res earch on investigating the regenerat ion of retinal pigment epithelium in the wound healing mice models. His scientific achievements include a first author article published in Experimental Eye Research in October of 20 11, a secondauthor article in Investi gative Ophthalmology & Visual Science in November of 2011 and another first author manuscript in preparation.