Visualization of choroidal vasculature in pigmented mouse eyes from experimental models of AMD.

A variety of techniques exist to investigate retinal and choroidal vascular changes in experimental mouse models of human ocular diseases. While all have specific advantages, a method for evaluating the choroidal vasculature in pigmented mouse eyes has been more challenging especially for whole mount visualization and morphometric analysis. Here we report a simple, reliable technique involving bleaching pigment prior to immunostaining the vasculature in whole mounts of pigmented mouse choroids. Eyes from healthy adult pigmented C57BL/6J mice were used to establish the methodology. The retina and anterior segment were separated from the choroid. The choroid with retinal pigment epithelial cells (RPE) and sclera was soaked in 1% ethylenediaminetetraacetic acid (EDTA) to remove the RPE. Tissues were fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Choroids were subjected to melanin bleaching with 10% hydrogen peroxide (H2O2) at 55 °C for 90 min, washed in PBS and then immunostained with anti-podocalyxin antibody to label vascular endothelium followed by Cy3-AffiniPure donkey anti-goat IgG at 4 °C overnight. Images of immunostained bleached choroids were captured using a Zeiss 710 confocal microscope. In addition to control eyes, this method was used to analyze the choroids from subretinal sodium iodate (NaIO3) RPE atrophy and laser-induced choroidal neovascularization (CNV) mouse models. The H2O2 pretreatment effectively bleached the melanin, resulting in a transparent choroid. Immunolabeling with podocalyxin antibody following bleaching provided excellent visualization of choroidal vasculature in the flat perspective. In control choroids, the choriocapillaris (CC) displayed different anatomical patterns in peripapillary (PP), mid peripheral (MP) and far peripheral (FP) choroid. Morphometric analysis of the vascular area (VA) revealed that the CC was most dense in the PP region (87.4 ± 4.3% VA) and least dense in FP (79.9 ± 6.7% VA). CC diameters also varied depending on location from 11.4 ± 1.97 mm in PP to 15.1 ± 3.15 mm in FP. In the NaIO3-injected eyes, CC density was significantly reduced in the RPE atrophic regions (50.7 ± 5.8% VA in PP and 45.8 ± 6.17% VA in MP) compared to the far peripheral non-atrophic regions (82.8 ± 3.8% VA). CC diameters were significantly reduced in atrophic regions (6.35 ± 1.02 mm in PP and 6.5 ± 1.2 mm in MP) compared to non-atrophic regions (14.16 ± 2.12 mm). In the laser-induced CNV model, CNV area was 0.26 ± 0.09 mm2 and luminal diameters of CNV vessels were 4.7 ± 0.9 mm. Immunostaining on bleached choroids with anti-podocalyxin antibody provides a simple and reliable tool for visualizing normal and pathologic choroidal vasculature in pigmented mouse eyes for quantitative morphometric analysis. This method will be beneficial for examining and evaluating the effects of various treatment modalities on the choroidal vasculature in mouse models of ocular diseases such as age-related macular degeneration, and degenerative genetic diseases.

A role for mast cells in geographic atrophy.

Mast cells (MCs) are the initial responders of innate immunity and their degranulation contribute to various etiologies. While the abundance of MCs in the choroid implies their fundamental importance in the eye, little is known about the significance of MCs and their degranulation in choroid. The cause of geographic atrophy (GA), a progressive dry form of age-related macular degeneration is elusive and there is currently no therapy for this blinding disorder. Here we demonstrate in both human GA and a rat model for GA, that MC degranulation and MC-derived tryptase are central to disease progression. Retinal pigment epithelium degeneration followed by retinal and choroidal thinning, characteristic phenotypes of GA, were driven by continuous choroidal MC stimulation and activation in a slow release fashion in the rat. Genetic manipulation of MCs, pharmacological intervention targeting MC degranulation with ketotifen fumarate or inhibition of MC-derived tryptase with APC 366 prevented all of GA-like phenotypes following MC degranulation in the rat model. Our results demonstrate the fundamental role of choroidal MC involvement in GA disease etiology, and will provide new opportunities for understanding GA pathology and identifying novel therapies targeting MCs.

Choriocapillaris dropout in early age-related macular degeneration.

Loss of choriocapillaris (CC) in advanced age-related macular degeneration (AMD) is well documented but changes in early AMD have not been quantified. Postmortem eyes from donors with clinically documented early AMD were examined in choroidal whole mounts to determine the area, pattern, and severity of CC loss. Choroids from postmortem human eyes without AMD (n = 7; mean age = 86.1) and from eyes with a Grade 2 clinical classification of early AMD (n = 7; mean age = 87) were immunolabeled with Ulex europaeus agglutinin (UEA) lectin-FITC to stain blood vessels. Whole mounts were imaged using confocal microscopy and image analysis was performed to determine the area of vascular changes and density of vasculature (percent vascular area, %VA). All areas evaluated had a complete RPE monolayer upon gross examination. In age-matched control eyes, the CC had broad lumens and a homogenous pattern of freely interconnecting capillaries. The mean %VA ± standard deviation in submacula of control subjects was 78.1 ± 3.25%. In eyes with early AMD, there was a significant decrease in mean %VA to 60.1 ± 10.4% (p < 0.0001). The paramacular %VA was not significantly different in eyes with or without AMD. The area of submacular choroid affected by CC dropout was 0.04 ± 0.09 mm2 in control eyes. In eyes with early AMD, the mean area affected by CC dropout was significantly increased (10.4 ± 6.1 mm2; p < 0.001). In some cases, incipient neovascular buds were observed at the border of regions with CC dropout in early AMD choroids. In conclusion, UEA lectin-labeled choroidal whole mounts from donors with clinically documented early AMD has provided a unique opportunity to examine regional changes in vascular pathology associated with choriocapillaris. The study demonstrated attenuation of submacular CC in early AMD subjects but no vascular pathology was observed outside the submacular region. While the affected area in some eyes was quite extensive histologically, these changes may not be detectable clinically using standard in vivo imaging.

Idiopathic preretinal glia in aging and age-related macular degeneration.

During analysis of glia in wholemount aged human retinas, frequent projections onto the vitreal surface of the inner limiting membrane (ILM) were noted. The present study characterized these preretinal glial structures. The amount of glial cells on the vitreal side of the ILM was compared between eyes with age-related macular degeneration (AMD) and age-matched control eyes. Retinal wholemounts were stained for markers of retinal astrocytes and activated Müller cells (glial fibrillary acidic protein, GFAP), Müller cells (vimentin, glutamine synthetase) and microglia/hyalocytes (IBA-1). Retinal vessels were labeled with UEA lectin. Images were collected using a Zeiss LSM 710 confocal microscope. Retinas were then cryopreserved. Laminin labeling of cryosections determined the location of glial structures in relation to the ILM. All retinas investigated herein had varied amounts of preretinal glia. These glial structures were classified into three groups based on size: sprouts, blooms, and membranes. The simplest of the glial structures observed were focal sprouts of singular GFAP-positive cells or processes on the vitreal surface of the ILM. The intermediate structures observed, glial blooms, were created by multiple cells/processes exiting from a single point and extending along the vitreoretinal surface. The most extensive structures, glial membranes, consisted of compact networks of cells and processes. Preretinal glia were observed in all areas of the retina but they were most prominent over large vessels. While all glial blooms and membranes contained vimentin and GFAP-positive cells, these proteins did not always co-localize. Many areas had no preretinal GFAP but had numerous vimentin only glial sprouts. In double labeled glial sprouts, vimentin staining extended beyond that of GFAP. Hyalocytes and microglia were detected along with glial sprouts, blooms, and membranes. They did not, however, concentrate in the retina below these structures. Cross sectional analysis identified small breaks in the ILM above large retinal vessels through which glial cells exited the retina. Preretinal glial structures of varied sizes are a common occurrence in aged retinas and, in most cases, are subclinical. While all retinal glia are found in blooms, vimentin labeling suggests that Müller cells form the leading edge. All retinas investigated from eyes with active choroidal neovascularization (CNV) had extensive glial membranes on the vitreal surface of the ILM. Although these structures may be benign, they may exert traction on the retina as they spread along the vitreoretinal interface. In cases with CNV, glial cells in the vitreous could bind intravitreally injected anti-vascular endothelial growth factor. These preretinal glial structures indicate the remodeling of both astrocytes and Müller cells in aged retinas, in particular those with advanced AMD.

Retinal macroglia changes in a triple transgenic mouse model of Alzheimer's disease.

The retinas of Alzheimer's disease (AD) patients and transgenic AD animal models display amyloid beta deposits and degeneration of ganglion cells. Little is known, however, about the glial changes in the AD retina. The present study used a triple transgenic mouse model (3xTG-AD), which carries mutated human amyloid precursor protein, tau, and presenilin 1 genes and closely mimics the human brain pathology, to investigate retinal glial changes in AD. AD cognitive symptoms are known to begin in the 3xTG-AD mice at four months of age but plaques and tangles are not seen until six to twelve months. Müller cells in 3xTG-AD animals were GFAP-positive, indicating activation, at the earliest time point investigated, nine months. Astrocyte activation was also suggested in the 3xTG-AD mice by an apparent increase in size and process number. Another glial marker, S100, was expressed by astrocytes in both the non-transgenic (NTG) controls and 3xTG-AD retinas. Labeling was predominantly nuclear in nine month non-transgenic (NTG) control mice but was also seen in the cytoplasm and processes at 18 months of age. Interestingly, the nuclear localization was not as prominent in the 3xTG-AD retina even at nine months with labeling observed in astrocyte processes. The diffusion of S100 suggests the possible secretion of this protein, as is seen in the brain, with age and, more profoundly, associated with AD. Several dense, abnormally shaped, opaque structures were noted in all 3xTG-AD mice investigated. These structures, which were enveloped by GFAP and S100-positive astrocytes and Müller cells, were positive for amyloid beta, suggesting that they are amyloid plaques. Staining control retinas with amyloid showed similar structures in 30% of NTG animals but these were fewer in number and not associated with glial activation. The results herein indicate retinal glia activation in the 3xTG-AD mouse retina.

Histologic and Immunohistochemical Characterization of GA-Like Pathology in the Rat Subretinal Sodium Iodate Model.

Purpose: Geographic atrophy (GA) is an advanced form of dry age-related macular degeneration with multifactorial etiology and no well-established treatment. A model recapitulating the hallmarks would serve as a key to understanding the underlying pathologic mechanisms better. In this report, we further characterized our previously reported subretinal sodium iodate model of GA. Methods: Retinal degeneration was induced in rats (6-8 weeks old) by subretinal injections of NaIO3 as described previously. Animals were sacrificed at 3, 8 and 12 weeks after injection and eyes were fixed or cryopreserved. Some choroids were processed as flatmounts while other eyes were cryopreserved, sectioned, and immunolabeled with a panel of antibodies. Finally, some eyes were prepared for transmission electron microscopic (TEM) analysis. Results: NaIO3 subretinal injection resulted in a well-defined focal area of retinal pigment epithelium (RPE) degeneration surrounded by viable RPE. These atrophic lesions expanded over time. RPE morphologic changes at the border consisted of hypertrophy, multilayering, and the possible development of a migrating phenotype. Immunostaining of retinal sections demonstrated external limiting membrane descent, outer retinal tubulation (ORT), and extension of Müller cells toward RPE forming a glial membrane in the subretinal space of the atrophic area. TEM findings demonstrated RPE autophagy, cellular constituents of ORT, glial membranes, basal laminar deposits, and defects in Bruch's membrane. Conclusions: In this study, we showed pathologic features of a rodent model resembling human GA in a temporal order through histology, immunofluorescence, and TEM analysis and gained insights into the cellular and subcellular levels of the GA-like phenotypes. Translational Relevance: Despite its acute nature, the expansion of atrophy and the GA-like border in this rat model makes it ideal for studying disease progression and provides a treatment window to test potential therapeutics for GA.

Loss of REP1 impacts choroidal melanogenesis and vasculogenesis in choroideremia.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, however, the involvement of the choroid in disease progression is not fully understood. CHM is caused by mutations in the CHM gene, encoding the ubiquitously expressed Rab escort protein 1 (REP1). REP1 plays an important role in intracellular trafficking of vesicles, including melanosomes. In this study, we examined the ultrastructure of the choroid in chmru848 fish and Chmnull/WT mouse models using transmission electron and confocal microscopy. Significant pigmentary disruptions were observed, with lack of melanosomes in the choroid of chmru848 fish from 4 days post fertilisation (4dpf), and a reduction in choroidal blood vessel diameter and interstitial pillars suggesting a defect in vasculogenesis. Total melanin and expression of melanogenesis genes tyr, tryp1a, mitf, dct and pmel were also reduced from 4dpf. In Chmnull/WT mice, choroidal melanosomes were significantly smaller at 1 month, with reduced eumelanin at 1 year. The choroid in CHM patients were also examined using spectral domain optical coherence tomography (SD-OCT) and OCT-angiography (OCT-A) and the area of preserved choriocapillaris (CC) was found to be smaller than that of overlying photoreceptors, suggesting that the choroid is degenerating at a faster rate. Histopathology of an enucleated eye from a 74-year-old CHM male patient revealed isolated areas of RPE but no associated underlying CC. Pigmentary disruptions in CHM animal models reveal an important role for REP1 in melanogenesis, and drugs that improve melanin production represent a potential novel therapeutic avenue.

NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development.

Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4(nmf192/nmf192) mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4(nmf192/nmf192) mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4(nmf192/nmf192) male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4(nmf192/nmf192) mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.

VEGF 165 b in the developing vasculatures of the fetal human eye.

VEGF(165) b is an anti-angiogenic form of VEGF(165) produced by alternative splicing. The localization of pro-angiogenic VEGF(165) and anti-angiogenic VEGF(165) b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF(165) immunoreactivity at 7WG and very little VEGF(165) b. Both forms were elevated at 12WG. VEGF(165) then decreased around 17WG when the TVL regresses but VEGF(165) b remained elevated. In choroid, VEGF(165) was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF(165) b was apical and VEGF(165) basal in RPE. Diffuse VEGF(165) immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF(165) b was present in most CXCR4(+) progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF(165) was present in nerve fibers and VEGF(165) b in the inner Muller cell process. The localization of VEGF(165) b was distinctly different from VEGF(165) both spatially and temporally and it was often associated with nucleus in progenitors.

Clinicopathologic Findings in Three Siblings With Geographic Atrophy.

Purpose: Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly worldwide. Clinical imaging and histopathologic studies are crucial to understanding disease pathology. This study combined clinical observations of three brothers with geographic atrophy (GA), followed for 20 years, with histopathologic analysis. Methods: For two of the three brothers, clinical images were taken in 2016, 2 years prior to death. Immunohistochemistry, on both flat-mounts and cross sections, histology, and transmission electron microscopy were used to compare the choroid and retina in GA eyes to those of age-matched controls. Results: Ulex europaeus agglutinin (UEA) lectin staining of the choroid demonstrated a significant reduction in the percent vascular area and vessel diameter. In one donor, histopathologic analysis demonstrated two separate areas with choroidal neovascularization (CNV). Reevaluation of swept-source optical coherence tomography angiography (SS-OCTA) images revealed CNV in two of the brothers. UEA lectin also revealed a significant reduction in retinal vasculature in the atrophic area. A subretinal glial membrane, composed of processes positive for glial fibrillary acidic protein and/or vimentin, occupied areas identical to those of retinal pigment epithelium (RPE) and choroidal atrophy in all three AMD donors. SS-OCTA also demonstrated presumed calcific drusen in the two donors imaged in 2016. Immunohistochemical analysis and alizarin red S staining verified calcium within drusen, which was ensheathed by glial processes. Conclusions: This study demonstrates the importance of clinicohistopathologic correlation studies. It emphasizes the need to better understand how the symbiotic relationship between choriocapillaris and RPE, glial response, and calcified drusen impact GA progression.

Rare intercellular material transfer as a confound to interpreting inner retinal neuronal transplantation following internal limiting membrane disruption.

Intercellular cytoplasmic material transfer (MT) occurs between transplanted and developing photoreceptors and ambiguates cell origin identification in developmental, transdifferentiation, and transplantation experiments. Whether MT is a photoreceptor-specific phenomenon is unclear. Retinal ganglion cell (RGC) replacement, through transdifferentiation or transplantation, holds potential for restoring vision in optic neuropathies. During careful assessment for MT following human stem cell-derived RGC transplantation into mice, we identified RGC xenografts occasionally giving rise to labeling of donor-derived cytoplasmic, nuclear, and mitochondrial proteins within recipient Müller glia. Critically, nuclear organization is distinct between human and murine retinal neurons, which enables unequivocal discrimination of donor from host cells. MT was greatly facilitated by internal limiting membrane disruption, which also augments retinal engraftment following transplantation. Our findings demonstrate that retinal MT is not unique to photoreceptors and challenge the isolated use of species-specific immunofluorescent markers for xenotransplant identification. Assessment for MT is critical when analyzing neuronal replacement interventions.

The deletion of Math5 disrupts retinal blood vessel and glial development in mice.

Retinal vascular development is a complex process that is not yet fully understood. The majority of research in this area has focused on astrocytes and the template they form in the inner retina, which precedes endothelial cells in the mouse retina. In humans and dogs, however, astrocyte migration follows behind development of blood vessels, suggesting that other cell types may guide this process. One such cell type is the ganglion cell, which differentiates before blood vessel formation and lies adjacent to the primary retinal vascular plexus. The present study investigated the potential role played by ganglion cells in vascular development using Math5(-/-) mice. It has previously been reported that Math5 regulates the differentiation of ganglion cells and Math5(-/-) mice have a 95% reduction in these cells. The development of blood vessels and glia was investigated using Griffonia simplicifolia isolectin B4 labeling and GFAP immunohistochemistry, respectively. JB-4 analysis demonstrated that the hyaloid vessels arose from choriovitreal vessels adjacent to the optic nerve area. As previously reported, Math5(-/-) mice had a rudimentary optic nerve. The primary retinal vessels did not develop post-natally in the Math5(-/-) mice, however, branches of the hyaloid vasculature eventually dove into the retina and formed the inner retinal capillary networks. An astrocyte template only formed in some areas of the Math5(-/-) retina. In addition, GFAP(+) Müller cells were seen throughout the retina that had long processes wrapped around the hyaloid vessels. Transmission electron microscopy confirmed Müller cell abnormalities and revealed disruptions in the inner limiting membrane. The present data demonstrates that the loss of ganglion cells in the Math5(-/-) mice is associated with a lack of retinal vascular development.

Glial remodeling and choroidal vascular pathology in eyes from two donors with Choroideremia.

Choroideremia (CHM) is a recessive, X-linked disease that affects 1 in 50,000 people worldwide. CHM causes night blindness in teenage years with vision loss progressing over the next two to three decades. While CHM is known to cause progressive loss of retinal pigment epithelial (RPE) cells, photoreceptors and choroidal vessels, little attention has been given to retinal glial changes in eyes with CHM. In addition, while choroidal loss has been observed clinically, no histopathologic assessment of choroidal loss has been done. We investigated glial remodeling and activation as well as choriocapillaris changes and their association with RPE loss in postmortem eyes from two donors with CHM. Eyes were fixed and cryopreserved or the retina and choroid/RPE were processed as flatmounts with a small piece cut for transmission electron microscopy. A dense glial membrane, made up of vimentin and GFAP double-positive cells, occupied the subretinal space in the area of RPE and photoreceptor loss of both eyes. The membranes did not extend into the far periphery, where RPE and photoreceptors were viable. A glial membrane was also found on the vitreoretinal surface. Transmission electron microscopy analysis demonstrated prominence and disorganization of glial cells, which contained exosome-like vesicles. UEA lectin demonstrated complete absence of choriocapillaris in areas with RPE loss while some large choroidal vessels remained viable. In the far periphery, where the RPE monolayer was intact, choriocapillaris appeared normal. The extensive glial remodeling present in eyes with CHM should be taken into account when therapies such as stem cell replacement are considered as it could impede cells entering the retina. This gliosis would also need to be reversed to some extent for Müller cells to perform their normal homeostatic functions in the retina. Future studies investigating donor eyes as well as clinical imaging from carriers or those with earlier stages of CHM will prove valuable in understanding the glial changes, which could affect disease progression if they occur early. This would also provide insights into the progression of disease in the photoreceptor/RPE/choriocapillaris complex, which is crucial for identifying new treatments and finding the windows for treatment.

Mutations in Lama1 disrupt retinal vascular development and inner limiting membrane formation.

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.

Lama1 mutations lead to vitreoretinal blood vessel formation, persistence of fetal vasculature, and epiretinal membrane formation in mice.

BACKGROUND: Valuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. Two such models are the recently described mouse lines with mutations in Lama1, an important component of the retinal internal limiting membrane (ILM). These mutants have a persistence of the fetal vasculature of vitreous (FVV) but lack a primary retinal vascular plexus. The present study provides a detailed analysis of astrocyte and vascular development in these Lama1 mutants. RESULTS: Although astrocytes and blood vessels initially migrate into Lama1 mutant retinas, both traverse the peripapillary ILM into the vitreous by P3. Once in the vitreous, blood vessels anastomose with vessels of the vasa hyaloidea propria, part of the FVV, and eventually re-enter the retina where they dive to form the inner and outer retinal capillary networks. Astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy. CONCLUSIONS: Lama1 and a fully intact ILM are required for normal retinal vascular development. Mutations in Lama1 allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand. Together, these vessels branch into the retina to form fairly normal inner retinal vascular capillary plexi. The Lama1 mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy.

Topical Ketotifen Fumarate Inhibits Choroidal Mast Cell Degranulation and Loss of Retinal Pigment Epithelial Cells in Rat Model for Geographic Atrophy.

Purpose: This study evaluates whether topical ketotifen fumarate (KTF) can prevent geographic atrophy (GA)-like phenotypes in a rat model. Methods: Pharmacokinetics (PKs) of KTF after topical administration twice daily for 5 days was analyzed in rat retina, retinal pigment epithelium (RPE)/choroid/sclera, and in plasma by an liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Rats were then given hydrogel implants +/- 48/80 in the superior subconjunctival space and topically treated with 1% and 0.25% of KTF or phosphate buffer saline (PBS) twice daily. Rats were euthanized at 1, 2, 4, and 8 weeks postinjection. Choroidal mast cells (MCs) were stained with nonspecific esterase and the RPE monolayer was labeled with RPE65 and ZO-1 in whole mount choroids. Retinal and choroidal areas were determined in cryosections stained with picrosirius red. Dark-adapted electroretinogram (ERG) was also performed to evaluate retinal function. Results: PK results showed the highest level of KTF (average 5.6 nM/mg) in the RPE/choroid/sclera in rats given topical 1% KTF. Topical 1% KTF significantly reduced choroidal MC degranulation at 1 week and 2 weeks (both P < 0.001) and RPE loss at 4 weeks (P < 0.001) as well as retinal and choroidal thinning (both P < 0.001) and reduction in ERG amplitude at 8 weeks (P < 0.05) compared to PBS. Similar results were obtained with 0.25% KTF. Conclusions: Both 1% and 0.25% KTF eye drops effectively reduced MC degranulation, RPE loss, and retinal and choroidal thinning while preventing the decline of ERG amplitude in a GA-like rat model. These data suggest that topical KTF might be a new therapeutic drug for treating GA. Translational Relevance: The results of this study demonstrate that topical KTF successfully reduced GA-like phenotypes in a rat model and may provide a novel therapy for GA.

Retinal Glial and Choroidal Vascular Pathology in Donors Clinically Diagnosed With Stargardt Disease.

Purpose: The present study investigated retinal glia and choroidal vessels in flatmounts and sections from individuals with clinically diagnosed Stargardt disease (STGD). Methods: Eyes from three donors clinically diagnosed with STGD were obtained through the Foundation Fighting Blindness (FFB). Genetic testing was performed to determine the disease-causing mutations. Eyes were enucleated and fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. After imaging, retinas were dissected and immunostained for glial fibrillary acidic protein, vimentin, and peanut agglutin. Following RPE removal, the choroid was immunostained with Ulex europaeus agglutinin lectin. For each choroid, the area of affected vasculature, percent vascular area, and choriocapillaris luminal diameters were measured. The retina from one donor was hemisected and cryopreserved or embedded in JB-4 for cross-section analysis. Results: Genetic testing confirmed the STGD diagnosis in donor 1, whereas a mutation in peripherin 2 was identified in donor 3. Genetic testing was not successful on donor 2. Therefore, only donor 1 can definitively be classified as having STGD. All donors had areas of RPE atrophy within the macular region, which correlated with underlying choriocapillaris loss. In addition, Müller cells formed pre- and subretinal membranes. Subretinal gliotic membranes correlated almost identically with RPE and choriocapillaris loss. Conclusions: Despite bearing different genetic mutations, all donors demonstrated choriocapillaris loss and Müller cell membranes correlating with RPE loss. Müller cell remodeling was most extensive in the donor with the peripherin mutation, whereas choriocapillaris loss was greatest in the confirmed STGD donor. This study emphasizes the importance of genetic testing when diagnosing macular disease.

An Acute Injury Model for the Phenotypic Characteristics of Geographic Atrophy.

Purpose: Geographic atrophy (GA) is the late stage of non-neovascular age-related macular degeneration. A lack of animal models for GA has hampered treatment efforts. Presented herein is a rat model for GA using subretinal injection of sodium iodate (NaIO3). Methods: Rats were given subretinal injections of NaIO3 (5 µg/µL) using a pico-injector. Fundus photographs and spectral domain optical coherent tomography scans were collected at 1, 3, 7, 14, and 28 days after injection, at which time rats were euthanized and eyes were enucleated. Eyes were either cryopreserved or dissected into retinal and choroidal flatmounts. Fluorescence immunohistochemistry was performed for retinal glial fibrillary acidic protein (activated Müller cells and astrocytes) and vimentin (Müller cells), as well as peanut agglutin lectin (photoreceptors) labeling. RPE/choroids were labeled for RPE65 and CD34. Images were collected on Zeiss confocal microscopes. Results: Fundus photos, spectral domain optical coherent tomography, and RPE65 staining revealed well-demarcated areas with focal loss of RPE and photoreceptors in NaIO3-treated rats. At 1 day after injection, RPE cells appeared normal. By 3 days, there was patchy RPE and photoreceptor loss in the injected area. RPE and photoreceptors were completely degenerated in the injected area by 7 days. A large subretinal glial membrane occupied the degenerated area. Choriocapillaris was highly attenuated in the injected area at 14 and 28 days. Conclusions: The rat model reported herein mimics the photoreceptor cell loss, RPE atrophy, glial membrane formation, and choriocapillaris degeneration seen in GA. This model will be valuable for developing and testing drugs and progenitor cell regenerative therapies for GA.

Subretinal Glial Membranes in Eyes With Geographic Atrophy.

Purpose: Müller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study evaluated the relationship between focal photoreceptors and RPE loss in geographic atrophy (GA) and Müller cell extension into the subretinal space. Methods: Human donor eyes with no retinal disease or geographic atrophy (GA) were fixed and the eye cups imaged. The retinal posterior pole was stained for glial fibrillary acidic protein (GFAP; astrocytes and activated Müller cells) and vimentin (Müller cells) while the submacular choroids were labeled with Ulex Europaeus Agglutinin lectin (blood vessels). Choroids and retinas were imaged using a Zeiss 710 confocal microscope. Additional eyes were cryopreserved or processed for transmission electron microscopy (TEM) to better visualize the Müller cells. Results: Vimentin staining of aged control retinas (n = 4) revealed a panretinal cobblestone-like ELM. While this pattern was also observed in the GA retinas (n = 7), each also had a distinct area in which vimentin+ and vimentin+/GFAP+ processes created a subretinal membrane. Subretinal glial membranes closely matched areas of RPE atrophy in the gross photos. Choroidal vascular loss was also evident in these atrophic areas. Smaller glial projections were noted, which correlated with drusen in gross photos. The presence of glia in the subretinal space was confirmed by TEM and cross cross-section immunohistochemistry. Conclusions: In eyes with GA, subretinal Müller cell membranes present in areas of RPE atrophy may be a Müller cell attempt to replace the ELM. These membranes could interfere with treatments such as stem cell therapy.

Mast Cell-Derived Tryptase in Geographic Atrophy.

Purpose: Our previous study demonstrated significantly more degranulating mast cells (MCs) in choroids from subjects with age-related macular degeneration compared to aged controls. This study examined the immunolocalization of tryptase, the most abundant MC secretory granule-derived serine protease, in aged control eyes and eyes with geographic atrophy (GA). Methods: Postmortem human eyes with and without GA were obtained from the National Disease Research Interchange. Tissue was fixed, cryopreserved, sectioned, and immunostained with a monoclonal antibody against tryptase. Sections were imaged on a Zeiss 710 Confocal Microscope. Results: In the posterior pole of all aged control eyes, tryptase was confined to choroidal MCs, which were located primarily in Sattler's layer. In eyes with GA, many MCs were located in the inner choroid near choriocapillaris and Bruch's membrane (BM). Tryptase was found not only in MCs but also diffusely around them in stroma, suggesting they had degranulated. In contrast with aged control eyes, eyes with GA also had strong tryptase staining in BM. Tryptase was observed within BM in regions of RPE atrophy, at the border of atrophy, and extending well into the nonatrophic region. Conclusions: Our results demonstrate that tryptase, released during choroidal MC degranulation, binds to BM in GA in advance of RPE atrophy. Tryptase activates MMPs that can degrade extracellular matrix (ECM) and basement membrane components found in BM. ECM modifications are likely to have a profound effect on the function and health of RPE and choroidal thinning in GA.