Down-expression of the NLRP3 inflammasome delays the progression of diabetic retinopathy.

The investigation aimed to evaluate the effects of Mcc950, an inhibitor of the NLRP3 inflammasome, on diabetic retinopathy (DR) mice. The general physiological condition of each group of mice was recorded. Retinal blood vessels were stained for observation of the density of blood vessels, and retinas were used for further morphological examination and fluorescent staining after the intravitreal injection of Mcc950. Mcc950 partially reversed hyperglycemia-induced vascular damage and had reduced histological changes compared to DR mice. IL-1ß production in mice retinas in the diabetic model (DM) group increased, but pretreatment with Mcc950 significantly reversed these changes. Additionally, Mcc950 engineered reduced FITC dextran extravasation and vascular leakage. Therefore, it played an apparent protective role in DR and could be a new treatment strategy for DR.

Role of CD 20+ T cells and related cytokines in mediating retinal microvascular changes and ocular complications in chronic-plaque type psoriasis.

OBJECTIVE: To assess the role of CD3+ CD20+ CD4- CD8- double-negative (DN) or CD3+CD20+ CD4/CD8+ T cells and the related pro-inflammatory cytokines in the humor aqueous, in mediating retinal microvascular changes in patients with chronic plaque-type moderate to severe psoriasis. DESIGN: A total of 76 patients (57.6 ± 11.7 years) with chronic plaque-type psoriasis were initially evaluated. Nineteen patients (19 eyes) and 19 healthy volunteers (19 eyes) were subjected to dermatological evaluation with Psoriasis Area Severity Index (PASI) and the Dermatology life quality index (DLQI). Retinal images were processed using an automatized software. On the same day, a venous sample was collected and analyzed using multiparametric flow cytometry. Three out of 6 patients who presented cataract, consented to perform surgery with humor aqueous collection. The samples were analyzed using a Multi-Analyte ELISA kit for the simultaneous quantification of IL1α, IL1ß, IL2, IL4, IL6, IL8, IL10, IL12, IL17A, IFNγ, TNF-α, GMCSF. RESULTS: The CD3+CD4+/CD8+CD20+CD56- T cells expression was greater in the psoriatic patients (+73.9%, P < 0.001) compared to controls, but not the DN T cells (-8.2%, P = 0.30). Ocular complications were diagnosed in 61.1% of patients, microvascular parameters including artero-venous ratio (P = 0.04), subfoveal choriocapillaris/Sattler's layer, and choroidal thickness (CT, both P < 0.001) were significantly altered in psoriasis subgroup. The increased circulating levels of the CD3+CD4+/CD8+CD20+CD56- T cells were associated with thinning of subfoveal CT (P = 0.03) and Haller's layer (P = 0.01). Instead, the DN T cells presented an inverse relationship with disease duration (P = 0.02), DLQI score (P = 0.02), and the use of biological therapy (P = 0.05). The related cytokine patterns possibly modified in this cellular context have been investigated. No significant differences were observed in cytokines levels between psoriasis and controls, the most significant difference was detected on IL-6, without reaching statistical significance (fold change of 1.4, P = 0.13). CONCLUSION: Our findings demonstrated that CD20+ T cell subpopulation is highly represented in psoriasis regardless of the use of immunomodulatory therapies, and the diffuse microvascular alterations suggested possible endothelial damage as mainstream for the genesis of psoriatic-mediated complications as further supported by the comparable concentrations of cytokines, at least as humor aqueous content, with respect to healthy eyes.

STAT3 activation in circulating myeloid-derived cells contributes to retinal microvascular dysfunction in diabetes.

BACKGROUND: Leukostasis is a key patho-physiological event responsible for capillary occlusion in diabetic retinopathy. Circulating monocytes are the main cell type entrapped in retinal vessels in diabetes. In this study, we investigated the role of the signal transducer and activator of transcription 3 (STAT3) pathway in diabetes-induced immune cell activation and its contribution to retinal microvascular degeneration. METHODS: Forty-one patients with type 1 diabetes (T1D) [mild non-proliferative diabetic retinopathy (mNPDR) (n = 13), active proliferative DR (aPDR) (n = 14), inactive PDR (iPDR) (n = 14)] and 13 age- and gender-matched healthy controls were recruited to the study. C57BL/6 J WT mice, SOCS3fl/fl and LysMCre/+SOCS3fl/fl mice were rendered diabetic by Streptozotocin injection. The expression of the phosphorylated human and mouse STAT3 (pSTAT3), mouse LFA-1, CD62L, CD11b and MHC-II in circulating immune cells was evaluated by flow cytometry. The expression of suppressor of cytokine signalling 3 (SOCS3) was examined by real-time RT-PCR. Mouse plasma levels of cytokines were measured by Cytometric Beads Array assay. Retinal leukostasis was examined following FITC-Concanavalin A perfusion and acellular capillary was examined following Isolectin B4 and Collagen IV staining. RESULTS: Compared to healthy controls, the expression of pSTAT3 in circulating leukocytes was statistically significantly higher in mNPDR but not aPDR and was negatively correlated with diabetes duration. The expression of pSTAT3 and its inhibitor SOCS3 was also significantly increased in leukocytes from diabetic mice. Diabetic mice had higher plasma levels of IL6 and CCL2 compared with control mice. LysMCre/+SOCS3fl/fl mice and SOCS3fl/fl mice developed comparative levels of diabetes, but leukocyte activation, retinal leukostasis and number of acellular capillaries were statistically significantly increased in LysMCre/+SOCS3fl/fl diabetic mice. CONCLUSION: STAT3 activation in circulating immune cells appears to contribute to retinal microvascular degeneration and may be involved in DR initiation in T1D.

Endothelial Cell-Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature.

Objective- Blood-CNS (central nervous system) barrier defects are implicated in retinopathies, neurodegenerative diseases, stroke, and epilepsy, yet, the pathological mechanisms downstream of barrier defects remain incompletely understood. Blood-retina barrier (BRB) formation and retinal angiogenesis require ß-catenin signaling induced by the ligand norrin (NDP [Norrie disease protein]), the receptor FZD4 (frizzled 4), coreceptor LRP5 (low-density lipoprotein receptor-like protein 5), and the tetraspanin TSPAN12 (tetraspanin 12). Impaired NDP/FZD4 signaling causes familial exudative vitreoretinopathy, which may lead to blindness. This study seeked to define cell type-specific functions of TSPAN12 in the retina. Approach and Results- A loxP-flanked Tspan12 allele was generated and recombined in endothelial cells using a tamoxifen-inducible Cdh5-CreERT2 driver. Resulting phenotypes were documented using confocal microscopy. RNA-Seq, histopathologic analysis, and electroretinogram were performed on retinas of aged mice. We show that TSPAN12 functions in endothelial cells to promote vascular morphogenesis and BRB formation in developing mice and BRB maintenance in adult mice. Early loss of TSPAN12 in endothelial cells causes lack of intraretinal capillaries and increased VE-cadherin (CDH5 [cadherin5 aka VE-cadherin]) expression, consistent with premature vascular quiescence. Late loss of TSPAN12 strongly impairs BRB maintenance without affecting vascular morphogenesis, pericyte coverage, or perfusion. Long-term BRB defects are associated with immunoglobulin extravasation, complement deposition, cystoid edema, and impaired b-wave in electroretinograms. RNA-sequencing reveals transcriptional responses to the perturbation of the BRB, including genes involved in vascular basement membrane alterations in diabetic retinopathy. Conclusions- This study establishes mice with late endothelial cell-specific loss of Tspan12 as a model to study pathological consequences of BRB impairment in an otherwise intact vasculature.

The alternative complement pathway aids in vascular regression during the early stages of a murine model of proliferative retinopathy.

Proliferative retinopathic diseases often progress in 2 phases: initial regression of retinal vasculature (phase 1) followed by subsequent neovascularization (NV) (phase 2). The immune system has been shown to aid in vascular pruning in such retinopathies; however, little is known about the role of the alternative complement pathway in the initial vascular regression phase. Using a mouse model of oxygen-induced retinopathy (OIR), we observed that alternative complement pathway-deficient mice (Fb(-/-)) exhibited a mild decrease in vascular loss at postnatal day (P)8 compared with age- and strain-matched controls (P = 0.035). Laser capture microdissection was used to isolate the retinal blood vessels. Expression of the complement inhibitors Cd55 and Cd59 was significantly decreased in blood vessels isolated from hyperoxic retinas compared with those from normoxic control mice. Vegf expression was measured at P8 and found to be significantly lower in OIR mice than in normoxic control mice (P = 0.0048). Further examination of specific Vegf isoform expression revealed a significant decrease in Vegf120 (P = 0.00032) and Vegf188 (P = 0.0092). In conjunction with the major modulating effects of Vegf during early retinal vascular development, our data suggest a modest involvement of the alternative complement pathway in targeting vessels for regression in the initial vaso-obliteration stage of OIR.

Angiogenesis mediated by toll-like receptor 4 in ischemic neural tissue.

OBJECTIVE: Activation of the immune system via toll-like receptors (TLRs) is implicated in atherosclerosis, microvascular complications, and angiogenesis. However, the involvement of TLRs in inflammation-associated angiogenesis in ischemic neural tissue has not been investigated. The goal of this study is to determine the role of TLR4 signaling in oxygen-induced neovascularization in retina, a neural tissue. METHODS AND RESULTS: In oxygen-induced retinopathy model, we found that retinal neovascularization was significantly attenuated in TLR4(-/-) mice. The further study revealed that the absence of TLR4 led to downregulation of proinflammatory factors in association with the attenuated activation of glia in the ischemic retina, which was also associated with reduced expression of high-mobility group box-1, an endogenous ligand for TLR4. The application of high-mobility group box-1 to the ischemic retina promoted the production of proinflammatory factors in wild-type but not TLR4(-/-) mice. High-mobility group box-1 treatment in vitro also significantly promoted the production of proinflammatory factors in retinal glial cells from wild-type mice, but much less from TLR4(-/-) mice. CONCLUSIONS: Our results suggest that the release of high-mobility group box-1 in ischemic neural tissue initiates TLR4-dependent responses that contribute to neovascularization. These findings represented a previously unrecognized effect of TLR4 on angiogenesis in association with the activation of glia in ischemic neural tissue.

CD40 mediates retinal inflammation and neurovascular degeneration.

Retinopathies are major causes of visual impairment. We used a model of ischemic retinopathy to examine the role of CD40 in the pathogenesis of retinal injury. Retinal inflammation, loss of ganglion cells, and capillary degeneration were markedly attenuated in ischemic retinas of CD40(-/-) mice. Up-regulation of NOS2 and COX2 after retinal ischemia were blunted in CD40(-/-) mice. NOS2-COX-2 up-regulation in ischemic retinas from wild-type mice was at least in part explained by recruitment of NOS2(+)COX-2(+) leukocytes. Up-regulation of KC/CXCL1 and ICAM-1 also required CD40. Retinal endothelial and Muller cells expressed CD40. Stimulation of these cells through CD40 caused ICAM-1 up-regulation and KC/CXCL1 production. Bone marrow transplant experiments revealed that leukocyte infiltration, ganglion cell loss, and up-regulation of proinflammatory molecules after retinal ischemia were dependent on CD40 expression in the retina and not peripheral blood leukocytes. These studies identified CD40 as a regulator of retinal inflammation and neurovascular degeneration. They support a model in which CD40 stimulation of endothelial and Muller cells triggers adhesion molecule up-regulation and chemokine production, promoting the recruitment of leukocytes that express NOS2/COX-2, molecules linked to neurovascular degeneration.

Label-free imaging of immune cell dynamics in the living retina using adaptive optics.

Our recent work characterized the movement of single blood cells within the retinal vasculature (Joseph et al. 2019) using adaptive optics ophthalmoscopy. Here, we apply this technique to the context of acute inflammation and discover both infiltrating and tissue-resident immune cells to be visible without any labeling in the living mouse retina using near-infrared light alone. Intravital imaging of immune cells can be negatively impacted by surgical manipulation, exogenous dyes, transgenic manipulation and phototoxicity. These confounds are now overcome, using phase contrast and time-lapse videography to reveal the dynamic behavior of myeloid cells as they interact, extravasate and survey the mouse retina. Cellular motility and differential vascular responses were measured noninvasively and in vivo across hours to months at the same retinal location, from initiation to the resolution of inflammation. As comparable systems are already available for clinical research, this approach could be readily translated to human application.

Neutrophil extracellular traps target senescent vasculature for tissue remodeling in retinopathy.

In developed countries, the leading causes of blindness such as diabetic retinopathy are characterized by disorganized vasculature that can become fibrotic. Although many such pathological vessels often naturally regress and spare sight-threatening complications, the underlying mechanisms remain unknown. Here, we used orthogonal approaches in human patients with proliferative diabetic retinopathy and a mouse model of ischemic retinopathies to identify an unconventional role for neutrophils in vascular remodeling during late-stage sterile inflammation. Senescent vasculature released a secretome that attracted neutrophils and triggered the production of neutrophil extracellular traps (NETs). NETs ultimately cleared diseased endothelial cells and remodeled unhealthy vessels. Genetic or pharmacological inhibition of NETosis prevented the regression of senescent vessels and prolonged disease. Thus, clearance of senescent retinal blood vessels leads to reparative vascular remodeling.

Vascular adhesion protein-1 regulates leukocyte transmigration rate in the retina during diabetes.

Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity and is involved in leukocyte recruitment. Leukocyte adhesion to retinal vessels is a predominant feature of experimentally induced diabetic retinopathy (DR). However, the role of VAP-1 in this process is unknown. Diabetes was induced by i.p. injection of Streptozotocin in Long-Evans rats. The specific inhibitor of VAP-1, UV-002, was administered by daily i.p. injections. The expression of VAP-1 mRNA in the retinal extracts of normal and diabetic animals was measured by real-time quantitative polymerase chain reaction (PCR). Firm leukocyte adhesion was quantified in retinal flatmounts after intravascular staining with concanavalin A (ConA). Leukocyte transmigration rate was quantified by in vivo acridine orange leukocyte staining (AOLS). In diabetic rats, the rate of leukocyte transmigration into the retinal tissues of live animals was significantly increased, as determined by AOLS. When diabetic animals were treated with daily injections of the VAP-1 inhibitor (0.3 mg/kg), leukocyte transmigration rate was significantly reduced (P < 0.05). However, firm adhesion of leukocytes in diabetic animals treated with the inhibitor did not differ significantly from vehicle-treated diabetic controls. This work provides evidence for an important role of VAP-1 in the recruitment of leukocyte to the retina in experimental DR. Our results reveal the critical contribution of VAP-1 to leukocyte transmigration, with little impact on firm leukocyte adhesion in the retinas of diabetic animals. VAP-1 inhibition might be beneficial in the treatment of DR.

The CD40-ATP-P2X 7 Receptor Pathway: Cell to Cell Cross-Talk to Promote Inflammation and Programmed Cell Death of Endothelial Cells.

Extracellular adenosine 5'-triphosphate (ATP) functions not only as a neurotransmitter but is also released by non-excitable cells and mediates cell-cell communication involving glia. In pathological conditions, extracellular ATP released by astrocytes may act as a "danger" signal that activates microglia and promotes neuroinflammation. This review summarizes in vitro and in vivo studies that identified CD40 as a novel trigger of ATP release and purinergic-induced inflammation. The use of transgenic mice with expression of CD40 restricted to retinal Müller glia and a model of diabetic retinopathy (a disease where the CD40 pathway is activated) established that CD40 induces release of ATP in Müller glia and triggers in microglia/macrophages purinergic receptor-dependent inflammatory responses that drive the development of retinopathy. The CD40-ATP-P2X7 pathway not only amplifies inflammation but also induces death of retinal endothelial cells, an event key to the development of capillary degeneration and retinal ischemia. Taken together, CD40 expressed in non-hematopoietic cells is sufficient to mediate inflammation and tissue pathology as well as cause death of retinal endothelial cells. This process likely contributes to development of degenerate capillaries, a hallmark of diabetic and ischemic retinopathies. Blockade of signaling pathways downstream of CD40 operative in non-hematopoietic cells may offer a novel means of treating diabetic and ischemic retinopathies.

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor ß (PDGFRß) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

Plasma Exosomes Contribute to Microvascular Damage in Diabetic Retinopathy by Activating the Classical Complement Pathway.

Diabetic retinopathy (DR) is a microvascular complication of diabetes and is the leading cause of vision loss in working-age adults. Recent studies have implicated the complement system as a player in the development of vascular damage and progression of DR. However, the role and activation of the complement system in DR are not well understood. Exosomes, small vesicles that are secreted into the extracellular environment, have a cargo of complement proteins in plasma, suggesting that they can participate in causing the vascular damage associated with DR. We demonstrate that IgG-laden exosomes in plasma activate the classical complement pathway and that the quantity of these exosomes is increased in diabetes. Moreover, we show that a lack of IgG in exosomes in diabetic mice results in a reduction in retinal vascular damage. The results of this study demonstrate that complement activation by IgG-laden plasma exosomes could contribute to the development of DR.

The effect of Toxoplasma gondii infection on expression of chemokines by rat retinal vascular endothelial cells.

Cells infected by Toxoplasma gondii undergo up-regulation of proinflammatory cytokines, organelle redistribution, and protection from apoptosis. During infection in man, the parasite encysts within the retina, a process that results in retinochoroiditis which can lead to permanent loss of sight. The reasons for the parasite to infect retinal tissue and the mechanisms by which it encysts are not clearly understood. We studied the effect of infection with T. gondii of retinal vascular endothelial cells using the Clontech Atlastrade mark array system in order to elucidate changes in gene expression. We compared hybridization of RNA to the array from infected and uninfected cells at two time points; 2 and 24 h. Exposure to T. gondii after 2 h resulted in change of expression of approximately 6% of genes on the array, including those involved in cell structure, protein and vesicle trafficking, cell-cycle regulation, transcriptional and translational machinery, and apoptosis. Among the genes involved in the inflammatory response, chemokine genes such as GRO1 (Growth Regulated Oncogene 1), MCP-1 (Monocyte Chemotactic Protein-1), FKN (Fractalkine) and RANTES (Regulated upon Activation, Normal T Cell Expressed and Secreted) were found to be up-regulated and protein production was confirmed by ELISA. However after 24 h of infection, GRO1, MCP-1 and FKN were down-regulated, confirmed by RT-PCR. Thus, invasion of retinal vascular endothelium (RVE) cells by T. gondii leads to the production of chemokines important in directing the traffic of inflammatory cells to the infected area.

Reduction in shear stress, activation of the endothelium, and leukocyte priming are all required for leukocyte passage across the blood--retina barrier.

The passage of leukocytes across the blood-retina barrier at the early stages of an inflammatory reaction is influenced by a complex series of interactions about which little is known. In particular, the relationship between hydrodynamic factors, such as shear stress and leukocyte velocity, to the adherence and subsequent extravasation of leukocytes into the retina is unclear. We have used a physiological method, scanning laser ophthalmoscopy, to track labeled leukocytes circulating in the retina, followed by confocal microscopy of retinal flatmounts to detect infiltrating cells at the early stage of experimental autoimmune uveitis. This has shown that retinal vessels are subjected to high shear stress under normal circumstances. During the inflammatory reaction, shear stress in retinal veins is reduced 24 h before leukocyte infiltration. This reduction is negatively correlated with leukocyte rolling and sticking in veins and postcapillary venules, the sites of leukocyte extravasation. Activation of vascular endothelial cells is also a prerequisite for leukocyte rolling and infiltration. In addition, antigen priming of leukocytes is influential at the early stage of inflammation, and this is seen clearly in the reduction in rolling velocity and adherence of the primed leukocytes in activated retinal venules, 9 days postimmunization.

Leukocyte trafficking in experimental autoimmune uveitis in vivo.

Leukocyte trafficking from blood into tissue is a fundamental process in immune surveillance and the immune response to stimuli. Experimental autoimmune uveitis (EAU) is an animal model for posterior uveitis and is mediated by T lymphocytes and macrophages that infiltrate the posterior segment of the eye. To analyze leukocyte migration into retinal tissue during the course of EAU, labeled cells were identified in vivo by scanning laser ophthalmoscopy and in retinal flatmounts by confocal microscopy. Adhesion of blood leukocytes to retinal endothelial cells in vivo was significantly raised 48 h before the appearance of clinical disease, and this correlated with the increased expression of CD54 on retinal vessels. Mitogen-activated spleen cells and CD4+ T cells only entered into retinal tissue in animals with clinical disease and not naive recipients. The disease status of the donor animal had no effect on leukocyte trafficking. These results, which identify leukocyte-endothelial cell interactions in vivo, suggest that the activation of the retinal endothelium is a prerequisite to leukocyte adhesion and extravasation into ocular tissue during EAU.

Diabetic Retinopathy: Vascular and Inflammatory Disease.

Diabetic retinopathy (DR) is the leading cause of visual impairment in the working-age population of the Western world. The pathogenesis of DR is complex and several vascular, inflammatory, and neuronal mechanisms are involved. Inflammation mediates structural and molecular alterations associated with DR. However, the molecular mechanisms underlying the inflammatory pathways associated with DR are not completely characterized. Previous studies indicate that tissue hypoxia and dysregulation of immune responses associated with diabetes mellitus can induce increased expression of numerous vitreous mediators responsible for DR development. Thus, analysis of vitreous humor obtained from diabetic patients has made it possible to identify some of the mediators (cytokines, chemokines, and other factors) responsible for DR pathogenesis. Further studies are needed to better understand the relationship between inflammation and DR. Herein the main vitreous-related factors triggering the occurrence of retinal complication in diabetes are highlighted.

Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).

The microvascular and cellular responses in the retina during acute EAE were characterized using whole-mount preparations. The earliest detectable event was the accumulation of monocytes and T cells within veins on day 7 postinduction (pi). Mild breakdown of the blood-retinal barrier (BRB), activation of microglia and infiltration of monocytes and T cells into the retinal parenchyma were first evident on days 7 to 8 pi. Monocyte adhesion to the vessel wall and breakdown of the BRB were colocalized in the same vessel segments and occurred predominantly in veins. The marked difference in response observed in the retina versus the myelinated region of the optic nerve suggests that two types of inflammatory cascades are initiated. A mild response, characterised by very low numbers of T cells and monocytes and an absence of expression of MHC class II by resident microglia, is initiated when only small amounts of the encephalitogenic antigen are present in the perivascular space or associated with perivascular antigen-presenting cells. A full blown inflammatory reaction, as observed in the optic nerve, is initiated in the presence of substantial amounts of encephalitogenic antigen. This severe response is characterised by the infiltration of large numbers of CD4+, CD8+ T cells and ED1+ monocytes, and by abundant MHC class II expression by resident microglia as well as other cell types. Thus, MHC class II expression by resident microglia may be a possible effective amplifier mechanism if the encephalitogenic antigen is encountered in the tissue parenchyma.

Müller cells in vascular and avascular retinae: a survey of seven mammals.

Eight monoclonal antibodies were used to label Müller cells in four mammals that have vascular retinae (cats, dogs, humans, and rats) and in three with avascular retinae (echidnas, guinea pigs, and rabbits). Müller cells were found to have a fairly uniform retinal distribution in seven species, with a mean density of 8,000-13,000 cells mm-2. Müller cells in avascular retinae differ from their vascular counterparts in four respects. First, they are shorter than those in vascular retinae. This difference is mainly due to a reduction in the thickness of the outer nuclear layer. Second, the trunks of Müller cells in avascular retinae tend to be thicker, although those in echidnas are an exception to this trend. Third, Müller cell rootlets in avascular retinae follow a more tortuous course than those in vascular retinae, reflecting the fact that photoreceptor nuclei in the two types of retina have different shapes and stacking patterns. Fourth, due to a reduction in the density of photoreceptors in avascular retinae, there are fewer neurones per Müller cell. Although these four features may enable Müller cells to assist the nutrition of neurones in the inner layers of avascular retinae, they are unlikely to be morphological specializations that have evolved for that purpose. Rather, these features appear to be a direct consequence of the fact that avascular retinae are thinner and have a differently organised outer nuclear layer. These features aside, Müller cells in avascular retinae closely resemble their counterparts in vascular retinae.