Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.
The polycystin family of transient receptor potential (TRP) channels form Ca2+ regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca2+ signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca2+ activated, had a maximum slope conductance of 114 pS and could be blocked in a dose-dependent manner by increasing concentrations of Mg2+. The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca2+ release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca2+ signaling.
polycystin; transient receptor potential channel; retinal ganglion cells; calcium; electrophysiology
Axotomy of central neurons leads to functional and structural alterations which largely revert when neural progenitor cells (NPCs) are implanted in the lesion site. The new microenvironment created by NPCs in the host tissue might modulate in the damaged neurons the expression of a high variety of molecules with relevant roles in the repair mechanisms, including neurotrophic factors. In the present work, we aimed to analyze changes in neurotrophic factor expression in axotomized neurons induced by NPC implants. For this purpose, we performed immunofluorescence followed by confocal microscopy analysis for the detection of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) on brainstem sections from rats with axotomy of abducens internuclear neurons that received NPC implants (implanted group) or vehicle injections (axotomized group) in the lesion site. Control abducens internuclear neurons were strongly immunoreactive to VEGF and BDNF but showed a weak staining for NT-3 and NGF. Comparisons between groups revealed that lesioned neurons from animals that received NPC implants showed a significant increase in VEGF content with respect to animals receiving vehicle injections. However, the immunoreactivity for BDNF, which was increased in the axotomized group as compared to control, was not modified in the implanted group. The modifications induced by NPC implants on VEGF and BDNF content were specific for the population of axotomized abducens internuclear neurons since the neighboring abducens motoneurons were not affected. Similar levels of NT-3 and NGF immunolabeling were obtained in injured neurons from axotomized and implanted animals. Among all the analyzed neurotrophic factors, only VEGF was expressed by the implanted cells in the lesion site. Our results point to a role of NPC implants in the modulation of neurotrophic factor expression by lesioned central neurons, which might contribute to the restorative effects of these implants.
Recently, multiple neurotrophic/growth factors have been proposed to play an important role in the therapeutic action of antidepressants. In this study, we prepared astrocyte- and neuron-enriched cultures from the neonatal rat cortex, and examined the changes in neurotrophic/growth factor expression by antidepressant treatment using real-time PCR. Treatment with amitriptyline (a tricyclic antidepressant) significantly increased the expression of fibroblast growth factor-2 (FGF-2), brain-derived neurotrophic factor, vascular endothelial growth factor and glial cell line-derived neurotrophic factor mRNA with a different time course in astrocyte cultures, but not in neuron-enriched cultures. Only the expression of FGF-2 was higher in astrocyte cultures than in neuron-enriched cultures. We focused on the FGF-2 production in astrocytes. Several different classes of antidepressants, but not non-antidepressants, also induced FGF-2 mRNA expression. Noradrenaline (NA) is known to induce FGF-2 expression in astrocyte cultures, as with antidepressants. Therefore, we also assessed the mechanism of NA-induced FGF-2 expression, in comparison to amitriptyline. NA increased the FGF-2 mRNA expression via α1 and β-adrenergic receptors; however, the amitriptyline-induced FGF-2 mRNA expression was not mediated via these adrenergic receptors. Furthermore, the amitriptyline-induced FGF-2 mRNA expression was completely blocked by cycloheximide (an inhibitor of protein synthesis), while the NA-induced FGF-2 mRNA was not. These data suggest that the regulation of FGF-2 mRNA expression by amitriptyline was distinct from that by NA. Taken together, antidepressant-stimulated astrocytes may therefore be important mediators that produce several neurotrophic/growth factors, especially FGF-2, through a monoamine-independent and a de novo protein synthesis-dependent mechanism.
Inherited retinal degenerations afflict 1 in 3,500 individuals and are a heterogeneous group of diseases that result in profound vision loss, usually the result of retinal neuronal apoptosis. Atrophic changes in the retinal vasculature are also observed in many of these degenerations. While it is thought that this atrophy is secondary to diminished metabolic demand in the face of retinal degeneration, the precise relationship between the retinal neuronal and vascular degeneration is not clear. In this study we demonstrate that whenever a fraction of mouse or human adult bone marrow–derived stem cells (lineage-negative hematopoietic stem cells [Lin– HSCs]) containing endothelial precursors stabilizes and rescues retinal blood vessels that would ordinarily completely degenerate, a dramatic neurotrophic rescue effect is also observed. Retinal nuclear layers are preserved in 2 mouse models of retinal degeneration, rd1 and rd10, and detectable, albeit severely abnormal, electroretinogram recordings are observed in rescued mice at times when they are never observed in control-treated or untreated eyes. The normal mouse retina consists predominantly of rods, but the rescued cells after treatment with Lin– HSCs are nearly all cones. Microarray analysis of rescued retinas demonstrates significant upregulation of many antiapoptotic genes, including small heat shock proteins and transcription factors. These results suggest a new paradigm for thinking about the relationship between vasculature and associated retinal neuronal tissue as well as a potential treatment for delaying the progression of vision loss associated with retinal degeneration regardless of the underlying genetic defect.
Polycystin-1 (PC1) is a large, membrane-bound protein that localizes to the cilia and is implicated in the common ciliopathy autosomal-dominant polycystic kidney disease. The physiological function of PC1 is dependent upon its subcellular localization as well as specific cleavages that release soluble fragments of its C-terminal tail. The techniques described here allow visualization and quantification of these aspects of the biology of the PC1 protein. To visualize PC1 at the plasma membrane, a live-cell surface labeling immunofluorescence protocol paired with the labeling of an internal antigen motif allows a robust detection of the surface population of this protein. This technique is modified to generate a surface enzyme-linked immunosorbent assay (ELISA), which quantitatively measures the amount of surface protein as a fraction of the total amount of the protein expressed in that cell population. These assays are powerful tools in the assessment of the small but biologically important pool of PC1 that reaches the cell surface. The C-terminal tail cleavage of PC1 constitutes an interesting modification that allows PC1 to extend its functional role into the nucleus. A reporter assay based on Gal4/VP16 luciferase can be used to quantitate the amount of PC1 C-terminal tail that reaches the nucleus. This assay can be paired with quantitative measurement of the protein expression in the cell, allowing a more complete understanding of the pattern of PC1 cleavage and the nuclear localization of the resultant.
Neuronal activity evokes localized changes in blood flow, a response termed neurovascular coupling. One widely recognized hypothesis of neurovascular coupling holds that glial cell depolarization evoked by neuronal activity leads to the release of K+ onto blood vessels (K+ siphoning) and to vessel relaxation. We now present two direct tests of this glial cell-K+ siphoning hypothesis of neurovascular coupling. Potassium efflux was evoked from glial cells in the rat retina by applying depolarizing current pulses to individual cells. Glial depolarizations as large as 100 mV produced no change in the diameter of adjacent arterioles. We also monitored light-evoked vascular responses in Kir4.1 knock-out mice, where functional Kir K+ channels are absent from retinal glial cells. The magnitude of light-evoked vasodilations was identical in Kir4.1 knock-out and wild-type animals. Contrary to the hypothesis, the results demonstrate that glial K+ siphoning in the retina does not contribute significantly to neurovascular coupling.
blood flow; glia; astrocytes; Müller cells; Kir4.1; potassium siphoning; retina
Iron accumulation is associated with age-related neurodegenerations and may contribute to age-related increased susceptibility of neurons to damage. We compared young and old rodent retinas to assess iron homeostasis during normal aging and the effects of increased iron on the susceptibility of retinal neurons to degeneration. Retinal iron was significantly increased with age. Quantitative RT-PCR showed that transferrin and ferritin genes were upregulated in the aged retina. At the protein level, we found decreased transferrin, and increased transferrin receptor, ferritin, ferroportin, and ceruloplasmin in the aged retina. These results support an increased steady state of iron with age in the retina. We tested susceptibility of retinal neurons with increased intracellular iron to damage in vitro. Exposure of RGC-5 cells to increased iron potentiated the neurotoxicity induced by paraquat, glutamate and TNF α. Our results demonstrate that iron homeostasis in the retina is altered with age and suggest that iron accumulation, due to altered levels of iron regulatory proteins in the aged retina, could be a susceptibility factor in age-related retinal diseases.
iron; aging; susceptibility; neurodegeneration; RGC-5; stress
Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.
RESEARCH DESIGN AND METHODS
After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 μg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1–30 μg/kg pHBSP or control peptide).
pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01–0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.
Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.
Although primary cilia are increasingly recognized to play sensory roles in several cellular systems, their role in vascular smooth muscle cells (VSMCs) has not been defined. We examined in situ position/orientation of primary cilia and ciliary proteins in VSMCs and tested the hypothesis that primary cilia of VSMCs exert sensory functions. By immunofluorescence and electron microscopic imaging, primary cilia of VSMCs were positioned with their long axis aligned at 58.3° angle in relation to the cross-sectional plane of the artery, projecting into the extracellular matrix (ECM). Polycystin-1, polycystin-2 and α3- and β1-integrins are present in cilia. In scratch wound experiments, the majority of cilia were repositioned to the cell-wound interface. Such repositioning was largely abolished by a β1-integrin blocker. Moreover, compared to non-ciliated/deciliated cells, ciliated VSMCs showed more efficient migration in wound repair. Lastly, when directly stimulated with collagen (an ECM component and cognate ligand for α3β1-integrins) or induced ciliary deflection, VSMCs responded with a rise in [Ca2+]i that is dependent on the presence of cilia. Taken together, primary cilia of VSMCs are preferentially oriented, possess proteins critical for cell-ECM interaction and mechanosensing and respond to ECM protein and mechanical stimulations. These observations suggest a role for primary cilia in mechanochemical sensing in vasculature.
Primary cilia; Vascular smooth muscle cells; Mechanosensing; Intracellular calcium; Integrins
Diabetic retinopathy is widely considered to be a neurovascular disease. This is in contrast to its previous identity as solely a vascular disease. Early in the disease progression of diabetes, the major cells in the neuronal component of the retina consist of retinal ganglion cells and glial cells, both of which have been found to be compromised. A number of retinal function tests also indicated a functional deficit in diabetic retina, which further supports dysfunction of neuronal cells. As an endocrinological disorder, diabetes alters metabolism both systemically and locally in several body organs, including the retina. A growing body of evidences indicates increased levels of excitotoxic metabolites, including glutamate, branched chain amino acids and homocysteine in cases of diabetic retinopathy. Also present, early in the disease, are decreased levels of folic acid and vitamin-B12, which are potential metabolites capable of damaging neurons. These altered levels of metabolites are found to activate several metabolic pathways, leading to increases in oxidative stress and decreases in the level of neurotrophic factors. As a consequence, they may damage retinal neurons in diabetic patients. In this review, we have discussed those potential excitotoxic metabolites and their implications in neuronal damage. Possible therapeutic targets to protect neurons are also discussed. However, further research is needed to understand the exact molecular mechanism of neurodegeneration so that effective neuroprotection strategies can be developed. By protecting retinal neurons early in diabetic retinopathy cases, damage of retinal vessels can be protected, thereby helping to ameliorate the progression of diabetic retinopathy, a leading cause of blindness worldwide.
diabetic retinopathy; drugs; neurodegeneration; retina; oxidative stress; glutamate
Toxoplasmic retinochoroiditis is a common blinding retinal infection caused by the parasite, Toxoplasma gondii. Basic processes relating to establishment of infection in the human eye by T. gondii tachyzoites have not been investigated. To evaluate the ability of tachyzoites to navigate the human retina, we developed an ex vivo assay, in which a suspension containing 1.5×107 parasites replaced vitreous in a posterior eyecup. After 8 hours, the retina was formalin-fixed and paraffin-embedded, and sections were immunostained to identify tachyzoites. To determine the preference of tachyzoites for human retinal neuronal versus glial populations, we infected dissociated retinal cultures, subsequently characterized by neuron-specific enolase or glial fibrillary acidic protein expression, and retinal cell lines, with YFP-expressing tachyzoites. In migration assays, retinas contained 110–250 live tachyzoites; 64.5–95.2% (mean = 79.6%) were localized to the nerve fiber layer, but some were detected in the outer retina. Epifluorescence imaging of dissociated retinal cultures 24 hours after infection indicated preferential infection of glia. This observation was confirmed in growth assays, with significantly higher (p≤0.005) numbers of tachyzoites measured in glial verus neuronal cell lines. Our translational studies indicate that, after entering retina, tachyzoites may navigate multiple tissue layers. Tachyzoites preferentially infect glial cells, which exist throughout the retina. These properties may contribute to the success of T. gondii as a human pathogen.
Recent studies have described a novel type of glial cell that is scattered across the inner layers of the avian retina and possibly the retinas of primates. These cells have been termed Non-astrocytic Inner Retinal Glial (NIRG) cells. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and become reactive. These changes in glial activity correlate with increased susceptibility of retinal neurons and Müller glia to excitotoxic damage. The purpose of this study was to further study the NIRG cells in retinas treated with IGF1 or acute damage. In response to IGF1, the reactivity, proliferation and migration of NIRG cells persists through 3 days after treatment. At 7 days after treatment, the numbers and distribution of NIRG cells returns to normal, suggesting that homeostatic mechanisms are in place within the retina to maintain the numbers and distribution of these glial cells. By comparison, IGF1-induced microglial reactivity persists for at least 7 days after treatment. In damaged retinas, we find a transient accumulation of NIRG cells, which parallels the accumulation of reactive microglia, suggesting that the reactivity of NIRG cells and microglia are linked. When the microglia are selectively ablated by the combination of interleukin 6 and clodronate-liposomes, the NIRG cells down-regulate transitin and perish within the following week, suggesting that the survival and phenotype of NIRG cells are somehow linked to the microglia. We conclude that the abundance, reactivity and retinal distribution of NIRG cells can be dynamic, are regulated by homoestatic mechanisms and are tethered to the microglia.
We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the vascular wall accompanies PAI-1, MCP-1, iNOS and Tissue Factor expression. Furthermore we show evidence that Ang II causes the upregulation of NF-kB in our model.
We started PDTC-treatment on four weeks old dTGR (200 mg/kg sc) and age-matched SD rats.. Blood-pressure- and albuminuria- measurements were monitored during the treatement period (four weeks). The seven weeks old animals were killed, hearts and kidneys were isolated and used for immunohistochemical-and electromobility shift assay analsis.
Chronic treatment with the antioxidant PDTC decreased blood pressure (162 ± 8 vs. 190 ± 7 mm Hg, p = 0.02). Cardiac hypertrophy index was significantly reduced (4.90 ± 0.1 vs. 5.77 ± 0.1 mg/g, p < 0.001) compared to dTGR. PDTC reduced 24 h albuminuria by 85 % (2.7 ± 0.5 vs. 18.0 ± 3.4 mg/d, p < 0.001) and prevented death significantly. Vascular injury was ameliorated in small renal and cardiac vessels. PDTC inhibited NF-κB binding activity in heart and kidney. Immunohistochemical analysis shows increased expression of the p65 NF-κB subunit in the endothelium, smooth muscles cells of damaged small vessels, infiltrated cells, glomeruli, tubuli and collecting ducts of dTGR. PDTC markedly reduced the immunoreactivity of p65.
Our data show that inhibition of NF-κB by PDTC markedly reduces inflammation, iNOS expression in the dTGR most likely leading to decreased cytotoxicity, and cell proliferation. Thus, NF-κB activation plays an important role in ANG II-induced end-organ damage.
To clarify the effects on the visual pathway that occur following retinal damage, we examined the morphological alterations present in the superior colliculus (SC) after N-methyl-D-aspartate (NMDA)-induced retinal damage in mice.
NMDA was injected into the vitreous body of the left eye in mice to induce retinal damage. The time-dependent neuronal degeneration in the SC was assessed using immunohistochemistry.
The number of neuronal nuclear specific protein (NeuN)-immunostained neurons showed a significant decrease in the contralateral SC at both 90 and 180 days after intravitreal NMDA injection. In contrast, the ipsilateral SC displayed no significant change in the number of NeuN-positive cells. An increase in glial fibrillary acid protein (GFAP) immunoreactivity was observed in the contralateral SC at 7, 30, and 90 days after NMDA injection and in the ipsilateral SC at 7 days, while brain-derived neurotrophic factor (BDNF) expression was increased in the contralateral SC at 30 and 90 days. In the contralateral SC, some GFAP-positive astroglial cells also exhibited BDNF at 30 days after NMDA injection.
Evidence of time-dependent morphological neuronal degeneration along the retinocollicular pathway from the retina to the SC was detected at 90 and 180 days, but not at 30 days, after NMDA-induced retinal damage. This neurodegeneration was preceded by an increase in BDNF expression in the SC, specifically at 30 and 90 days after NMDA injection. Hence, these findings may provide useful information concerning the pathological mechanisms of several disorders accompanied by retinal degeneration.
The vasodegenerative phase of diabetic retinopathy is characterized by not only retinal vascular degeneration but also inadequate vascular repair due to compromised bone marrow derived endothelial progenitor cells (EPCs). We propose that n-3 polyunsaturated fatty acid (PUFA) deficiency in diabetes results in activation of the central enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM) and that ASM represents a molecular metabolic link connecting the initial damage in the retina and the dysfunction of EPCs.
Research Design and Methods
Type 2 diabetic rats on control or docosahexaenoic acid (DHA)-rich diet were studied. The number of acellular capillaries in the retinas was assessed by trypsin digest. mRNA levels of interleukin (IL)-1β, IL-6, intracellular adhesion molecule (ICAM)-1 in the retinas from diabetic animals were compared to controls and ASM protein was assessed by western analysis. EPCs were isolated from blood and bone marrow and their numbers and ability to form colonies in vitro, ASM activity and lipid profiles were determined.
DHA-rich diet prevented diabetes-induced increase in the number of retinal acellular capillaries and significantly enhanced the life span of type 2 diabetic animals. DHA-rich diet blocked upregulation of ASM and other inflammatory markers in diabetic retina and prevented the increase in ASM activity in EPCs, normalized the numbers of circulating EPCs and improved EPC colony formation.
In a type 2 diabetes animal model, DHA-rich diet fully prevented retinal vascular pathology through inhibition of ASM in both retina and EPCs, leading to a concomitant suppression of retinal inflammation and correction of EPC number and function.
The damaged zebrafish retina replaces lost neurons through a regenerative response that initiates with the asymmetric cell division of Müller glia to produce neuronal progenitor cells that proliferate and migrate to the damaged retinal layer, where they differentiate into the lost neuronal cell types. Because Müller glia are known to phagocytose apoptotic retinal cells during development, we tested if Müller glia engulfed apoptotic rod cell bodies in light-damaged retinas. After 24 hours of constant intense light, damaged retinas revealed both a strong nuclear TUNEL signal in photoreceptors and a weak cytoplasmic TUNEL signal in Müller glia, although Müller glial apoptosis is not observed in the light-damaged retina. Light damage of a rod-specific transgenic reporter line, Tg(XlRho:EGFP)fl1, resulted in some Müller glia containing both TUNEL signal and EGFP, which indicated that this subset of Müller glia engulfed apoptotic photoreceptor cell bodies.
To determine if phagocytosis induced the Müller glial proliferative response in the light-damaged retina, we utilized O-phospho-L-serine (L-SOP), a molecule that mimics the phosphatidylserine head group and partially blocks microglial phagocytosis of apoptotic cells. Intravitreal injection of L-SOP immediately prior to beginning constant intense light treatment: i) did not significantly reduce light-induced photoreceptor cell death, ii) significantly reduced the number of PCNA-positive Müller glia, and iii) significantly reduced the number of cone photoreceptors in the regenerated retina relative to control retinas. Because L-SOP is also a specific group III metabotropic glutamate receptor (mGluR) agonist, we also tested if the more potent specific group III agonist, L-2-amino-4-phosphonobutyrate (L-AP4), the specific group III antagonist (RS)-α-Methylserine-O-phosphate (MSOP) or the specific group I antagonist, L-2-amino-3-phophonopropanoic acid (L-AP3) affected Müller glial proliferation. We found no changes with any of these factors compared to control retinas, revealing that metabotropic glutamate receptors were not necessary in the Müller glia proliferative response. Furthermore, ascl1a and stat3 expression were unaffected in either the L-SOP or MSOP-injected retinas relative to controls, suggesting L-SOP disrupts Müller glia proliferation subsequent to or in parallel with ascl1a and stat3 activation. This implies that at least one signaling mechanism, in addition to the process disrupted by L-SOP, is required to activate Müller glia proliferation in the light-damaged retina.
regeneration; Müller glia; retina; phosphatidylserine; type III metabotropic glutamate receptor; phagocytosis
To investigate the anatomic and functional changes triggered by light exposure in the albino mouse retina and compare them with those observed in the albino rat.
BALB/c albino mice were exposed to 3,000 lx of white light during 24 h and their retinas analyzed from 1 to 180 days after light exposure (ALE). Left pupil mydriasis was induced with topical atropine. Retinal function was analyzed by electroretinographic (ERG) recording. To assess retinal degeneration, hematoxylin and eosin staining, the TdT-mediated dUTP nick-end labeling (TUNEL) technique, and quantitative immunohistofluorescence for synaptophysin and protein kinase Cα (PKCα) were used in cross sections. Intravenous injection of horseradish peroxidase and Fluoro-Gold™ tracing were used in whole-mounted retinas to study the retinal vasculature and the retinal ganglion cell (RGC) population, respectively.
Light exposure caused apoptotic photoreceptor death in the central retina. This death was more severe in the dorsal than in the ventral retina, sparing the periphery. Neither retinal vascular leakage nor retinal ganglion cell death was observed ALE. The electroretinographic a-wave was permanently impaired, while the b-wave decreased but recovered gradually by 180 days ALE. The scotopic threshold responses, associated with the inner retinal function, diminished at first but recovered completely by 14 days ALE. This functional recovery was concomitant with the upregulation of protein kinase Cα and synaptophysin. Similar results were obtained in both eyes, irrespective of mydriasis.
In albino mice, light exposure induces substantial retinal damage, but the surviving photoreceptors, together with compensatory morphological/molecular changes, allow an important restoration of the retinal function.
Ciliary neurotrophic factor (CNTF), a member of the interleukin-6 cytokine family, has been implicated in the development, differentiation and survival of retinal neurons. The mechanisms of CNTF action as well as its cellular targets in the retina are poorly understood. It has been postulated that some of the biological effects of CNTF are mediated through its action via retinal glial cells; however, molecular changes in retinal glia induced by CNTF have not been elucidated. We have, therefore, examined gene expression dynamics of purified Müller (glial) cells exposed to CNTF in vivo.
Müller cells were flow-sorted from mgfap-egfp transgenic mice one or three days after intravitreal injection of CNTF. Microarray analysis using RNA from purified Müller cells showed differential expression of almost 1,000 transcripts with two- to seventeen-fold change in response to CNTF. A comparison of transcriptional profiles from Müller cells at one or three days after CNTF treatment showed an increase in the number of transcribed genes as well as a change in the expression pattern. Ingenuity Pathway Analysis showed that the differentially regulated genes belong to distinct functional types such as cytokines, growth factors, G-protein coupled receptors, transporters and ion channels. Interestingly, many genes induced by CNTF were also highly expressed in reactive Müller cells from mice with inherited or experimentally induced retinal degeneration. Further analysis of gene profiles revealed 20–30% overlap in the transcription pattern among Müller cells, astrocytes and the RPE.
Our studies provide novel molecular insights into biological functions of Müller glial cells in mediating cytokine response. We suggest that CNTF remodels the gene expression profile of Müller cells leading to induction of networks associated with transcription, cell cycle regulation and inflammatory response. CNTF also appears to function as an inducer of gliosis in the retina.
The purpose of this study was to determine whether the expression of ER stress-related factors IRE1α, apoptosis signal-regulating kinase 1 (ASK1), SAPK/ERK kinase 1 (SEK1) and c-Jun N-terminal kinase (JNK) is associated with the damaged retinal neurons induced by ischemia-reperfusion injury. After 60 minutes of ischemia, the rat retinas were reperfused, and retinas were isolated and fixed after 6, 9, 12, 18, and 24 hours, and 2, 5, and 9 days of reperfusion. Cryosections were immunostained with Fluoro-Jade B, a degenerating neuron marker to label degenerating neurons. Semi-quantitative analysis of the expression of IRE1α, ASK1, SEK1, and JNK were performed in both control and ischemic retinas. In ischemic retinas, the intensities of IRE1α immunoreactivity in the ganglion cell layer (GCL) were significantly higher than in the control retinas. In ischemic retinas, the numbers of SEK1-, ASK1-, and JNK-positive cells were significantly increased in the GCL compared to those in the control retinas. In addition, the cells that were positive for SEK1-, ASK1-, and JNK were also positive for Fluoro-Jade B-positive cells. These results indicate that the increased expression of ER stress-related factors was, in part, associated with the retinal neuronal abnormalities after ischemia-reperfusion injury in rat retinas.
endoplasmic reticulum; IRE1α; apoptosis signal-regulating kinase 1; SAPK/ERK kinase 1; c-Jun N-terminal kinase; Fluoro-Jade B; ischemia-reperfusion injury
Glial cell line-derived neurotrophic factor (GDNF) is neuroprotective of retinal neurons, and transduced retinal progenitor cells (RPCs) can deliver this cytokine for the treatment of retinal diseases, yet the potential effects of GDNF on RPCs have received little attention.
Murine RPCs were assessed under multiple conditions in the presence or absence of epidermal growth factor (EGF, 20 ng/ml) and/or GDNF (10 ng/ml) using a variety of techniques, including live-cell imaging, caspase-3 activity assay, whole genome microarray, quantitative polymerase chain reaction (qPCR), and western blotting.
Live monitoring revealed that formation of initial aggregates resulted largely from the collision and adherence of dissociated RPCs, as opposed to clonal proliferation. Spheres enlarged in size and number, with more reaching the threshold criteria for cross-sectional areas in the EGF+GDNF condition. Proliferation was measurably augmented in association with EGF+GDNF, and Ki-67 expression was modestly increased (1.07 fold), as were hairy and enhancer of split 5 (Hes5), mammalian achaete-scute homolog 1 (Mash1), and Vimentin. However, global gene expression did not reveal a notable treatment-related response, and the expression of the majority of progenitor and lineage markers examined remained stable. GDNF reduced RPC apoptosis, compared to complete growth-factor withdrawal, although it could not by itself sustain mitotic activity.
These data support the feasibility of developing GDNF-transduced RPCs as potential therapeutic agents for use in retinal diseases.
Cardiovascular complications such as hypertension are a continuous concern in patients with autosomal dominant polycystic kidney disease (ADPKD). The PKD2 encoding for polycystin-2 is mutated in ≈15% of ADPKD patients. Here, we show that polycystin-2 is localized to the cilia of mouse and human vascular endothelial cells. We demonstrate that the normal expression level and localization of polycystin-2 to cilia is required for the endothelial cilia to sense fluid shear stress through a complex biochemical cascade, involving calcium, calmodulin, Akt/PKB, and protein kinase C. In response to fluid shear stress, mouse endothelial cells with knockdown or knockout of Pkd2 lose the ability to generate nitric oxide (NO). Consistent with mouse data, endothelial cells generated from ADPKD patients do not show polycystin-2 in the cilia and are unable to sense fluid flow. In the isolated artery, we further show that ciliary polycystin-2 responds specifically to shear stress and not to mechanical stretch, a pressurized biomechanical force that involves purinergic receptor activation. We propose a new role for polycystin-2 in transmitting extracellular shear stress to intracellular NO biosynthesis. Thus, aberrant expression or localization of polycystin-2 to cilia could promote high blood pressure because of inability to synthesize NO in response to an increase in shear stress (blood flow).
biophysical force; endothelia; mechanotransduction; primary cilium; shear stress
Neuronal activity leads to arteriole dilation and increased blood flow in retinal vessels. This response, termed functional hyperemia, is diminished in the retinas of diabetic patients, possibly contributing to the development of diabetic retinopathy. The mechanism responsible for this loss is unknown. Here we show that light-evoked arteriole dilation was reduced by 58% in a streptozotocin-induced rat model of type 1 diabetes. Functional hyperemia is believed to be mediated by glial cells and we found that glial-evoked vasodilation was reduced by 60% in diabetic animals. The diabetic retinas showed neither a decrease in the thickness of the retinal layers nor an increase in neuronal loss, although signs of early glial reactivity and an upregulation of inducible nitric oxide synthase (iNOS) were detected. Inhibition of iNOS restored both light- and glial-evoked dilations to control levels. These findings suggest that high NO levels resulting from iNOS upregulation alters glial control of vessel diameter and may underlie the loss of functional hyperemia observed in diabetic retinopathy. Restoring functional hyperemia by iNOS inhibition may limit the progression of retinopathy in diabetic patients.
iNOS; glial cells; neurovascular coupling; diabetes; retina
The authors examined the neuroprotective effect of BDNF-secreting mesenchymal stem cells on retina and optic nerve function and structure in hypertensive eyes.
To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH).
COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells (RGCs) and evaluating optic nerve cross-sections.
After transplantation into COH eyes, BDNF-MSCs preserved significantly more retina and optic nerve function than GFP-MSC–treated eyes when pupil light reflex (PLR) and ERG function were evaluated. PLR analysis showed significantly better function (P = 0.03) in BDNF-MSC–treated eyes (operated/control ratio = 63.00% ± 11.39%) than GFP-MSC–treated eyes (operated/control ratio = 31.81% ± 9.63%) at 42 days after surgery. The BDNF-MSC–transplanted eyes also displayed a greater level of RGC preservation than eyes that received the GFP-MSCs only (RGC cell counts: BDNF-MSC–treated COH eyes, 112.2 ± 19.39 cells/section; GFP-MSC–treated COH eyes, 52.21 ± 11.54 cells/section; P = 0.01).
The authors have demonstrated that lentiviral-transduced BDNF-producing MSCs can survive in eyes with chronic hypertension and can provide retina and optic nerve functional and structural protection. Transplantation of BDNF-producing stem cells may be a viable treatment strategy for glaucoma.