Methods to Identify Rat and Mouse Retinal Ganglion Cells in Retinal Flat-Mounts.

The identification of distinct retinal ganglion cell (RGC) populations in flat-mounted retinas is key to investigating pathological or pharmacological effects in these cells. In this chapter, we review the main techniques for detecting the total population of RGCs and various of their subtypes in whole-mounted retinas of pigmented and albino rats and mice, four of the animal strains most studied by the scientific community in the retina field. These methods are based on the studies published by the Vidal-Sanz's laboratory.

7,8-Dihydroxiflavone Maintains Retinal Functionality and Protects Various Types of RGCs in Adult Rats with Optic Nerve Transection.

To analyze the neuroprotective effects of 7,8-Dihydroxyflavone (DHF) in vivo and ex vivo, adult albino Sprague-Dawley rats were given a left intraorbital optic nerve transection (IONT) and were divided in two groups: One was treated daily with intraperitoneal (ip) DHF (5 mg/kg) (n = 24) and the other (n = 18) received ip vehicle (1% DMSO in 0.9% NaCl) from one day before IONT until processing. At 5, 7, 10, 12, 14, and 21 days (d) after IONT, full field electroretinograms (ERG) were recorded from both experimental and one additional naïve-control group (n = 6). Treated rats were analyzed 7 (n = 14), 14 (n = 14) or 21 d (n = 14) after IONT, and the retinas immune stained against Brn3a, Osteopontin (OPN) and the T-box transcription factor T-brain 2 (Tbr2) to identify surviving retinal ganglion cells (RGCs) (Brn3a+), α-like (OPN+), α-OFF like (OPN+Brn3a+) or M4-like/α-ON sustained RGCs (OPN+Tbr+). Naïve and right treated retinas showed normal ERG recordings. Left vehicle-treated retinas showed decreased amplitudes of the scotopic threshold response (pSTR) (as early as 5 d), the rod b-wave, the mixed response and the cone response (as early as 10 d), which did not recover with time. In these retinas, by day 7 the total numbers of Brn3a+RGCs, OPN+RGCs and OPN+Tbr2+RGCs decreased to less than one half and OPN+Brn3a+RGCs decreased to approximately 0.5%, and Brn3a+RGCs showed a progressive loss with time, while OPN+RGCs and OPN+Tbr2+RGCs did not diminish after seven days. Compared to vehicle-treated, the left DHF-treated retinas showed significantly greater amplitudes of the pSTR, normal b-wave values and significantly greater numbers of OPN+RGCs and OPN+Tbr2+RGCs for up to 14 d and of Brn3a+RGCs for up to 21 days. DHF affords significant rescue of Brn3a+RGCs, OPN+RGCs and OPN+Tbr2+RGCs, but not OPN+Brn3a+RGCs, and preserves functional ERG responses after IONT.

MMP2 Modulates Inflammatory Response during Axonal Regeneration in the Murine Visual System.

Neuroinflammation has been put forward as a mechanism triggering axonal regrowth in the mammalian central nervous system (CNS), yet little is known about the underlying cellular and molecular players connecting these two processes. In this study, we provide evidence that MMP2 is an essential factor linking inflammation to axonal regeneration by using an in vivo mouse model of inflammation-induced axonal regeneration in the optic nerve. We show that infiltrating myeloid cells abundantly express MMP2 and that MMP2 deficiency results in reduced long-distance axonal regeneration. However, this phenotype can be rescued by restoring MMP2 expression in myeloid cells via a heterologous bone marrow transplantation. Furthermore, while MMP2 deficiency does not affect the number of infiltrating myeloid cells, it does determine the coordinated expression of pro- and anti-inflammatory molecules. Altogether, in addition to its role in axonal regeneration via resolution of the glial scar, here, we reveal a new mechanism via which MMP2 facilitates axonal regeneration, namely orchestrating the expression of pro- and anti-inflammatory molecules by infiltrating innate immune cells.

Tightening the retinal glia limitans attenuates neuroinflammation after optic nerve injury.

Increasing evidence suggests that functional impairments at the level of the neurovascular unit (NVU) underlie many neurodegenerative and neuroinflammatory diseases. While being part of the NVU, astrocytes have been largely overlooked in this context and only recently, tightening of the glia limitans has been put forward as an important neuroprotective response to limit these injurious processes. In this study, using the retina as a central nervous system (CNS) model organ, we investigated the structure and function of the glia limitans, and reveal that the blood-retina barrier and glia limitans function as a coordinated double barrier to limit infiltration of leukocytes and immune molecules. We provide in vitro and in vivo evidence for a protective response at the NVU upon CNS injury, which evokes inflammation-induced glia limitans tightening. Matrix metalloproteinase-3 (MMP-3) was found to be a crucial regulator of this process, thereby revealing its beneficial and immunomodulatory role in the CNS. in vivo experiments in which MMP-3 activity was deleted via genetic and pharmacological approaches, combined with a comprehensive study of tight junction molecules, glial end feet markers, myeloid cell infiltration, cytokine expression and neurodegeneration, show that MMP-3 attenuates neuroinflammation and neurodegeneration by tightening the glia limitans, thereby pointing to a prominent role of MMP-3 in preserving the integrity of the NVU upon injury. Finally, we gathered promising evidence to suggest that IL1b, which is also regulated by MMP-3, is at least one of the molecular messengers that induces glia limitans tightening in the injured CNS.

Activation of adenosine A3 receptor protects retinal ganglion cells from degeneration induced by ocular hypertension.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness. This disease is characterized by optic nerve damage and retinal ganglion cell (RGC) death. The current treatments available target the lowering of intraocular pressure (IOP), the main risk factor for disease onset and development. However, in some patients, vision loss progresses despite successful IOP control, indicating that new and effective treatments are needed, such as those targeting the neuroprotection of RGCs. Adenosine A3 receptor (A3R) activation confers protection to RGCs following an excitotoxic stimulus. In this work, we investigated whether the activation of A3R could also afford protection to RGCs in the laser-induced ocular hypertension (OHT) model, a well-characterized animal model of glaucoma. The intravitreal injection of 2-Cl-IB-MECA, a selective A3R agonist, abolished the alterations induced by OHT in the negative and positive components of scotopic threshold response (STR) without changing a- and b-wave amplitudes both in scotopic and photopic conditions. Moreover, the treatment of OHT eyes with the A3R agonist promoted the survival of RGCs, attenuated the impairment in retrograde axonal transport, and improved the structure of the optic nerve. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA, we can envisage that A3R activation can be considered a good therapeutic strategy to protect RGCs from glaucomatous damage.

Tracing the retina to analyze the integrity and phagocytic capacity of the retinal pigment epithelium.

We have developed a new technique to study the integrity, morphology and functionality of the retinal neurons and the retinal pigment epithelium (RPE). Young and old control albino (Sprague-Dawley) and pigmented (Piebald Virol Glaxo) rats, and dystrophic albino (P23H-1) and pigmented (Royal College of Surgeons) rats received a single intravitreal injection of 3% Fluorogold (FG) and their retinas were analyzed from 5 minutes to 30 days later. Retinas were imaged in vivo with SD-OCT and ex vivo in flat-mounts and in cross-sections. Fifteen minutes and 24 hours after intravitreal administration of FG retinal neurons and the RPE, but no glial cells, were labeled with FG-filled vesicles. The tracer reached the RPE 15 minutes after FG administration, and this labeling remained up to 30 days. Tracing for 15 minutes or 24 hours did not cause oxidative stress. Intraretinal tracing delineated the pathological retinal remodelling occurring in the dystrophic strains. The RPE of the P23H-1 strain was highly altered in aged animals, while the RPE of the RCS strain, which is unable to phagocytose, did not accumulate the tracer even at young ages when the retinal neural circuit is still preserved. In both dystrophic strains, the RPE cells were pleomorphic and polymegathic.

Systemic and Intravitreal Antagonism of the TNFR1 Signaling Pathway Delays Axotomy-Induced Retinal Ganglion Cell Loss.

Here, we have blocked the signaling pathway of tumor necrosis factor α (TNFα) in a mouse model of traumatic neuropathy using a small cell permeable molecule (R7050) that inhibits TNFα/TNF receptor 1 (TNFR1) complex internalization. Adult pigmented mice were subjected to intraorbital optic nerve crush (ONC). Animals received daily intraperitoneal injections of R7050, and/or a single intravitreal administration the day of the surgery. Some animals received a combinatorial treatment with R7050 (systemic or local) and a single intravitreal injection of brain derived neurotrophic factor (BDNF). As controls, untreated animals were used. Retinas were analyzed for RGC survival 5 and 14 days after the lesion i.e., during the quick and slow phase of axotomy-induced RGC death. qPCR analyses were done to verify that Tnfr1 and TNFα were up-regulated after ONC. At 5 days post-lesion, R7050 intravitreal or systemic treatment neuroprotected RGCs as much as BDNF alone. At 14 days, RGC rescue by systemic or intravitreal administration of R7050 was similar. At this time point, intravitreal treatment with BDNF was significantly better than intravitreal R7050. Combinatory treatment was not better than BDNF alone, although at both time points, the mean number of surviving RGCs was higher. In conclusion, antagonism of the extrinsic pathway of apoptosis rescues axotomized RGCs as it does the activation of survival pathways by BDNF. However, manipulation of both pathways at the same time, does not improve RGC survival.

Mesenchymal stromal cell therapy for damaged retinal ganglion cells, is gold all that glitters?

Mesenchymal stromal cells are an excellent source of stem cells because they are isolated from adult tissues or perinatal derivatives, avoiding the ethical concerns that encumber embryonic stem cells. In preclinical models, it has been shown that mesenchymal stromal cells have neuroprotective and immunomodulatory properties, both of which are ideal for central nervous system treatment and repair. Here we will review the current literature on mesenchymal stromal cells, focusing on bone marrow mesenchymal stromal cells, adipose-derived mesenchymal stromal cells and mesenchymal stromal cells from the umbilical cord stroma, i.e., Wharton's jelly mesenchymal stromal cells. Finally, we will discuss the use of these cells to alleviate retinal ganglion cell degeneration following axonal trauma.

Optogenetic Stimulation of the Superior Colliculus Confers Retinal Neuroprotection in a Mouse Glaucoma Model.

Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) in the eye, which ultimately results in visual impairment or even blindness. Because current therapies often fail to halt disease progression, there is an unmet need for novel neuroprotective therapies to support RGC survival. Various research lines suggest that visual target centers in the brain support RGC functioning and survival. Here, we explored whether increasing neuronal activity in one of these projection areas could improve survival of RGCs in a mouse glaucoma model. Prolonged activation of an important murine RGC target area, the superior colliculus (SC), was established via a novel optogenetic stimulation paradigm. By leveraging the unique channel kinetics of the stabilized step function opsin (SSFO), protracted stimulation of the SC was achieved with only a brief light pulse. SSFO-mediated collicular stimulation was confirmed by immunohistochemistry for the immediate-early gene c-Fos and behavioral tracking, which both demonstrated consistent neuronal activity upon repeated stimulation. Finally, the neuroprotective potential of optogenetic collicular stimulation was investigated in mice of either sex subjected to a glaucoma model and a 63% reduction in RGC loss was found. This work describes a new paradigm for optogenetic collicular stimulation and a first demonstration that increasing target neuron activity can increase survival of the projecting neurons.SIGNIFICANCE STATEMENT Despite glaucoma being a leading cause of blindness and visual impairment worldwide, no curative therapies exist. This study describes a novel paradigm to reduce retinal ganglion cell (RGC) degeneration underlying glaucoma. Building on previous observations that RGC survival is supported by the target neurons to which they project and using an innovative optogenetic approach, we increased neuronal activity in the mouse superior colliculus, a main projection target of rodent RGCs. This proved to be efficient in reducing RGC loss in a glaucoma model. Our findings establish a new optogenetic paradigm for target stimulation and encourage further exploration of the molecular signaling pathways mediating retrograde neuroprotective communication.

A Hydroxypyrone-Based Inhibitor of Metalloproteinase-12 Displays Neuroprotective Properties in Both Status Epilepticus and Optic Nerve Crush Animal Models.

Recently, we showed that matrix metalloproteinase-12 (MMP-12) is highly expressed in microglia and myeloid infiltrates, which are presumably involved in blood⁻brain barrier (BBB) leakage and subsequent neuronal cell death that follows status epilepticus (SE). Here, we assessed the effects of a hydroxypyrone-based inhibitor selective for MMP-12 in the pilocarpine-induced SE rat model to determine hippocampal cell survival. In the hippocampus of rats treated with pilocarpine, intra-hippocampal injections of the MMP-12 inhibitor protected Cornu Ammonis 3 (CA3) and hilus of dentate gyrus neurons against cell death and limited the development of the ischemic-like lesion that typically develops in the CA3 stratum lacunosum-moleculare of the hippocampus. Furthermore, we showed that MMP-12 inhibition limited immunoglobulin G and albumin extravasation after SE, suggesting a reduction in BBB leakage. Finally, to rule out any possible involvement of seizure modulation in the neuroprotective effects of MMP-12 inhibition, neuroprotection was also observed in the retina of treated animals after optic nerve crush. Overall, these results support the hypothesis that MMP-12 inhibition can directly counteract neuronal cell death and that the specific hydroxypyrone-based inhibitor used in this study could be a potential therapeutic agent against neurological diseases/disorders characterized by an important inflammatory response and/or neuronal cell loss.

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system.

Due to the lack of axonal regeneration, age-related deterioration in the central nervous system (CNS) poses a significant burden on the wellbeing of a growing number of elderly. To overcome this regenerative failure and to improve the patient's life quality, the search for novel regenerative treatment strategies requires valuable (animal) models and techniques. As an extension of the CNS, the retinofugal system, consisting of retinal ganglion cells that send their axons along the optic nerve to the visual brain areas, has importantly contributed to the current knowledge on mechanisms underlying the restricted regenerative capacities and to the development of novel strategies to enhance axonal regeneration. It provides an extensively used research tool, not only in amniote vertebrates including rodents, but also in anamniote vertebrates, such as zebrafish. Indeed, the latter show robust regeneration capacities, thereby providing insights into the factors that contribute to axonal regrowth and proper guidance, complementing studies in mammals. This review provides an integrative and critical overview of the classical and state-of-the-art models and methods that have been employed in the retinofugal system to advance our knowledge on the signaling pathways underlying the restricted versus robust axonal regeneration in rodents and zebrafish, respectively. In vitro, ex vivo and in vivo models and techniques to improve the visualization and analysis of regenerating axons are summarized. As such, the retinofugal system is presented as a valuable model to further facilitate research on axonal regeneration and to open novel therapeutic avenues for CNS pathologies.

Microglial dynamics after axotomy-induced retinal ganglion cell death.

BACKGROUND: Microglial cells (MCs) are the sentries of the central nervous system. In health, they are known as surveying MCs because they examine the tissue to maintain the homeostasis. In disease, they activate and, among other functions, become phagocytic to clean the cellular debris. In this work, we have studied the behavior of rat retinal MCs in two models of unilateral complete intraorbital optic nerve axotomy which elicit a different time course of retinal ganglion cell (RGC) loss. METHODS: Albino Sprague-Dawley rats were divided into these groups: (a) intact (no surgery), (b) fluorogold (FG) tracing from the superior colliculi, and (c) FG tracing + crush or transection of the left optic nerve. The retinas were dissected from 2 days to 2 months after the lesions (n = 4-12 group/lesion and time point) and then were subjected to Brn3a and Iba1 double immunodetection. In each intact retina, the total number of Brn3a+RGCs and Iba+MCs was quantified. In each traced retina (b and c groups), FG-traced RGCs and phagocytic microglial cells (PMCs, FG+Iba+) were also quantified. Topographical distribution was assessed by neighbor maps. RESULTS: In intact retinas, surveying MCs are homogenously distributed in the ganglion cell layer and the inner plexiform layer. Independently of the axotomy model, RGC death occurs in two phases, one quick and one protracted, and there is a lineal and topographical correlation between the appearance of PMCs and the loss of traced RGCs. Furthermore, the clearance of FG+RGCs by PMCs occurs 3 days after the actual loss of Brn3a expression that marks RGC death. In addition, almost 50% of MCs from the inner plexiform layer migrate to the ganglion cell layer during the quick phase of RGC loss, returning to the inner plexiform layer during the slow degeneration phase. Finally, in contrast to what happens in mice, in rats, there is no microglial phagocytosis in the contralateral uninjured retina. CONCLUSIONS: Axotomy-induced RGC death occurs earlier than RGC clearance and there is an inverse correlation between RGC loss and PMC appearance, both numerically and topographically, suggesting that phagocytosis occurs as a direct response to RGC death rather than to axonal damage.

Shared and Differential Retinal Responses against Optic Nerve Injury and Ocular Hypertension.

Glaucoma, one of the leading causes of blindness worldwide, affects primarily retinal ganglion cells (RGCs) and their axons. The pathophysiology of glaucoma is not fully understood, but it is currently believed that damage to RGC axons at the optic nerve head plays a major role. Rodent models to study glaucoma include those that mimic either ocular hypertension or optic nerve injury. Here we review the anatomical loss of the general population of RGCs (that express Brn3a; Brn3a+RGCs) and of the intrinsically photosensitive RGCs (that express melanopsin; m+RGCs) after chronic (LP-OHT) or acute (A-OHT) ocular hypertension and after complete intraorbital optic nerve transection (ONT) or crush (ONC). Our studies show that all of these insults trigger RGC death. Compared to Brn3a+RGCs, m+RGCs are more resilient to ONT, ONC, and A-OHT but not to LP-OHT. There are differences in the course of RGC loss both between these RGC types and among injuries. An important difference between the damage caused by ocular hypertension or optic nerve injury appears in the outer retina. Both axotomy and LP-OHT induce selective loss of RGCs but LP-OHT also induces a protracted loss of cone photoreceptors. This review outlines our current understanding of the anatomical changes occurring in rodent models of glaucoma and discusses the advantages of each one and their translational value.

Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury.

Axonal injury is a common feature of central nervous system insults that culminates with the death of the affected neurons, and an irreversible loss of function. Inflammation is an important component of the neurodegenerative process, where the microglia plays an important role by releasing proinflammatory factors as well as clearing the death neurons by phagocytosis. Here we have identified the purinergic signaling through the P2X7 receptor as an important component for the neuronal death in a model of optic nerve axotomy. We have found that in P2X7 receptor deficient mice there is a delayed loss of retinal ganglion cells and a decrease of phagocytic microglia at early times points after axotomy. In contralateral to the axotomy retinas, P2X7 receptor controlled the numbers of phagocytic microglia, suggesting that extracellular ATP could act as a danger signal activating the P2X7 receptor in mediating the loss of neurons in contralateral retinas. Finally, we show that intravitreal administration of the selective P2X7 receptor antagonist A438079 also delays axotomy-induced retinal ganglion cell death in retinas from wild type mice. Thus, our work demonstrates that P2X7 receptor signaling is involved in neuronal cell death after axonal injury, being P2X7 receptor antagonism a potential therapeutic strategy.

MMP-3 Deficiency Alleviates Endotoxin-Induced Acute Inflammation in the Posterior Eye Segment.

Matrix metalloproteinase-3 (MMP-3) is known to mediate neuroinflammatory processes by activating microglia, disrupting blood-central nervous system barriers and supporting neutrophil influx into the brain. In addition, the posterior part of the eye, more specifically the retina, the retinal pigment epithelium (RPE) and the blood-retinal barrier, is affected upon neuroinflammation, but a role for MMP-3 during ocular inflammation remains elusive. We investigated whether MMP-3 contributes to acute inflammation in the eye using the endotoxin-induced uveitis (EIU) model. Systemic administration of lipopolysaccharide induced an increase in MMP-3 mRNA and protein expression level in the posterior part of the eye. MMP-3 deficiency or knockdown suppressed retinal leukocyte adhesion and leukocyte infiltration into the vitreous cavity in mice subjected to EIU. Moreover, retinal and RPE mRNA levels of intercellular adhesion molecule 1 (Icam1), interleukin 6 (Il6), cytokine-inducible nitrogen oxide synthase (Nos2) and tumor necrosis factor α (Tnfα), which are key molecules involved in EIU, were clearly reduced in MMP-3 deficient mice. In addition, loss of MMP-3 repressed the upregulation of the chemokines monocyte chemoattractant protein (MCP)-1 and (C-X-C motif) ligand 1 (CXCL1). These findings suggest a contribution of MMP-3 during EIU, and its potential use as a therapeutic drug target in reducing ocular inflammation.

Differential visual system organization and susceptibility to experimental models of optic neuropathies in three commonly used mouse strains.

Mouse disease models have proven indispensable in glaucoma research, yet the complexity of the vast number of models and mouse strains has also led to confusing findings. In this study, we evaluated baseline intraocular pressure, retinal histology, and retinofugal projections in three mouse strains commonly used in glaucoma research, i.e. C57Bl/6, C57Bl/6-Tyr(c), and CD-1 mice. We found that the mouse strains under study do not only display moderate variations in their intraocular pressure, retinal architecture, and retinal ganglion cell density, also the retinofugal projections to the dorsal lateral geniculate nucleus and the superior colliculus revealed striking differences, potentially underlying diverging optokinetic tracking responses and visual acuity. Next, we reviewed the success rate of three models of (glaucomatous) optic neuropathies (intravitreal N-methyl-d-aspartic acid injection, optic nerve crush, and laser photocoagulation-induced ocular hypertension), looking for differences in disease susceptibility between these mouse strains. Different genetic backgrounds and albinism led to differential susceptibility to experimentally induced retinal ganglion cell death among these three mouse strains. Overall, CD-1 mice appeared to have the highest sensitivity to retinal ganglion cell damage, while the C57Bl/6 background was more resistant in the three models used.

Tackling Glaucoma from within the Brain: An Unfortunate Interplay of BDNF and TrkB.

According to the neurotrophin deprivation hypothesis, diminished retrograde delivery of neurotrophic support during an early stage of glaucoma pathogenesis is one of the main triggers that induce retinal ganglion cell (RGC) degeneration. Therefore, interfering with neurotrophic signaling seems an attractive strategy to achieve neuroprotection. Indeed, exogenous neurotrophin administration to the eye has been shown to reduce loss of RGCs in animal models of glaucoma; however, the neuroprotective effect was mostly insufficient for sustained RGC survival. We hypothesized that treatment at the level of neurotrophin-releasing brain areas might be beneficial, as signaling pathways activated by target-derived neurotrophins are suggested to differ from pathways that are initiated at the soma membrane. In our study, first, the spatiotemporal course of RGC degeneration was characterized in mice subjected to optic nerve crush (ONC) or laser induced ocular hypertension (OHT). Subsequently, the well-known neurotrophin brain-derived neurotrophic factor (BDNF) was chosen as the lead molecule, and the levels of BDNF and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), were examined in the mouse retina and superior colliculus (SC) upon ONC and OHT. Both models differentially influenced BDNF and TrkB levels. Next, we aimed for RGC protection through viral vector-mediated upregulation of collicular BDNF, thought to boost the retrograde neurotrophin delivery. Although the previously reported temporary neuroprotective effect of intravitreally delivered recombinant BDNF was confirmed, viral vector-induced BDNF overexpression in the SC did not result in protection of the RGCs in the glaucoma models used. These findings most likely relate to decreased neurotrophin responsiveness upon vector-mediated BDNF overexpression. Our results highlight important insights concerning the complexity of neurotrophic factor treatments that should surely be considered in future neuroprotective strategies.

Retinal neurodegeneration in experimental glaucoma.

In rats and mice, limbar tissues of the left eye were laser-photocoagulated (LP) and ocular hypertension (OHT) effects were investigated 1 week to 6 months later. To investigate the innermost layers, retinas were examined in wholemounts using tracing from the superior colliculi to identify retinal ganglion cells (RGCs) with intact retrograde axonal transport, melanopsin immunodetection to identify intrinsically photosensitive RGCs (m(+)RGC), Brn3a immunodetection to identify most RGCs but not m(+)RGCs, RECA1 immunodetection to examine the inner retinal vessels, and DAPI staining to detect all nuclei in the GC layer. The outer retinal layers (ORLs) were examined in cross sections analyzed morphometrically or in wholemounts to study S- and L-cones. Innervation of the superior colliculi was examined 10 days to 14 weeks after LP with orthogradely transported cholera toxin subunit B. By 2 weeks, OHT resulted in pie-shaped sectors devoid of FG(+)RGCs or Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. Brn3a(+)RGCs were significantly greater than FG(+)RGCs, indicating the survival of large numbers of RGCs with their axonal transport impaired. The inner retinal vasculature showed no abnormalities that could account for the sectorial loss of RGCs. m(+)RGCs decreased to approximately 50-51% in a diffuse loss across the retina. Cross sections showed focal areas of degeneration in the ORLs. RGC loss at 1m diminished to 20-25% and did not progress further with time, whereas the S- and L-cone populations diminished progressively up to 6m. The retinotectal projection was reduced by 10 days and did not progress further. LP-induced OHT results in retrograde degeneration of RGCs and m(+)RGCs, severe damage to the ORL, and loss of retinotectal terminals.

Decreased TNF Levels and Improved Retinal Ganglion Cell Survival in MMP-2 Null Mice Suggest a Role for MMP-2 as TNF Sheddase.

Matrix metalloproteinases (MMPs) have been designated as both friend and foe in the central nervous system (CNS): while being involved in many neurodegenerative and neuroinflammatory diseases, their actions appear to be indispensable to a healthy CNS. Pathological conditions in the CNS are therefore often related to imbalanced MMP activities and disturbances of the complex MMP-dependent protease network. Likewise, in the retina, various studies in animal models and human patients suggested MMPs to be involved in glaucoma. In this study, we sought to determine the spatiotemporal expression profile of MMP-2 in the excitotoxic retina and to unravel its role during glaucoma pathogenesis. We reveal that intravitreal NMDA injection induces MMP-2 expression to be upregulated in the Müller glia. Moreover, MMP-2 null mice display attenuated retinal ganglion cell death upon excitotoxic insult to the retina, which is accompanied by normal glial reactivity, yet reduced TNF levels. Hence, we propose a novel in vivo function for MMP-2, as an activating sheddase of tumor necrosis factor (TNF). Given the pivotal role of TNF as a proinflammatory cytokine and neurodegeneration-exacerbating mediator, these findings generate important novel insights into the pathological processes contributing to glaucomatous neurodegeneration and into the interplay of neuroinflammation and neurodegeneration in the CNS.

Transient Downregulation of Melanopsin Expression After Retrograde Tracing or Optic Nerve Injury in Adult Rats.

PURPOSE: To investigate the effect of retrograde tracing or axotomy on melanopsin mRNA expression and immunodetection in albino and pigmented rat retinas. METHODS: Groups were (1) intact-naïve retinas; (2) optic nerve crush (ONC) analyzed at 7 days (7d) or 2 months (2m); (3) Fluorogold (FG) tracing from the superior colliculi (SCi) analyzed at 7d or 2m; (4) tracing from the intact optic nerve (ON) with FG or hydroxystilbamidine methanesulfonate (OHSt), analyzed 3d later; and (5) sham tracing from the ON or sham surgery. Brn3a and melanopsin were double stained in whole mounts to quantify and assess the distribution of orthotopic and displaced Brn3a(+) retinal ganglion cells (Brn3a(+)RGCs) and melanopsin(+)RGCs (m(+)RGCs). Freshly dissected retinas were used for melanopsin mRNA quantitative PCR. RESULTS: Tracing from the SCi did not affect the number of Brn3a(+)RGCs or m(+)RGCs counted in pigmented rats. However, only 55% of m(+)RGCs were immunodetected in albinos at 7d, although by 2m the m(+)RGCs counts returned to normal. Optic nerve tracing had a more dramatic effect (38% or 77% of m(+)RGCs were immunodetected in albino or pigmented rats) that occurred irrespectively of the tracer (OHSt or FG). This effect was not observed in the sham groups. After ONC, Brn3a(+)RGCs decreased to 37% and 8% by 7d and 2m, respectively. Melanopsin (+)RGC counts diminished to 30% at 7d, but recovered to 49% of controls by 2m. Melanopsin mRNA was downregulated after ON tracing or 7d after ONC, but did not differ from intact values 2m after ONC. CONCLUSIONS: Following ON injury or retrograde tracing there is a transient melanopsin downregulation that should be taken into account when assessing m(+)RGC survival.