A corneo-retinal hypercitrullination axis underlies ocular injury to nitrogen mustard.

The vesicant sulfur mustard (SM) is a chemical warfare agent that causes acute and chronic injury to the cornea and proximal anterior segment structures. Despite clinical evidence of SM-exposure causing unexplained retinal deficits, there have been no animal studies conducted to examine the retinal toxicity of this vesciant. The cardinal hallmark of retinal response to stressors or injury is the activation of reactive gliosis, a cellular process largely governed by Müller glia. Previously we showed that corneal exposure to sodium hydroxide elicits rapid induction of reactive gliosis and results in retinal degeneration in a dose-related manner. Based on this evidence, we hypothesized that the vesicant nitrogen mustard (NM), an analog of SM, may also elicit reactive gliosis. To test this idea, we developed a mouse model of NM ocular injury and investigated corneal and retinal effects focusing on citrullination, a posttranslational modification (PTM) of proteins. This PTM was recently linked to alkali injury and has also been shown to occur in retinal degenerative conditions. Here, we demonstrate that corneal exposure to 1% NM causes a synchronous activation of citrullination in both the cornea and retina with hypercitrullination becoming apparent temporally and manifesting with altered cellular expression characteristics. A key finding is that ocular citrullination occurs acutely as early as 1-h post-injury in both the cornea and retina, which underscores a need for expeditious interception of this acute corneal and retinal response. Moreover, exploiting dose response and temporal studies, we uncoupled NM-induced retinal citrullination from its induction of retinal gliosis. Our findings demonstrate that hypercitrullination is a common corneo-retinal mechanism that sensitizes the eye to NM injury and suggests that counteracting hypercitrullination may provide a suitable countermeasure to vesicant injury.

Raloxifene, a cannabinoid type-2 receptor inverse agonist, mitigates visual deficits and pathology and modulates microglia after ocular blast.

Visual deficits after ocular blast injury (OBI) are common, but pharmacological approaches to improve long-term outcomes have not been identified. Blast forces frequently damage the retina and optic nerves, and work on experimental animals has shown the pro-inflammatory actions of microglia can further exacerbate such injuries. Cannabinoid type-2 receptor (CB2) inverse agonists specifically target activated microglia, biasing them away from the harmful pro-inflammatory M1 state toward the helpful reparative M2 state. We previously found that treating mice with CB2 inverse agonists after traumatic brain injury, produced by either focal cranial air blast or dorsal cranial impact, greatly attenuated the visual deficits and pathology that otherwise resulted. Here we examined the consequences of single and repeat OBI and the benefit provided by raloxifene, an FDA-approved estrogen receptor drug that possesses noteworthy CB2 inverse agonism. After single OBI, although the amplitudes of the A- and B-waves of the electroretinogram and pupil light response appeared to be normal, the mice showed hints of deficits in contrast sensitivity and visual acuity, a trend toward optic nerve axon loss, and significantly increased light aversion, which were reversed by 2 weeks of daily treatment with raloxifene. Mice subjected to repeat OBI (5 blasts spaced 1 min apart), exhibited more severe visual deficits, including decreases in contrast sensitivity, visual acuity, the amplitudes of the A- and B-waves of the electroretinogram, light aversion, and resting pupil diameter (i.e. hyperconstriction), accompanied by the loss of photoreceptor cells and optic nerve axons, nearly all of which were mitigated by raloxifene. Interestingly, optic nerve axon abundance was strongly correlated with contrast sensitivity and visual acuity across all groups of experimental mice in the repeat OBI study, suggesting optic nerve axon loss with repeat OBI and its attenuation with raloxifene are associated with the extent of these two deficits while photoreceptor abundance was highly correlated with A-wave amplitude and resting pupil size, suggesting a prominent role for photoreceptors in these two deficits. Quantitative PCR (qPCR) showed levels of M1-type microglial markers (e.g. iNOS, IL1ß, TNFα, and CD32) in retina, optic nerve, and thalamus were increased 3 days after repeat OBI. With raloxifene treatment, the overall expression of M1 markers was more similar to that in sham mice. Raloxifene treatment was also associated with the elevation of IL10 transcripts in all three tissues compared to repeat OBI alone, but the results for the three other M2 microglial markers we examined were more varied. Taken together, the qPCR results suggest that raloxifene benefit for visual function and pathology was associated with a lessening of the pro-inflammatory actions of microglia. The benefit we find for raloxifene following OBI provides a strong basis for phase-2 efficacy testing in human clinical trials for treating ocular injury.

Regulation of mitophagy by metformin improves the structure and function of retinal ganglion cells following excitotoxicity-induced retinal injury.

Excitotoxicity-induced retinal neuronal death is characterized by the progressive retinal ganglion cell (RGC) apoptosis. Strategies are needed to reduce neurodegeneration. Recent investigations have indicated the potential effects of metformin on multiple systems, especially in the networks. However, it also remains unclear whether mitophagy contributes to the neuroprotective effect of metformin on the retina. In this study, excitotoxicity-induced retinal injury models were constructed. In vitro, R28 cells were treated with calcium ionophore and metformin/phosphate-buffer saline (PBS). Cell viability, lactate dehydrogenase release, and the cellular apoptosis rate were assessed. In vivo, rats received intravitreal injection of N-methyl-D-aspartate and metformin/PBS. Comprehensive examinations including retrograde fluorescent gold labelling, Nissl's staining, full-field electroretinography, photopic negative response, optic coherence tomography and retinal imaging, transmission electron microscopy, western blotting, and quantitative polymerase chain reaction were conducted during the observation period. The viability of R28 cells was significantly increased in the metformin-treated group compared with the negative control group, while, the release of lactate dehydrogenase and R28 cell apoptosis showed a significant decrease. In vivo, metformin treatment significantly increased the number of surviving RGCs, the b/NR wave amplitude and the thickness of the inner retina but had no obvious adverse effects on the fundus. In the metformin-treated group, the morphology and number of mitochondria were better preserved, as observed for RGCs; mitochondrial autophagosomes were located in RGCs, as indicated by transmission electron microscopy; and the expression of mitophagy-related genes and proteins presented was significant regulated. These data indicated that the regulation of mitophagy by metformin improved the structure and function of RGCs.

Research models of sulfur mustard- and nitrogen mustard-induced ocular injuries and potential therapeutics.

Sulfur mustard (SM) is a notorious, bifunctional alkylating vesicant that was first used in warfare during World War I in 1917 and since then has been deployed in numerous skirmishes with its most recent documented use being during the Middle Eastern conflicts. Apart from its use in combat and terrorist activities, continual threat of accidental exposure from old stockpiles and improperly discarded munitions is ever present, especially to the innocent and unassuming civilian populations. SM can cause devastating injuries, depending on the dosage of SM exposure, route of exposure, as well as the physiological conditions of the individuals exposed. The most common routes of exposure are ocular, dermal, and exposure to the lungs and respiratory tissues through inhalation. Eyes are the most susceptible organ to SM-induced toxicities owing to their high moisture content and rapidly dividing cells. Additionally, ocular injury causes the most expeditious disablement of individuals even upon whole-body exposures. Therefore, it is imperative to understand the mechanisms underlying SM-induced ocular toxicity and design therapeutic interventions to prevent/mitigate ocular injuries. Ocular SM exposure may cause a wide range of symptoms such as inflammation, lacrimation, itching, dryness, photophobia, edema of the cornea/sclera/retina/iris, conjunctivitis, degradation of the corneal layer, fusion of two or more ocular layers, neovascularization, fibrosis, and temporary or permanent structural damage to one or more ocular layers. These symptoms may lead to vision impairments, resulting in partial or complete blindness that may be permanent. The highly toxic and exceedingly notorious nature of SM makes it a highly regulated chemical, requiring very expensive licensing, security, and safety requirements; thus, the more easily accessible analogue, nitrogen mustard (NM) that mimics SM-induced toxicity and injuries is employed in plethora of studies conducted in different animal models and culture systems. This review provides a comprehensive account of the injuries and symptoms that occur upon ocular SM exposures in human patients as well as studies in animal (in vivo, ex vivo) and cell (in vitro) models of SM and NM ocular exposures. Special emphasis has been laid on highlighting the strengths and lacunae in the research as well as the possible unexplored avenues of mechanisms underlying mustard-induced ocular injury that can be explored in future research endeavors. Furthermore, development of therapeutic interventions and targets of interest in the ocular system exposed to SM and NM, based on studies in human patients as well as in vivo, ex vivo, and in vitro models has been discussed in great depth, providing a valuable knowledge database to delineate pathways associated with vesicant-induced toxicity, and strategies/diagnostic tools against SM-induced toxicity.

Effect of trans-resveratrol on glutamate clearance and visual behaviour in rats with glutamate induced retinal injury.

Glutamate-induced excitotoxic injury is widely described as a prominent pathophysiological mechanism in several neurodegenerative diseases including glaucoma. Glaucoma, the leading cause of irreversible blindness, is characterized by loss of retinal ganglion cells (RGC). Currently, the treatment focuses on lowering intraocular pressure (IOP) and no neuroprotective therapies are available. Since excessive glutamate-mediated neurotransmission underlies glaucomatous RGC apoptosis, enhancing synaptic glutamate clearance by glutamate transporters in glial cells is expected to protect against excitotoxic injury. Trans-resveratrol is known for its neuroprotective effects; however, its effects on the expression of glutamate transporters and glutamate clearance in retina remain unclear. Hence, in the current study, we investigated the protective effects of trans-resveratrol against glutamate-induced retinal injury in rats. Rats were intravitreally injected with glutamate alone or glutamate with trans-resveratrol as pre- and post-treatment. Animals were subjected to Open Field Test (OFT) on day six and a two-chamber mirror test on day seven post-injection. Subsequently, rats were sacrificed and retinal expression of excitatory amino acid transporter (EAAT)1 and EAAT2 gene and protein was determined using PCR and ELISA, respectively. Retinal glutamate concentration was measured using ELISA and retinal morphology was studied on H&E-stained retinal sections. It was observed that pre-treatment with trans-resveratrol causes gene expression for EAAT1 and EAAT2 to increase by 2.51 and 1.93 folds compared to glutamate-treated group (p < 0.001 and p < 0.01, respectively); while the same in trans-resveratrol post-treatment group showed a 1.58- and 1.44 folds upregulation (p < 0.05).The retinal EAAT1 and EAAT2 protein expression was significantly greater in trans-resveratrol pre-treatment group compared to glutamate-treated group (p < 0.05) but not in post-treatment group. Retinal glutamate concentration was1.64 folds greater in glutamate-treated group than the vehicle-treated group (p < 0.01) but the same was 1.27-fold lower in trans-resveratrol pre-treatment group compared to glutamate-treated group (p < 0.01). Corresponding to these findings, we observed preservation of retinal morphology and visual behaviour in trans-resveratrol pre-treatment group compared to glutamate-treated group. We did not observe similar effects of trans-resveratrol when it was given as post-treatment after glutamate administration. In conclusion, current study showed that pre-treatment with trans-resveratrol protects against glutamate induced changes in retinal morphology and visual behaviour by increasing the expression of EAAT1 and EAAT2 and increasing glutamate clearance in rat retinas. The results of this study may be relevant to disease conditions involving excitotoxic neuronal injury.

Mesenchymal stem cell secretome protects against oxidative stress-induced ocular blast visual pathologies.

Visual deficits are a common concern among subjects with head trauma. Stem cell therapies have gained recent attention in treating visual deficits following head trauma. Previously, we have shown that adipose-derived stem cell (ASC) concentrated conditioned medium (ASC-CCM), when delivered via an intravitreal route, yielded a significant improvement in vision accompanied by a decrease in retinal neuroinflammation in a focal cranial blast model that indirectly injures the retina. The purpose of the current study is to extend our previous studies to a direct ocular blast injury model to further establish the preclinical efficacy of ASC-CCM. Adult C57BL/6J mice were subjected to repetitive ocular blast injury (rOBI) of 25 psi to the left eye, followed by intravitreal delivery of ASC-CCM (∼200 ng protein/2 µl) or saline within 2-3 h. Visual function and histological changes were measured 4 weeks after injury and treatment. In vitro, Müller cells were used to evaluate the antioxidant effect of ASC-CCM. Visual acuity, contrast sensitivity, and b-wave amplitudes in rOBI mice receiving saline were significantly decreased compared with age-matched sham blast mice. Immunohistological analyses demonstrated a significant increase in glial fibrillary acidic protein (a retinal injury marker) in Müller cell processes, DNA/RNA damage, and nitrotyrosine (indicative of oxidative stress) in the retina, while qPCR analysis revealed a >2-fold increase in pro-inflammatory cytokines (TNF-α, ICAM1, and Ccl2) in the retina, as well as markers for microglia/macrophage activation (IL-1ß and CD86). Remarkably, rOBI mice that received ASC-CCM demonstrated a significant improvement in visual function compared to saline-treated rOBI mice, with visual acuity, contrast sensitivity, and b-wave amplitudes that were not different from those in sham mice. This improvement in visual function also was associated with a significant reduction in retinal GFAP, neuroinflammation markers, and oxidative stress compared to saline-treated rOBI mice. In vitro, Müller cells exposed to oxidative stress via increasing doses of hydrogen peroxide demonstrated decreased viability, increased GFAP mRNA expression, and reduced activity for the antioxidant catalase. On the other hand, oxidatively stressed Müller cells pre-incubated with ASC-CCM showed normalized GFAP, viability, and catalase activity. In conclusion, our study demonstrates that a single intravitreal injection of ASC-CCM in the rOBI can significantly rescue retinal injury and provide significant restoration of visual function. Our in vitro studies suggest that the antioxidant catalase may play a major role in the protective effects of ASC-CCM, uncovering yet another aspect of the multifaceted benefits of ASC secretome therapies in neurotrauma.

A, B, C's of Trk Receptors and Their Ligands in Ocular Repair.

Neurotrophins are a family of closely related secreted proteins that promote differentiation, development, and survival of neurons, which include nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. All neurotrophins signal through tropomyosin receptor kinases (TrkA, TrkB, and TrkC) which are more selective to NGF, brain-derived neurotrophic factor, and neurotrophin-3, respectively. NGF is the most studied neurotrophin in the ocular surface and a human recombinant NGF has reached clinics, having been approved to treat neurotrophic keratitis. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 are less studied neurotrophins in the ocular surface, even though brain-derived neurotrophic factor is well characterized in glaucoma, retina, and neuroscience. Recently, neurotrophin analogs with panTrk activity and TrkC selectivity have shown promise as novel drugs for treating dry eye disease. In this review, we discuss the biology of the neurotrophin family, its role in corneal homeostasis, and its use in treating ocular surface diseases. There is an unmet need to investigate parenteral neurotrophins and its analogs that activate TrkB and TrkC selectively.

Assessment of necroptosis in the retina in a repeated primary ocular blast injury mouse model.

Primary blast injury (caused by the initial rapid increase in pressure following an explosive blast) to the retina and optic nerve (ON) causes progressive visual loss and neurodegeneration. Military personnel are exposed to multiple low-overpressure blast waves, which may be in quick succession, such as during breacher training or in combat. We investigated the necroptotic cell death pathway in the retina in a mouse repeated primary ocular blast injury (rPBI) model using immunohistochemistry. We further evaluated whether intravitreal injections of a potent necroptosis inhibitor, Necrostatin-1s (Nec-1s), protects the retina and ON axons by retinal ganglion cells (RGC) counts, ON axonal counting and optical coherence tomography (OCT) analysis of vitreous haze. Receptor interacting protein kinase (RIPK) 3, increased in the inner plexiform layer 2 days post injury (dpi) and persisted until 14 dpi, whilst RIPK1 protein expression did not change after injury. The number of degenerating ON axons was increased at 28 dpi but there was no evidence of a reduction in the number of intact ON axons or RNA-binding protein with multiple splicing (RBPMS)+ RGC in the retina by 28 dpi in animals not receiving any intravitreal injections. But, when intravitreal injections (vehicle or Nec-1s) were given there was a significant reduction in RBPMS+ RGC numbers, suggesting that rPBI with intraocular injections is damaging to RGC. There were fewer RGC lost after Nec-1s than vehicle injection, but there was no effect of Nec-1s or vehicle treatment on the number of degenerating axons. OCT analysis demonstrated no effect of rPBI on vitreous haze, but intravitreal injection combined with rPBI increased vitreous haze (P = 0.004). Whilst necroptosis may be an active cell death signalling pathway after rPBI, its inhibition did not prevent cell death, and intravitreal injections in combination with rPBI increased vitreous inflammation and reduced RBPMS+ RGC numbers, implying intravitreal injection is not an ideal method for drug delivery after rPBI.

Proteomic evidence that ABCA4 is vital for traumatic proliferative vitreoretinopathy formation and development.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment failure. The mechanism of PVR development is complex and still not completely elucidated. There are no proven methods for early prevention or clinical treatment. Retinal proteins are abnormally expressed during the entire PVR disease process. Due to the limitations of research methods and techniques, we do not fully understand the retinal protein changes in PVR. This proteomics study systemically analyzed and identified differential protein expression between retinas of PVR and non-PVR (normal) eyes. Retinal samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) coupled with mass spectrometry. Raw data were processed and analyzed by Maxquant software and then searched against the human UniProKB (201510) protein database. Differentially expressed proteins were selected and further validated in a human retinal pigment epithelial (RPE) cell line. The effects of dysregulated proteins on cell proliferation, apoptosis, and migration were studied. Systemic proteomics analysis identified several PVR-enriched proteins. The differentially expressed proteins were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation to find abnormal pathways involved in PVR. Retinal-specific ATP-binding cassette transporter (ABCA4) expression was one of the most increased proteins in PVR tissue. ABCA4 knockdown significantly reduced proliferation and affected the cell cycle in the human RPE cell line. ABCA4 knockdown also induced apoptosis and inhibited retinal cell migration. In conclusion, systemic proteomics analysis identified differentially expressed proteins in traumatic PVR, with ABCA4 being highly expressed. Disruption of ABCA4 expression induced apoptosis and inhibited cell proliferation and migration in a human RPE cell line.

TLR4-MyD88 pathway promotes the imbalanced activation of NLRP3/NLRP6 via caspase-8 stimulation after alkali burn injury.

Alkali burn (AB) is one of the most serious ocular traumas in the world, characterized by extreme ocular surface disorders, critical secondary dry eye and irreversible vision loss. The exact mechanisms involved are unknown. Innate immunity, including the involvement of Toll-like receptors (TLRs) and NOD-like receptors (NLRs), is believed to participate in the pathogenesis of the epithelia, but the exact mechanisms by which TLRs transduce signals to NLRs and downstream molecules to initiate innate immunity remain poorly defined. In this present study, we used murine models of AB and AB concomitant desiccating stress (DS) to investigate the potential functions and mechanisms of TLR4 in regulating NLRP3 and NLRP6 during AB injury and secondary dry eye. We demonstrated that AB injury induced activation of the TLR4-MyD88 pathway, leading to imbalanced NLRP3 and NLRP6 via the activation of caspase-8 signaling. DS worsened ocular surface disorders post-AB injury by magnifying this phenomenon. Caspase-8 signaling promoted NLRP3 upregulation via the nuclear factor (NF)-κB pathway, while NLRP6 suppressed NF-κB activation. Our findings also revealed that TLR4-MyD88 knockout can alleviate AB-induced or DS-worsened ocular surface disorders, shedding light on the potential therapeutic strategies in the future for AB injury. Taken together, our findings demonstrate that AB promotes the TLR4-MyD88-caspase-8 axis to cause imbalanced NLRP3/NLRP6, and DS exacerbates ocular surface damage via magnifying this imbalance.

Imaging rhodopsin degeneration in vivo in a new model of ocular ischemia in living mice.

Delivery of antibodies to monitor key biomarkers of retinopathy in vivo represents a significant challenge because living cells do not take up immunoglobulins to cellular antigens. We met this challenge by developing novel contrast agents for retinopathy, which we used with magnetic resonance imaging (MRI). Biotinylated rabbit polyclonal to chick IgY (rIgPxcIgY) and phosphorylthioate-modified oligoDNA (sODN) with random sequence (bio-sODN-Ran) were conjugated with NeutrAvidin-activated superparamagnetic iron oxide nanoparticles (SPION). The resulting Ran-SPION-rIgPxcIgY carries chick polyclonal to microtubule-associated protein 2 (MAP2) as Ran-SPION-rIgP/cIgY-MAP2, or to rhodopsin (Rho) as anti-Rho-SPION-Ran. We examined the uptake of Ran-SPION-rIgP/cIgY-MAP2 or SPION-rIgP/cIgY-MAP2 in normal C57black6 mice (n = 3 each, 40 µg/kg, i.c.v.); we found retention of Ran-SPION-rIgP/cIgY-MAP2 using molecular contrast-enhanced MRI in vivo and validated neuronal uptake using Cy5-goat IgPxcIgY ex vivo. Applying this novel method to monitor retinopathy in a bilateral carotid artery occlusion-induced ocular ischemia, we observed pericytes (at d 2, using Gd-nestin, by eyedrop solution), significant photoreceptor degeneration (at d 20, using anti-Rho-SPION-Ran, eyedrops, P = 0.03, Student's t test), and gliosis in Müller cells (at 6 mo, using SPION-glial fibrillary acidic protein administered by intraperitoneal injection) in surviving mice (n ≥ 5). Molecular contrast-enhanced MRI results were confirmed by optical and electron microscopy. We conclude that chimera and molecular contrast-enhanced MRI provide sufficient sensitivity for monitoring retinopathy and for theranostic applications.

Carbenoxolone prevents chemical eye ischemia-reperfusion-induced cell death via 11ß-hydroxysteroid dehydrogenase type 1 inhibition.

Glaucoma is one of the leading causes of preventable blindness diseases, affecting more than 2 million people in the United States. Recently, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors were found to exert preventive effects against glaucoma. Therefore, we investigated whether carbenoxolone (CBX), an 11ß-HSD1 inhibitor, prevents chemical ischemia-reperfusion-induced cell death in human trabecular meshwork (HTM) cells. The present study demonstrated that CBX inhibited cell death caused by iodoacetic acid (IAA)-induced ischemia-reperfusion, and its effect was associated with the inhibition of 11ß-HSD1 expression and activity. Furthermore, CBX reversed the IAA-induced structural damage on filamentous actin in HTM cells. In IAA-treated cells, the levels of 11ß-HSD1 and the apoptosis-related factors Bax and FASL were increased throughout the reperfusion period, and CBX was able to attenuate the expression of 11ß-HSD1 and the apoptosis-related factors. CBX also effectively suppressed IAA-induced intracellular ROS formation and cytochrome c release, which are involved in the mitochondrial apoptosis pathway. In addition, IAA-induced chemical ischemia-reperfusion stimulated TNF-α expression and NF-κB p65 phosphorylation, and these effects were attenuated by CBX. 11ß-HSD1 RNAi also suppressed IAA-induced cell apoptosis via reduction of oxidative stress and inhibition of the pro-inflammatory pathway. Taken together, the present study demonstrated that the inhibition of 11ß-HSD1 protected the TM against chemical ischemia-reperfusion injury, suggesting that the use of 11ß-HSD1 inhibitors could be a useful strategy for glaucoma therapy.

Erythropoietin either Prevents or Exacerbates Retinal Damage from Eye Trauma Depending on Treatment Timing.

PURPOSE: Erythropoietin (EPO) is a promising neuroprotective agent and is currently in Phase III clinical trials for the treatment of traumatic brain injury. The goal of this study was to determine if EPO is also protective in traumatic eye injury. METHODS: The left eyes of anesthetized DBA/2J or Balb/c mice were exposed to a single 26 psi overpressure air-wave while the rest of the body was shielded. DBA/2J mice were given intraperitoneal injections of EPO or buffer and analyses were performed at 3 or 7 days post-blast. Balb/c mice were given intramuscular injections of rAAV.EpoR76E or rAAV.eGFP either pre- or post-blast and analyses were performed at 1 month post-blast. RESULTS: EPO had a bimodal effect on cell death, glial reactivity, and oxidative stress. All measures were increased at 3 days post-blast and decreased at 7-days post-blast. Increased retinal ferritin and NADPH oxygenases were detected in retinas from EPO-treated mice. The gene therapy approach protected against axon degeneration, cell death, and oxidative stress when given after blast, but not before. CONCLUSIONS: Systemic, exogenous EPO and EPO-R76E protects the retina after trauma even when initiation of treatment is delayed by up to 3 weeks. Systemic treatment with EPO or EPO-R76E beginning before or soon after trauma may exacerbate protective effects of EPO within the retina as a result of increased iron levels from erythropoiesis and, thus, increased oxidative stress within the retina. This is likely overcome with time as a result of an increase in levels of antioxidant enzymes. Either intraocular delivery of EPO or treatment with non-erythropoietic forms of EPO may be more efficacious.

Seasonal and post-trauma remodeling in cone-dominant ground squirrel retina.

With a photoreceptor mosaic containing ∼85% cones, the ground squirrel is one of the richest known mammalian sources of these important retinal cells. It also has a visual ecology much like the human's. While the ground squirrel retina is understandably prominent in the cone biochemistry, physiology, and circuitry literature, far less is known about the remodeling potential of its retinal pigment epithelium, neurons, macroglia, or microglia. This review aims to summarize the data from ground squirrel retina to this point in time, and to relate them to data from other brain areas where appropriate. We begin with a survey of the ground squirrel visual system, making comparisons with traditional rodent models and with human. Because this animal's status as a hibernator often goes unnoticed in the vision literature, we then present a brief primer on hibernation biology. Next we review what is known about ground squirrel retinal remodeling concurrent with deep torpor and with rapid recovery upon re-warming. Notable here is rapidly-reversible, temperature-dependent structural plasticity of cone ribbon synapses, as well as pre- and post-synaptic plasticity throughout diverse brain regions. It is not yet clear if retinal cell types other than cones engage in torpor-associated synaptic remodeling. We end with the small but intriguing literature on the ground squirrel retina's remodeling responses to insult by retinal detachment. Notable for widespread loss of (cone) photoreceptors, there is surprisingly little remodeling of the RPE or Müller cells. Microglial activation appears minimal, and remodeling of surviving second- and third-order neurons seems absent, but both require further study. In contrast, traumatic brain injury in the ground squirrel elicits typical macroglial and microglial responses. Overall, the data to date strongly suggest a heretofore unrecognized, natural checkpoint between retinal deafferentiation and RPE and Müller cell remodeling events. As we continue to discover them, the unique ways by which ground squirrel retina responds to hibernation or injury may be adaptable to therapeutic use.

[Reparative Neurogenesis in the Brain and Changes in the Optic Nerve of Adult Trout Oncorhynchus mykiss after Mechanical Damage of the Eye].

Reparative proliferation and neurogenesis in the brain integrative centers after mechanical eye injury in an adult trout Oncorhynchus mykiss have been studied. We have found that proliferation and neurogenesis in proliferative brain regions, the cerebellum, and the optic tectum were significantly enhanced after the eye injury. The cerebellum showed a significant increase in the proliferative activity of the cells of the dorsal proliferative zone and parenchymal cells of the molecular and granular layers. One week after the injury, PCNA-positive radial glia cells have been identified in the tectum. We have found for the first time that the eye trauma resulted in the development of local clusters of undifferentiated cells forming so called neurogenic niches in the tectum and cerebellum. The differentiation of neuronal cells detected by labeling cells with antibodies against the protein HuC/D occurred in the proliferative zones of the telencephalon, the optic tectum, cerebellum, and medulla of a trout within 2 days after the injury. We have shown that the HuC/D expression is higher in the proliferative brain regions than in the definitive neurons of a trout. In addition, we have examined cell proliferation, migration, and apoptosis caused by the eye injury in the contra- and ipsilateral optic nerves and adjacent muscle fibers 2 days after the trauma. The qualitative and quantitative assessment of proliferation and apoptosis in the cells of the optic nerve of a trout has been made using antibodies against PCNA and the TUNEL method.

Epithelial basement membrane proteins perlecan and nidogen-2 are up-regulated in stromal cells after epithelial injury in human corneas.

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-min epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins-important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 were expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.

Pathophysiology of blast-induced ocular trauma in rats after repeated exposure to low-level blast overpressure.

BACKGROUND: The incidence of blast-induced ocular injury has dramatically increased due to advances in weaponry and military tactics. A single exposure to blast overpressure (BOP) has been shown to cause damage to the eye in animal models; however, on the battlefield, military personnel are exposed to BOP multiple times. The effects of repeated exposures to BOP on ocular tissues have not been investigated. The purpose of this study is to characterize the effects of single or repeated exposure on ocular tissues. METHODS: A compressed air shock tube was used to deliver 70 ± 7 KPa BOP to rats, once (single blast overpressure [SBOP]) or once daily for 5 days (repeated blast overpressure [RBOP]). Immunohistochemistry was performed to characterize the pathophysiology of ocular injuries induced by SBOP and RBOP. Apoptosis was determined by quantification activated caspase 3. Gliosis was examined by detection of glial fibrillary acidic protein (GFAP). Inflammation was examined by detection of CD68. RESULTS: Activated caspase 3 was detected in ocular tissues from all animals subjected to BOP, while those exposed to RBOP had more activated caspase 3 in the optic nerve than those exposed to SBOP. GFAP was detected in the retinas from all animals subjected to BOP. CD68 was detected in optic nerves from all animals exposed to BOP. CONCLUSION: SBOP and RBOP induced retinal damage. RBOP caused more apoptosis in the optic nerve than SBOP, suggesting that RBOP causes more severe optic neuropathy than SBOP. SBOP and RBOP caused gliosis in the retina and increased inflammation in the optic nerve.

Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.

We reported the expression of phosphorylated HSP27 during epithelial wound healing in murine corneas (Jain et al., 2012) in July of 2012. This in vivo investigation demonstrated that the expression levels of phosphorylated HSP27 were greater in wounded corneal epithelial cells than in unwounded controls and that the localization of phosphorylated HSP27 was in the basal and superficial epithelia three days following corneal epithelial wounding. We suggested that phosphorylated HSP27 had a role in the early phase of corneal epithelial wound healing. The purpose of this study was to investigate the exact role of heat shock protein 27 (HSP27) phosphorylation for the wound healing of cultured human corneal epithelial cells (HCECs). HSP27-specific siRNAs and control-siRNAs, with no known homologous targets in HCECs, were created. The cultured HCECs were divided into two groups: Scrambled control-siRNA-transfected group vs. HSP27-specific siRNA-transfected group. The scratch-induced directional wounding assay, Western blotting, using antibodies against non-phosphorylated and phosphorylated HSP27, non-phosphorylated and phosphorylated Akt, and Bcl-2-associated X protein (Bax), immunofluorescence staining to determine the filament actin, flow cytometry to measure apoptosis, and proliferation assay were performed to determine the role of HSP27. Western blot assay showed that the expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding, compared with those in unwounded HCECs (all p < 0.05). Western blot assay also showed HSP27-specific siRNAs effectively blocked the expression of non-phosphorylated HSP27. The HSP27-specific siRNA-transfected group had more Bax expression, less phosphorylated Akt expression, and less non-phosphorylated and phosphorylated HSP27 expression (all p < 0.05). The scratch-induced directional wounding assay showed the HSP27-specific siRNA-transfected group with a less migrating cell number than the control-siRNA-transfected group (p < 0.05). Immunofluorescence staining showed that reorganization of actin cytoskeleton prominently decreased in the HSP27-specific siRNA-transfected group, compared with the control siRNA-tranfected group. Flow cytometry revealed that the HSP27-specific siRNA-transfected group had more HCEC apoptosis. Proliferation assay showed no difference between the two groups. In conclusion, the role of HSP27 in corneal epithelial wound healing can be epithelial cell apoptosis, as well as epithelial migration. HSP27 is involved in HCEC migration by the reorganization of actin cytoskeleton.

Integrin-dependent neutrophil migration in the injured mouse cornea.

As an early responder to an inflammatory stimulus, neutrophils (PMNs) must exit the vasculature and migrate through the extravascular tissue to the site of insult, which is often remote from the point of extravasation. Following a central epithelial corneal abrasion, PMNs recruited from the peripheral limbal vasculature migrate into the avascular corneal stroma. In vitro studies suggest PMN locomotion over 2-D surfaces is dependent on integrin binding while migration within 3-D matrices can be integrin-independent. Electron micrographs of injured mouse corneas show migrating PMNs make extensive surface contact not only with collagen fibrils in the extracellular matrix (ECM), but also keratocytes. Evidence supporting involvement of integrins in corneal inflammation has prompted research and development of integrin blocking agents for use as anti-inflammatory therapies. However, the role of integrin binding (cell-cell; cell-ECM) during stromal migration in the inflamed cornea has previously not been clearly defined. In this study in vivo time lapse imaging sequences provided the means to quantify cell motility while observing PMN interactions with keratocytes and other stromal components in the living eye. The relative contribution of ß1, ß2 and ß3 integrins to PMN locomotion in the inflamed mouse cornea was investigated using blocking antibodies against the respective integrins. Of the 3 integrin families (ß1, ß2 and ß3) investigated for their potential role in PMN migration, only ß1 antibody blockade produced a significant, but partial, reduction in PMN motility. The preferential migration of PMNs along the keratocyte network was not affected by integrin blockade. Hence, the dominant mechanism for PMN motility within the corneal stroma appears to be integrin-independent as does the restriction of PMN migration paths to the keratocyte network.

A method to generate enhanced GFP+ chimeric mice to study the role of bone marrow-derived cells in the eye.

GFP-chimeric mice are important tools to study the role of bone marrow-derived cells in eye physiology. A method is described to generate GFP-chimeric mice using whole-body, sub-lethal radiation (600 rad) of wild-type C57BL/6 recipients followed by tail vein injection of bone marrow cells derived from GFP+ (GFP-transgenic C57/BL/6-Tg(UBC-GFP)30 Scha/J) mice. This method yields stable GFP+ chimeras with greater than 95% chimerism (range 95-99%), achieved within one month of bone marrow transfer confirmed by microscopy and fluorescence-assisted cell sorting (FACS) analysis, with lower mortality after irradiation than prior methods. To demonstrate the efficacy of GFP+ bone marrow chimeric mice, the role of circulating GFP+ bone marrow-derived cells in myofibroblast generation after irregular photo-therapeutic keratectomy (PTK) was analyzed. Many SMA+ myofibroblasts that were generated at one month after PTK were derived from GFP+ bone marrow-derived cells. The GFP+ bone marrow chimeric mouse provides an excellent model for studying the role of bone marrow-derived cells in corneal wound healing, glaucoma surgery, optic nerve head pathology and retinal pathophysiology and wound healing.