High-Mobility Group Box 1 Inhibitor BoxA Alleviates Neuroinflammation-Induced Retinal Ganglion Cell Damage in Traumatic Optic Neuropathy

Traumatic optic neuropathy (TON) is a significant cause of vision loss and irreversible blindness worldwide. It is defined as retinal ganglion cell death and axon degeneration caused by injury. Optic nerve crush (ONC), a well-validated model of TON, activates retinal microglia and initiates neuroinflammation. High-mobility group box 1 (HMGB1), a non-histone chromosomal binding protein in the nucleus of eukaryotic cells, is an important inducer of microglial activation and pro-inflammatory cytokine release. The purpose of this study was to examine the protective effects and mechanism of the HMGB1 inhibitor BoxA to neuroinflammation-induced retinal ganglion cells (RGCs) damage in traumatic optic neuropathy. For that purpose, an optic nerve crush model was established in C57BL/6J mice at 10–12 weeks. Model mice received an intravitreal injection of PBS and the HMGB1 inhibitor BoxA. Our data demonstrated that HMGB1 expression increased after optic nerve crush. Retinal ganglion cell function and morphology were damaged, and retinal ganglion cell numbers were reduced after optic nerve crush. Intravitreal injection of BoxA after ONC can alleviate damage. Furthermore, BoxA reduced microglial activation and expression levels of nuclear factor κB (NF-kB), nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC) in experimental ONC mice. In summary, HMGB1 mediates NLRP3 inflammasome via NF-kB to participate in retinal inflammatory injury after ONC. Thus, intravitreal injection of BoxA has potential therapeutic benefits for the effective treatment of RGC death to prevent TON.     

Roscovitine,a Cyclin-Dependent Kinase-5 Inhibitor,Decreases Phosphorylated Tau Formation and Death of Retinal Ganglion Cells of Rats after Optic Nerve Crush

Tauopathies are neurodegenerative diseases characterized by abnormal metabolism of misfolded tau proteins and are progressive. Pathological phosphorylation of tau occurs in the retinal ganglion cells (RGCs) after optic nerve injuries. Cyclin-dependent kinase-5 (Cdk5) causes hyperphosphorylation of tau. To determine the roles played by Cdk5 in retinal degeneration, roscovitine, a Cdk5 inhibitor, was injected intravitreally after optic nerve crush (ONC). The neuroprotective effect of roscovitine was determined by the number of Tuj-1-stained RGCs on day 7. The change in the levels of phosphorylated tau, calpain-1, and cleaved α-fodrin was determined by immunoblots on day 3. The expression of P35/P25, a Cdk5 activator, in the RGCs was determined by immunohistochemistry. The results showed that roscovitine reduced the level of phosphorylated tau by 3.5- to 1.6-fold. Calpain-1 (2.1-fold) and cleaved α-fodrin (1.5-fold) were increased on day 3, suggesting that the calpain signaling pathway was activated. P35/P25 was accumulated in the RGCs that were poorly stained by Tuj-1. Calpain inhibition also reduced the increase in phosphorylated tau. The number of RGCs decreased from 2191 ± 178 (sham) to 1216 ± 122 cells/mm² on day 7, and roscovitine preserved the level at 1622 ± 130 cells/mm². We conclude that the calpain-mediated activation of Cdk5 is associated with the pathologic phosphorylation of tau.     

Human Pluripotent Stem Cell-Derived Neural Progenitor Cells Promote Retinal Ganglion Cell Survival and Axon Recovery in an Optic Nerve Compression Animal Model

Human pluripotent stem cell-derived neural progenitor cells (NPCs) have the potential to recover from nerve injury. We previously reported that human placenta-derived mesenchymal stem cells (PSCs) have neuroprotective effects. To evaluate the potential benefit of NPCs, we compared them to PSCs using R28 cells under hypoxic conditions and a rat model of optic nerve injury. NPCs and PSCs (2 × 106 cells) were injected into the subtenon space. After 1, 2, and 4 weeks, we examined changes in target proteins in the retina and optic nerve. NPCs significantly induced vascular endothelial growth factor (Vegf) compared to age-matched shams and PSC groups at 2 weeks; they also induced neurofilaments in the retina compared to the sham group at 4 weeks. In addition, the expression of brain-derived neurotrophic factor (Bdnf) was high in the retina in the NPC group at 2 weeks, while expression in the optic nerve was high in both the NPC and PSC groups. The low expression of ionized calcium-binding adapter molecule 1 (Iba1) in the retina had recovered at 2 weeks after NPC injection and at 4 weeks after PSC injection. The expression of the inflammatory protein NLR family, pyrin domain containing 3 (Nlrp3) was significantly reduced at 1 week, and that of tumor necrosis factor-α (Tnf-α) in the optic nerves of the NPC group was lower at 2 weeks. Regarding retinal ganglion cells, the expressions of Brn3a and Tuj1 in the retina were enhanced in the NPC group compared to sham controls at 4 weeks. NPC injections increased Gap43 expression from 2 weeks and reduced Iba1 expression in the optic nerves during the recovery period. In addition, R28 cells exposed to hypoxic conditions showed increased cell survival when cocultured with NPCs compared to PSCs. Both Wnt/β-catenin signaling and increased Nf-ĸb could contribute to the rescue of damaged retinal ganglion cells via upregulation of neuroprotective factors, microglial engagement, and anti-inflammatory regulation by NPCs. This study suggests that NPCs could be useful for the cellular treatment of various optic neuropathies, together with cell therapy using mesenchymal stem cells.     

Erythropoietin in Optic Neuropathies: Current Future Strategies for Optic Nerve Protection and Repair

Erythropoietin (EPO) is known as a hormone for erythropoiesis in response to anemia and hypoxia. However, the effect of EPO is not only limited to hematopoietic tissue. Several studies have highlighted the neuroprotective function of EPO in extra-hematopoietic tissues, especially the retina. EPO could interact with its heterodimer receptor (EPOR/βcR) to exert its anti-apoptosis, anti-inflammation and anti-oxidation effects in preventing retinal ganglion cells death through different intracellular signaling pathways. In this review, we summarized the available pre-clinical studies of EPO in treating glaucomatous optic neuropathy, optic neuritis, non-arteritic anterior ischemic optic neuropathy and traumatic optic neuropathy. In addition, we explore the future strategies of EPO for optic nerve protection and repair, including advances in EPO derivates, and EPO deliveries. These strategies will lead to a new chapter in the treatment of optic neuropathy.     

Effect of PACAP in Central and Peripheral Nerve Injuries

Pituitary adenylate cyclase activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. In addition to its more classic role as a neuromodulator, PACAP functions as a neurotrophic factor. Several neurotrophic factors have been shown to play an important role in the endogenous response following both cerebral ischemia and traumatic brain injury and to be effective when given exogenously. A number of studies have shown the neuroprotective effect of PACAP in different models of ischemia, neurodegenerative diseases and retinal degeneration. The aim of this review is to summarize the findings on the neuroprotective potential of PACAP in models of different traumatic nerve injuries. Expression of endogenous PACAP and its specific PAC1 receptor is elevated in different parts of the central and peripheral nervous system after traumatic injuries. Some experiments demonstrate the protective effect of exogenous PACAP treatment in different traumatic brain injury models, in facial nerve and optic nerve trauma. The upregulation of endogenous PACAP and its receptors and the protective effect of exogenous PACAP after different central and peripheral nerve injuries show the important function of PACAP in neuronal regeneration indicating that PACAP may also be a promising therapeutic agent in injuries of the nervous system.     

Intravitreal Injection of Liposomes Loaded with a Histone Deacetylase Inhibitor Promotes Retinal Ganglion Cell Survival in a Mouse Model of Optic Nerve Crush

Various neuroprotective agents have been studied for the treatment of retinal ganglion cell (RGC) diseases, but issues concerning the side effects of systemically administered drugs and the short retention time of intravitreally injected drugs limit their clinical applications. The current study aimed to evaluate the neuroprotective effects of intravitreally injected trichostatin A (TSA)-loaded liposomes in a mouse model of optic nerve crush (ONC) and determine whether TSA-loaded liposomes have therapeutic potential in RGC diseases. The histone deacetylase inhibitor, TSA, was incorporated into polyethylene glycolylated liposomes. C57BL/6J mice were treated with an intravitreal injection of TSA-loaded liposomes and liposomes loaded with a lipophilic fluorescent dye for tracking, immediately after ONC injury. The expression of macroglial and microglial cell markers (glial fibrillary acidic protein and ionized calcium binding adaptor molecule-1), RGC survival, and apoptosis were assessed. We found that the liposomes reached the inner retina. Their fluorescence was detected for up to 10 days after the intravitreal injection, with peak intensity at 3 days postinjection. Intravitreally administered TSA-loaded liposomes significantly decreased reactive gliosis and RGC apoptosis and increased RGC survival in a mouse model of ONC. Our results suggest that TSA-loaded liposomes may help in the treatment of various RGC diseases.     

Neuroprotection,Growth Factors and BDNF-TrkB Signalling in Retinal Degeneration

Neurotrophic factors play key roles in the development and survival of neurons. The potent neuroprotective effects of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF) and nerve growth factor (NGF), suggest that they are good therapeutic candidates for neurodegenerative diseases. Glaucoma is a neurodegenerative disease of the eye that causes irreversible blindness. It is characterized by damage to the optic nerve, usually due to high intraocular pressure (IOP), and progressive degeneration of retinal neurons called retinal ganglion cells (RGCs). Current therapy for glaucoma focuses on reduction of IOP, but neuroprotection may also be beneficial. BDNF is a powerful neuroprotective agent especially for RGCs. Exogenous application of BDNF to the retina and increased BDNF expression in retinal neurons using viral vector systems are both effective in protecting RGCs from damage. Furthermore, induction of BDNF expression by agents such as valproic acid has also been beneficial in promoting RGC survival. In this review, we discuss the therapeutic potential of neurotrophic factors in retinal diseases and focus on the differential roles of glial and neuronal TrkB in neuroprotection. We also discuss the role of neurotrophic factors in neuroregeneration.     

Intraocular Delivery of a Collagen Mimetic Peptide Repairs Retinal Ganglion Cell Axons in Chronic and Acute Injury Models

Vision loss through the degeneration of retinal ganglion cell (RGC) axons occurs in both chronic and acute conditions that target the optic nerve. These include glaucoma, in which sensitivity to intraocular pressure (IOP) causes early RGC axonal dysfunction, and optic nerve trauma, which causes rapid axon degeneration from the site of injury. In each case, degeneration is irreversible, necessitating new therapeutics that protect, repair, and regenerate RGC axons. Recently, we demonstrated the reparative capacity of using collagen mimetic peptides (CMPs) to heal fragmented collagen in the neuronal extracellular milieu. This was an important step in the development of neuronal-based therapies since neurodegeneration involves matrix metalloproteinase (MMP)-mediated remodeling of the collagen-rich environment in which neurons and their axons exist. We found that intraocular delivery of a CMP comprising single-strand fractions of triple helix human type I collagen prevented early RGC axon dysfunction in an inducible glaucoma model. Additionally, CMPs also promoted neurite outgrowth from dorsal root ganglia, challenged in vitro by partial digestion of collagen. Here, we compared the ability of a CMP sequence to protect RGC axons in both inducible glaucoma and optic nerve crush. A three-week +40% elevation in IOP caused a 67% degradation in anterograde transport to the superior colliculus, the primary retinal projection target in rodents. We found that a single intravitreal injection of CMP during the period of IOP elevation significantly reduced this degradation. The same CMP delivered shortly after optic nerve crush promoted significant axonal recovery during the two-week period following injury. Together, these findings support a novel protective and reparative role for the use of CMPs in both chronic and acute conditions affecting the survival of RGC axons in the optic projection to the brain.     

Exosomes Secreted by Adipose-Derived Stem Cells Following FK506 Stimulation Reduce Autophagy of Macrophages in Spine after Nerve Crush Injury

Macrophages emerge in the milieu around innervated neurons after nerve injuries. Following nerve injury, autophagy is induced in macrophages and affects the regulation of inflammatory responses. It is closely linked to neuroinflammation, while the immunosuppressive drug tacrolimus (FK506) enhances nerve regeneration following nerve crush injury and nerve allotransplantation with additional neuroprotective and neurotrophic functions. The combined use of FK506 and adipose-derived stem cells (ADSCs) was employed in cell therapy for organ transplantation and vascularized composite allotransplantation. This study aimed to investigate the topical application of exosomes secreted by ADSCs following FK506 treatment (ADSC-F-exo) to the injured nerve in a mouse model of sciatic nerve crush injury. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile the potential exosomal proteins involved in autophagy. Immunohistochemical analysis revealed that nerve crush injuries significantly induced autophagy in the dorsal root ganglia and dorsal horn of the spinal segments. Locally applied ADSC-F-exo significantly reduced autophagy of macrophages in the spinal segments after nerve crush injury. Proteomic analysis showed that of the 22 abundant exosomal proteins detected in ADSC-F-exo, heat shock protein family A member 8 (HSPA8) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) are involved in exosome-mediated autophagy reduction.     

The Protective Effects of n-Butylidenephthalide on Retinal Ganglion Cells during Ischemic Injury

Clinically, acute ischemic symptoms in the eyes are one of the main causes of vision loss, with the associated inflammatory response and oxidative stress being the key factors that cause injury. Nonarteritic anterior ischemic optic neuropathy (NAION) is the most common type of ischemic optic neuropathy (ION); however, there are still no effective or safe treatment options to date. In this study, we investigated the neuroprotective effects of n-butylidenephthalide (BP) treatment in an experimental NAION rodent model (rAION). BP (10 mg/kg) or PBS (control group) were administered on seven consecutive days in the rAION model. Rats were evaluated for visual function by flash visual evoked potentials (FVEPs) at 4 weeks after NAION induction. The retina and optic nerve were removed for histological examination after the rats were euthanized. The molecular machinery of BP treatment in the rAION model was analyzed using Western blotting. We discovered that BP effectively improves retinal ganglion cell survival rates by preventing apoptotic processes after AION induction and reducing the inflammatory response through which blood-borne macrophages infiltrate the optic nerve. In addition, BP significantly preserved the integrity of the myelin sheath in the rAION model, demonstrating that BP can prevent the development of demyelination. Our immunoblotting results revealed the molecular mechanism through which BP mitigates the neuroinflammatory response through inhibition of the NF-κB signaling pathway. Taken together, these results demonstrate that BP can be used as an exceptional neuroprotective agent for ischemic injury.     

Human Multipotent Mesenchymal Stromal Cell–Derived Extracellular Vesicles Enhance Neuroregeneration in a Rat Model of Sciatic Nerve Crush Injury

Peripheral nerve injury remains a serious problem for medicine, with no effective method of treatment at the moment. The most prominent example of this problem is neonatal brachial plexus palsy, which results from the stretching of the brachial plexus nerves in the birth or perinatal period. Multipotent mesenchymal cells (MSCs) and the extracellular vesicles (EVs) they produce are known to have a marked neuroprotective effect in central nervous system injuries. We suggested that the use of MSCs-derived EVs may be an effective approach to the regeneration of peripheral nerves after injury. Sciatic nerve injury was modeled in rats via crushing, and then a gel containing MSCs–EVs was applied to the injured area. After 15 and 30 days, a histological, physiological, and functional assessment of nerve, dorsal root ganglia (DRG), and innervated muscles’ recovery was performed. Transplantation of EVs to the area of sciatic nerve injury significantly reduced muscle atrophy as compared to the control group. Functional recovery of the innervated muscles, as measured by the extensor postural thrust test, was revealed 30 days after the surgery. We associate the obtained results with EVs-induced neuroprotective mechanisms, which were expressed in a decrease in apoptotic neuronal death and an increase in regeneration-associated proteins NF-200 and GAP-43, as well as in DRG and damaged nerve. We suggest that the therapeutic scheme we used is efficient for the treatment of acute peripheral nervous system injuries and can be transferred to the clinics. However, additional studies are required for a more detailed analysis of neuroprotection mechanisms.     

Ligand-Induced GPR110 Activation Facilitates Axon Growth after Injury

Recovery from axonal injury is extremely difficult, especially for adult neurons. Here, we demonstrate that the activation of G-protein coupled receptor 110 (GPR110, ADGRF1) is a mechanism to stimulate axon growth after injury. N-docosahexaenoylethanolamine (synaptamide), an endogenous ligand of GPR110 that promotes neurite outgrowth and synaptogenesis in developing neurons, and a synthetic GPR110 ligand stimulated neurite growth in axotomized cortical neurons and in retinal explant cultures. Intravitreal injection of GPR110 ligands following optic nerve crush injury promoted axon extension in adult wild-type, but not in gpr110 knockout, mice. In vitro axotomy or in vivo optic nerve injury rapidly induced the neuronal expression of gpr110. Activating the developmental mechanism of neurite outgrowth by specifically targeting GPR110 that is upregulated upon injury may provide a novel strategy for stimulating axon growth after nerve injury in adults.     

Involvement of Anoikis in Dissociated Optic Nerve Fiber Layer Appearance

Dissociated optic nerve fiber layer (DONFL) appearance is characterized by dimpling of the fundus when observed after vitrectomy with the internal limiting membrane (ILM) peeling in macular diseases. However, the cause of DONFL remains largely unknown. Optical coherence tomography (OCT) findings have indicated that the nerve fiber layer (NFL) and ganglion cells are likely to have been damaged in patients with DONFL appearance. Since DONFL appearance occurs at a certain postoperative period, it is unlikely to be retinal damage directly caused by ILM peeling because apoptosis occurs at a certain period after tissue damage and/or injury. However, it may be due to ILM peeling-induced apoptosis in the retinal tissue. Anoikis is a type of apoptosis that occurs in anchorage-dependent cells upon detachment of those cells from the surrounding extracellular matrix (i.e., the loss of cell anchorage). The anoikis-related proteins βA3/A1 crystallin and E-cadherin are reportedly expressed in retinal ganglion cells. Thus, we theorize that one possible cause of DONFL appearance is ILM peeling-induced anoikis in retinal ganglion cells.     

Using Noninvasive Electrophysiology to Determine Time Windows of Neuroprotection in Optic Neuropathies

The goal of neuroprotection in optic neuropathies is to prevent loss of retinal ganglion cells (RGCs) and spare their function. The ideal time window for initiating neuroprotective treatments should be the preclinical period at which RGCs start losing their functional integrity before dying. Noninvasive electrophysiological tests such as the Pattern Electroretinogram (PERG) can assess the ability of RGCs to generate electrical signals under a protracted degenerative process in both clinical conditions and experimental models, which may have both diagnostic and prognostic values and provide the rationale for early treatment. The PERG can be used to longitudinally monitor the acute and chronic effects of neuroprotective treatments. User-friendly versions of the PERG technology are now commercially available for both clinical and experimental use.     

Dimethyl Fumarate Promotes the Survival of Retinal Ganglion Cells after Optic Nerve Injury,Possibly through the Nrf2/HO-1 Pathway

This study aimed to verify whether dimethyl fumarate (DMF) promotes the survival of retinal ganglion cells (RGCs) after optic nerve crush (ONC) accompanied by activation of the NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. We examined changes in the densities of tubulin β3 (TUBB3)-positive RGCs and the amplitudes of the positive scotopic threshold response (pSTR), reflecting the functional activity of RGCs, recorded on an electroretinogram, with daily administration of DMF, on day 7 after ONC. Furthermore, immunohistochemical and immunoblotting analyses were performed to study the activation of the Nrf2/HO-1 pathway using retinas treated with daily administration of DMF. Daily administration of DMF increasedthe density of TUBB3-positive RGCs in a dose-dependent fashion and significantly increased the amplitude of the pSTR. Immunohistochemical analysis showed that DMF administration increased the immunoreactivity for Nrf2 and HO-1, a potent antioxidant enzyme, in RGCs immunolabeled with RNA-binding protein with multiple splicing (RBPMS). Immunoblotting analysis revealed an increase in the nuclear expression of Nrf2 and marked upregulation of HO-1 after DMF administration. These results suggest that DMF has survival-promoting effects in RGC after ONC, possibly via the Nrf2/HO-1 pathway.     

The Role of Axonal Transport in Glaucoma

Glaucoma is a neurodegenerative disease that affects the retinal ganglion cells (RGCs) and leads to progressive vision loss. The first pathological signs can be seen at the optic nerve head (ONH), the structure where RGC axons leave the retina to compose the optic nerve. Besides damage of the axonal cytoskeleton, axonal transport deficits at the ONH have been described as an important feature of glaucoma. Axonal transport is essential for proper neuronal function, including transport of organelles, synaptic components, vesicles, and neurotrophic factors. Impairment of axonal transport has been related to several neurodegenerative conditions. Studies on axonal transport in glaucoma include analysis in different animal models and in humans, and indicate that its failure happens mainly in the ONH and early in disease progression, preceding axonal and somal degeneration. Thus, a better understanding of the role of axonal transport in glaucoma is not only pivotal to decipher disease mechanisms but could also enable early therapies that might prevent irreversible neuronal damage at an early time point. In this review we present the current evidence of axonal transport impairment in glaucomatous neurodegeneration and summarize the methods employed to evaluate transport in this disease.     

Comparison of the Response to the CXCR4 Antagonist AMD3100 during the Development of Retinal Organoids Derived from ES Cells and Zebrafish Retina

Retinal organoids generated from human embryonic stem cells or iPSCs recreate the key structural and functional features of mammalian retinal tissue in vitro. However, the differences in the development of retinal organoids and normal retina in vivo are not well defined. Thus, in the present study, we analyzed the development of retinal organoids and zebrafish retina after inhibition of CXCR4, a key role in neurogenesis and optic nerve development, with the antagonist AMD3100. Our data indicated that CXCR4 was mainly expressed in ganglion cells in retinal organoids and was rarely expressed in amacrine or photoreceptor cells. AMD3100 treatment reduced the retinal organoid generation ratio, impaired differentiation, and induced morphological changes. Ganglion cells, amacrine cells, and photoreceptors were decreased and abnormal locations were observed in organoids treated with AMD3100. Neuronal axon outgrowth was also damaged in retinal organoids. Similarly, a decrease of ganglion cells, amacrine cells, and photoreceptors and the distribution of neural outgrowth was induced by AMD3100 treatment in zebrafish retina. However, abnormal photoreceptor ensembles induced by AMD3100 treatment in the organoids were not detected in zebrafish retina. Therefore, our study suggests that although retinal organoids might provide a reliable model for reproducing a retinal developmental model, there is a difference between the organoids and the retina in vivo.     

Peripheral Nerve Regeneration and Muscle Reinnervation

Injured peripheral nerves but not central nerves have the capacity to regenerate and reinnervate their target organs. After the two most severe peripheral nerve injuries of six types, crush and transection injuries, nerve fibers distal to the injury site undergo Wallerian degeneration. The denervated Schwann cells (SCs) proliferate, elongate and line the endoneurial tubes to guide and support regenerating axons. The axons emerge from the stump of the viable nerve attached to the neuronal soma. The SCs downregulate myelin-associated genes and concurrently, upregulate growth-associated genes that include neurotrophic factors as do the injured neurons. However, the gene expression is transient and progressively fails to support axon regeneration within the SC-containing endoneurial tubes. Moreover, despite some preference of regenerating motor and sensory axons to “find” their appropriate pathways, the axons fail to enter their original endoneurial tubes and to reinnervate original target organs, obstacles to functional recovery that confront nerve surgeons. Several surgical manipulations in clinical use, including nerve and tendon transfers, the potential for brief low-frequency electrical stimulation proximal to nerve repair, and local FK506 application to accelerate axon outgrowth, are encouraging as is the continuing research to elucidate the molecular basis of nerve regeneration.     

Neuroprotective Effect of Statins in a Rat Model of Chronic Ocular Hypertension

Glaucoma is an optic neuropathy in which the degeneration of retinal ganglion cells (RGCs) results in irreversible vison loss. Therefore, neuroprotection of RGCs from glaucomatous afflictions is crucial for glaucoma treatment. In this study, we aimed to investigate the beneficial effects of statins in the protection of RGCs using a rat model. Glaucomatous injury was induced in rats by chronic ocular hypertension (OHT) achieved after performing a circumlimbal suture. The rats were given either statins such as simvastatin and atorvastatin or a solvent weekly for 6 weeks. Retina sections underwent hematoxylin and eosin, Brn3a, or cleaved casepase-3 staining to evaluate RGC survival. In addition, modulation of glial activation was assessed. While the retinas without statin treatment exhibited increased RGC death due to chronic OHT, statins promoted the survival of RGCs and reduced apoptosis. Statins also suppressed chronic OHT-mediated glial activation in the retina. Our results demonstrate that statins exert neuroprotective effects in rat retinas exposed to chronic OHT, which may support the prospect of statins being a glaucoma treatment.     

Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics

To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R² = 0.97; nasal, R² = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.