Treatment Of Optic Nerve Injury Research Articles

CX3CL1-CX3CR1 axis protects retinal ganglion cells by inhibiting microglia activation in a distal optic nerve trauma model

BackgroundThe chemokine CX3CL1 has been reported to play an important role in optic nerve protection, but the underlying mechanism is still unclear. CX3CR1, the only receptor of CX3CL1, is specifically expressed on retinal microglia, whose activation plays a role in the pathological process of optic nerve injury. This study aimed to evaluate whether CX3CL1 exerts optic neuroprotection by affecting the activation of microglia by combining with CX3CR1.MethodsA mouse model of distal optic nerve trauma (ONT) was used to evaluate the effects of the CX3CL1-CX3CR1 axis on the activation of microglia and survival or axonal regeneration of retinal ganglion cells (RGCs). The activation of microglia, loss of RGCs, and damage to visual function were detected weekly till 4 weeks after modeling. CX3CL1 was injected intravitreally immediately or delayed after injury and the status of microglia and RGCs were examined.ResultsIncreases in microglia activation and optic nerve damage were accompanied by a reduced production of the CX3CL1-CX3CR1 axis after the distal ONT modeling. Both immediate and delayed intravitreal injection of CX3CL1 inhibited microglia activation, promoted survival of RGCs, and improved axonal regenerative capacity. Injection with CX3CL1 was no longer effective after 48 h post ONT. The CX3CL1-CX3CR1 axis promotes survival and axonal regeneration, as indicated by GAP43 protein and gene expression, of RGCs by inhibiting the microglial activation after ONT.ConclusionsThe CX3CL1-CX3CR1 axis could promote survival and axonal regeneration of RGCs by inhibiting the microglial activation after optic nerve injury. The CX3CL1-CX3CR1 axis may become a potential target for the treatment of optic nerve injury. Forty-eight hours is the longest time window for effective treatment after injury. The study is expected to provide new ideas for the development of targeted drugs for the repair of optic nerve.

Adipose mesenchymal stem cell-derived extracellular vesicles reduce glutamate-induced excitotoxicity in the retina.

Adipose mesenchymal stem cells (ADSCs) have protective effects against glutamate-induced excitotoxicity, but ADSCs are limited in use for treatment of optic nerve injury. Studies have shown that the extracellular vesicles (EVs) secreted by ADSCs (ADSC-EVs) not only have the function of ADSCs, but also have unique advantages including non-immunogenicity, low probability of abnormal growth, and easy access to target cells. In the present study, we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography. In addition, R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium, downregulation of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) subunit GluA2, and phosphorylation of GluA2 and protein kinase C alpha in vitro. A protein kinase C alpha agonist, 12-O-tetradecanoylphorbol 13-acetate, inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells. These findings suggest that ADSC-EVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.

Effect of papaverine on axonal outgrowth of primary retinal ganglion cells of Sprague Dawley rats

Increasing the level of cyclic adenosine 3, 5′-monophosphate is an important mechanism for axon outgrowth and recovery of central nervous system function. This study aimed to investigate the effects of papaverine, a non-specific phosphodiesterase inhibitor, on axon outgrowth of primary retinal ganglion cells from Sprague Dawley rats. Experiments were performed on primary retinal ganglion cells extracted from Sprague Dawley rat pups within 48–72 h of birth. At 24 h after seeding, immunofluorescence was used to identify and calculate the purity of retinal ganglion cells isolated by an improved two-step immunopanning method developed by author Sujia Ma. The effects of a range of papaverine concentrations on axon outgrowth of primary retinal ganglion cells cultures were observed by immunofluorescence and measured by ImageJ software at three different time points: 24, 48, and 72 h. The ability of papaverine to enable retinal ganglion cells to overcome the inhibitory effects of glial scar component chondroitin sulfate proteoglycans was examined using chondroitin sulfate proteoglycans-coated culture plates. Rp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt, a blocking agent of cyclic adenosine 3, 5′-monophosphate, and dibutyryl cyclic adenosine 3, 5′-monophosphate, an analogue of cyclic adenosine 3, 5′-monophosphate, were used to explore the mechanism of papaverine in promoting retinal ganglion cells axon outgrowth. Our study shows 2 μg/mL papaverine concentration significantly promoted axon outgrowth in primary retinal ganglion cells and restored axon outgrowth of these cells on chondroitin sulfate proteoglycans. Axon outgrowth was blocked by Rp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt and obviously promoted by dibutyryl cyclic adenosine 3, 5′-monophosphate. Our study is the first to describe the use of papaverine to promote axon outgrowth of retinal ganglion cells. These results may help to expand the application of papaverine, and they provide a cytological basis for papaverine in the treatment of optic nerve injury caused by glaucoma and other diseases.

Gadd45α affects retinal ganglion cell injury in chronic ocular hypertension rats by regulating p38MAPK pathway.

To investigate the impact and the mechanism of Gadd45α mediating p38MAPK pathway on the retinal ganglion cells (RGCs) injury in chronic ocular hypertension (COH) rats. COH model in rats were established and intraocular pressure (IOP) was tested. Retrograde labeling was used for counting RGCs and TUNEL staining was performed for RGCs apoptosis. Western Blotting was conducted to examine the expression of Gadd45α and p38MAPK pathway. Besides, RGC-5 cells cultured in vitro were treated with H2O2. Cell viability was detected by CCK-8, ROS level tested by DCFH-DA assay, and cell apoptosis examined by flow cytometry. COH rats had increased expression of Gadd45α and p-p38/p38 protein 1-4weeks after surgery. Rats in COH group enhanced obviously in IOP, RGC apoptosis rate and the protein expression of Gadd45α, p-p38/p38, Bax/Bcl-2 and cleaved caspase-3, but declined appreciably in RGC counting. However, the above indicators of COH rats were effectively improved by Gadd45α shRNA treatment. Additionally, RGC-5 cells in H2O2 group reduced in cell viability and went up in ROS level and apoptosis rate. The H2O2-induced RGC-5 cells treated with Gadd45α shRNA were improved apparently in those indicators, and cells treated with pcDNA Gadd45α showed an opposite trend. Moreover, p38 MAPK inhibitor SB203580 can effectively reverse the damage of pcDNA Gadd45α from H2O2-induced RGC-5 cells. Silencing Gadd45α can reduce the RGC damage in COH rats by inhibiting p38MAPK pathway and such a protective role may be associated with the suppression of RGC apoptosis and the mitigation of oxidative stress.

Nrn1 Overexpression Attenuates Retinal Ganglion Cell Apoptosis, Promotes Axonal Regeneration, and Improves Visual Function Following Optic Nerve Crush in Rats.

Neuritin (Nrn1) is a small highly conserved extracellular membrane protein involved in the process of neural cell survival and differentiation, axonal and dendritic growth, and synapse formation and maturation. Previous studies have demonstrated that intravitreal injection of recombinant Nrn1 as a gene therapy could alleviate retinal ganglion cell (RGC) apoptosis and promote optic nerve axon regeneration after optic nerve crush (ONC). However, the mechanism underlying the repairing effect of Nrn1 against optic never injury remains elusive. In this study, a rAAV2-mediated Nrn1 overexpression vector (AAV2-Nrn1) was applied to treat ONC through intravitreal injection for the purpose of further exploring the effect and mechanism of Nrn1 in repairing the injured optic nerve. The results showed that AAV2-Nrn1 was mainly transfected into RGCs without affecting astrocytes. Nrn1 overexpression effectively reduced RGC apoptosis and promoted optic nerve regeneration and visual function restoration as demonstrated by retinal imaging, histopathological analysis, and physiological function detection in vivo following ONC. Immunoblot assay revealed that functional molecules of Nrn1 activated the Akt1 and Stat3 pathways and inhibited the mitochondrial apoptotic pathway. The results of the present study may provide experimental evidence for further application of Nrn1 to the clinical treatment of optic nerve injury.

Pim-1 Protects Retinal Ganglion Cells by Enhancing Their Regenerative Ability Following Optic Nerve Crush.

Provirus integration site Moloney murine leukemia virus (Pim-1) is a proto-oncogene reported to be associated with cell proliferation, differentiation and survival. This study was to explore the neuroprotective role of Pim-1 in a rat model subjected to optic nerve crush (ONC), and discuss its related molecules in improving the intrinsic regeneration ability of retinal ganglion cells (RGCs). Immunofluorescence staining showed that AAV2-Pim-1 infected 71% RGCs and some amacrine cells in the retina. Real-time PCR and Western blotting showed that retina infection with AAV2-Pim-1 up-regulated the Pim-1 mRNA and protein expressions compared with AAV2-GFP group. Hematoxylin-Eosin (HE) staining, γ-synuclein immunohistochemistry, Cholera toxin B (CTB) tracing and TUNEL showed that RGCs transduction with AAV2-Pim-1 prior to ONC promoted the survival of damaged RGCs and decreased cell apoptosis. RITC anterograde labeling showed that Pim-1 overexpression increased axon regeneration and promoted the recovery of visual function by pupillary light reflex and flash visual evoked potential. Western blotting showed that Pim-1 overexpression up-regulated the expression of Stat3, p-Stat3, Akt1, p-Akt1, Akt2 and p-Akt2, as well as βIII-tubulin, GAP-43 and 4E-BP1, and downregulated the expression of SOCS1 and SOCS3, Cleaved caspase 3, Bad and Bax. These results demonstrate that Pim-1 exerted a neuroprotective effect by promoting nerve regeneration and functional recovery of RGCs. In addition, it enhanced the intrinsic regeneration capacity of RGCs after ONC by activating Stat3, Akt1 and Akt2 pathways, and inhibiting the mitochondrial apoptosis pathways. These findings suggest that Pim-1 may prove to be a potential therapeutic target for the clinical treatment of optic nerve injury.

Gene modification induces differentiation of bone marrow mesenchymal stem cells into neurons for treatment of optic nerve injury

Gene modification induces differentiation of bone marrow mesenchymal stem cells into neurons for treatment of optic nerve injury

Branched-Chain Amino Acids Enhance Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Transection in Rats

ABSTRACT Purpose This study’s aim was to investigate the beneficial effects of branched-chain amino acids (BCAAs) on the neuronal survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) transection. Method The experimental rats received daily BCAA injections through the caudal vein after left intra-orbital ON transection. Neuroprotection was evaluated by counting Fluorogold-labeled RGCs. The role of mammalian target of rapamycin (mTOR) pathway activation in promoting RGC survival was studied after rapamycin administration. Moreover, a peripheral nerve (PN) graft was transplanted onto the transected ON to study the effects of BCAAs on axon regeneration of injured RGCs. Results Our results showed that BCAAs alleviated the death of RGCs 7 and 14 days after ON transection, accompanied by an activation of mTOR pathway in RGCs. Blocking mTOR pathway with rapamycin eliminated such neuroprotective effects of BCAAs. Moreover, BCAAs also promoted axon regeneration of injured RGCs into a PN graft. Conclusion Our results suggest a neuroprotection of BCAAs through the activation of mTOR pathway. BCAAs also have a beneficial effect on axon regeneration of injured RGCs. Therefore, BCAAs could be considered for the clinical treatment of ON injury.

Urgent requirements for clinical translational studies in restoration of injured optic nerve

The optic nerve belongs to the central nervous system (CNS). Because of the lack of neurotrophic factors in the micro environment of the CNS and the presence of myelin and glial scar-related inhibitory molecules, and the inherent low renewal potentials of CNS neurons comparing to the peripheral nerve system, it is difficult to spontaneously regenerate the optic nerve after injury. Protecting damaged retinal ganglion cells (RGCs), supplementing neurotrophic factor, antagonizing axon regeneration inhibitory factor, and regulating the inherent regeneration potential of RGCs can effectively promote the regeneration and repair of optic nerve. Basic research has made important progress, including the restoration of visual function, but there are still a lot of unsolved problems in clinical translation of these achievements, so far there is no ideal method of treatment of optic nerve injury. Therefore, it is rather urgent to strengthen the cooperation between basic and clinical research, to promote the transformation of basic research to the clinical applications as soon as possible, which will change the unsatisfactory clinical application status. Key words: Optic nerve injury; Retinal ganglion cells; Axons; Editorial

Sirtuin 1 regulates lipid metabolism associated with optic nerve regeneration.

Injury to the optic nerve may lead to axonal degeneration, followed by the gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. In the present study the mechanism of optic nerve injury, and the following regeneration and repair processes associated with sirtuin 1 (SIRT1)‑regulated lipid metabolism were analyzed. In addition, the treatment of optic nerve injury using resveratrol was investigated. A rat model of optic nerve damage was prepared, and rats were divided into control, model and resveratrol groups. The model rats (with optic nerve damage) were treated with phosphate‑buffered saline or resveratrol once a day for seven, 14 and 21 days. The rats were then sacrificed and the optic nerve was dissected. The expression levels of SIRT1, sterol regulatory element‑binding protein 2 (SREBP‑2) and 3‑hydroxy‑3‑methylglutaryl‑CoA reductase (HMGCR) mRNA in the optic nerve were measured by reverse transcription‑quantitative polymerase chain reaction, and the protein expression of SIRT1, SREBP2 and HMGCR was evaluated by western blotting. In addition, the cholesterol level of the optic nerve was assessed. The retina was excised and the surviving RGCs were observed and counted. The morphology of the RGCs in the rat model of optic nerve injury changed; however, the degree of damage in rats treated with resveratrol was relatively small. The number of surviving RGCs and the cholesterol level in the RGCs from the model rats was observed to be restored by treatment with resveratrol following optic nerve damage. Additionally, the expression levels of SIRT1, SREBP‑2 and HMGCR mRNA and protein were restored by resveratrol treatment in the rats with optic nerve damage. Thus, resveratrol reversed certain features of the optic nerve injury damage via SIRT1 and SREBP‑2‑associated signaling pathways and the downstream regulated gene, HMGCR. Furthermore, resveratrol promoted cholesterol synthesis in, and repair of, RGCs. Therefore, SIRT1 may serve as a promising novel target for the treatment of optic nerve damage.

MicroRNA-30b promotes axon outgrowth of retinal ganglion cells by inhibiting Semaphorin3A expression

Semaphorin3A (Sema3A) is a major inhibitory factor of optic nerve (ON) regeneration post-injury. Many microRNAs (miRNAs) are expressed specifically in the mammalian brain and retina and are dynamically regulated during development, suggesting that this group of miRNAs may be associated with neural development. We found that microRNA-30b (miR-30b) bound to the three prime untranslated region (3′ UTR) of Sema3A and inhibited the expression of Sema3A mRNA. The mRNA expression level of miR-30b and the protein expression levels of Sema3A, Neuropilin1 (NRP1), PlexinA1 (PlexA1), phosphorylated p38MAPK (p-p38MAPK), and active caspase-3 were all upregulated in retinas from rats with a damaged ON relative to those with an intact ON. Transfection of cultured retinal ganglion cells (RGCs) with an miR-30b mimic led to decreased levels of Sema3A, NRP1, PlexA1, p-p38MAPK, and active caspase-3 protein expression, as well as axon elongation and reduced levels of apoptosis. These findings provide evidence that miR-30b inhibits Sema3A expression. Decreased Sema3A expression promotes axon outgrowth in RGCs due to reduced levels of Sema3A binding to NRP1 and PlexA1 and simultaneously reduces apoptosis by inhibiting the p38MAPK and caspase-3 pathways. Our findings provide the first evidence that miR-30b-mediated Sema3A downregulation may serve as a new strategy for the clinical treatment of ON injury.

Oncomodulin/truncated protamine-mediated Nogo-66 receptor small interference RNA delivery promotes axon regeneration in retinal ganglion cells.

The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ∼12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.

α-Crystallin protects RGC survival and inhibits microglial activation after optic nerve crush

AimsActivation of retinal microglial cells (RMCs) is known to contribute to retinal ganglion cell (RGC) death after optic nerve injury. The purpose of this study was to investigate the effects of intravenous injection of α-crystallin on RGC survival and RMC activation in a rat model of optic nerve crush. Main methodsRGCs were retrogradely labeled with fluorogold. Rats were intravenously injected with normal saline or α-crystallin (0.05g/kg, 0.5g/kg, and 5g/kg) at 2, 4, 6, 8, 10, and 12days after the optic nerve crush. Activated RMCs were characterized using immunofluorescence labeling with CD11b, and TNF-α and iNOS expression was detected using immunoblot analyses. We analyzed the morphology and numbers of RGC and RMC 2 and 4weeks after injury using fluorescence and confocal microscopy. Key findingsThe number of RGCs decreased after optic nerve injury, accompanied by significantly increased numbers of activated RMCs. Intravenous injection of α-crystallin decreased the number of RMCs, and enhanced the number of RGCs compared to saline injection. α-Crystallin administration inhibited TNF-α and iNOS protein expression induced by optic nerve injury. SignificanceOur results suggest that α-crystallin promotes RGC survival and inhibits RMC activation. Intravenous injection of α-crystallin could be a possible strategy for the treatment of optic nerve injury.

Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury

Optic nerve injury (ONI) induces retinal ganglion cell (RGC) death and optic nerve atrophy that lead to visual loss. Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced RGC apoptosis. In this study, we found that ONI-induced p38 activation and RGC loss were suppressed in ASK1-deficient mice. Sequential in vivo retinal imaging revealed that post-ONI treatment with a p38 inhibitor into the eyeball was effective for RGC protection. ONI-induced monocyte chemotactic protein-1 production in RGCs and microglial accumulation around RGCs were suppressed in ASK1-deficient mice. In addition, the productions of tumor necrosis factor and inducible nitric oxide synthase in microglia were decreased when the ASK1-p38 pathway was blocked. These results suggest that ASK1 activation in both neural and glial cells is involved in neural cell death, and that pharmacological interruption of ASK1-p38 pathways could be beneficial in the treatment of ONI.

Endoscopic decompression of optic canal

To evaluate the effectiveness of endoscope in the treatment of optic nerve injury. Thirteen patients with fracture at the inner and lower walls of optical canal underwent endoscopic decompression. The posterior 1/3 of the middle concha, the posterior ethmoid sinus and sphenoid sinus were opened, chips of bone and old hematocele were scavenged, and the optic ring of the optic canal was polished; however, the sheath of the optic nerve remained intact. In 2 cases with the dura broken at the cranial part of optic nerve, the broken site was covered with mucosa of middle concha, and the sphenoid sinus and the middle nasal meatus were covered with oil ribbon gauze. Three days after the operation, the ribbon gauze was pulled put, however, if cerebro-spinal fluid leakage was suspected, the ribbon gauze was pulled out 7-10 days after. After the operation the visual acuity improvement reaching one grade or more and improvement of visual field were defined as effective. Five of the 6 patients without light perception before the operation showed improvement in visual acuity. The other 7 patients with residual visual acuity all recovered to different extents. A mini-invasive surgery, decompression of optic canal with endoscope is effective in decompressing optic nerve, decreasing the injury of optic nerve, and the patients recover quickly after operation.

Transcranial optic nerve decompression for optic nerve injury

To discuss the operative indications and advantages of transcranial optic nerve decompression in treatment of optic nerve injury resulted from skull base fracture. The data, such as the site of impact, vision, ocular movement, characteristic of CT, and pathologic changes during operation, and the extent of operative decompression of 118 patients with optic nerve injury. According the site of impact on the head, 87 of lateral superciliary arch type, 18 of medial superciliary arch type, and 13 of zygomatic type, undergoing transcranial optic nerve decompression were analyzed retrospectively. The patients were followed up for 6 months after operation. For the purpose of evaluation, the postoperative outcome of visual acuity was classified into five grades: blindness, hand movement, finger count, light perception and visual acuity > 0.05. The visual acuity improvement reaching one grade or more was defined as effective. The improvement of visual field was also considered effective. After follow-up of 6 months, effect was shown in 35 out of the 72 patients with pre-operative blindness (48.6%), and all the 46 patients with residual vision (100%). The total effective rate was 68.6%. The post-operative effective rate was 64.4% in patients with lateral superciliary arch type, 83.3% in patients with medial superciliary arch type injury and 76.9% in patients with zygomatic type injury. Transcranial optic nerve decompression is worthy recommending to the patients with traumatic optic neuropathy. The operative indications include patients with residual vision; patients with bilateral optic nerve injury; and patients with blindness less than 3 days.