Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

Several studies have found that transplantation of neural progenitor cells(NPCs)promotes the survival of injured neurons.However,a poor integration rate and high risk of tumorigenicity after cell transplantation limits their clinical application.Small extracellular vesicles(sEVs)contain bioactive molecules for neuronal protection and regeneration.Previous studies have shown that stem/progenitor cell-derived sEVs can promote neuronal survival and recovery of neurological function in neurodegenerative eye diseases and other eye diseases.In this study,we intravitreally transplanted sEVs derived from human induced pluripotent stem cells(hiPSCs)and hiPSCs-differentiated NPCs(hiPSC-NPC)in a mouse model of optic nerve crush.Our results show that these intravitreally injected sEVs were ingested by retinal cells,especially those localized in the ganglion cell layer.Treatment with hiPSC-NPC-derived sEVs mitigated optic nerve crush-induced retinal ganglion cell degeneration,and regulated the retinal microenvironment by inhibiting excessive activation of microglia.Component analysis further revealed that hiPSC-NPC derived sEVs transported neuroprotective and anti-inflammatory miRNA cargos to target cells,which had protective effects on RGCs after optic nerve injury.These findings suggest that sEVs derived from hiPSC-NPC are a promising cell-free therapeutic strategy for optic neuropathy.

Optic nerve sheath diameters in nontraumatic brain injury:A scoping review and role in the intensive care unit

BACKGROUND Neuromonitoring in medical intensive care units is challenging as most patients are unfit for invasive intracranial pressure(ICP)modalities or unstable to transport for imaging.Ultrasonography-based optic nerve sheath diameter(ONSD)is an attractive option as it is reliable,repeatable and easily performed at the bedside.It has been sufficiently validated in traumatic brain injury(TBI)to be incorporated into the guidelines.However,currently the data for non-TBI patients is inconsistent for a scientific recommendation to be made.AIM To compile the existing evidence for understanding the scope of ONSD in measuring ICP in adult non-traumatic neuro-critical patients.METHODS PubMed,Google Scholar and research citation analysis databases were searched for studies in adult patients with non-traumatic causes of raised ICP.Studies from 2010 to 2024 in English languages were included.RESULTS We found 37 articles relevant to our search.The cutoff for ONSD in predicting ICP varied from 4.1 to 6.3 mm.Most of the articles used cerebrospinal fluid opening pressure followed by raised ICP on computed tomography/magnetic resonance imaging as the comparator parameter.ONSD was also found to be a reliable outcome measure in cases of acute ischaemic stroke,intracerebral bleeding and intracranial infection.However,ONSD is of doubtful utility in septic metabolic encephalopathy,dysnatremias and aneurysmal subarachnoid haemorrhage.CONCLUSION ONSD is a useful tool for the diagnosis of raised ICP in non-traumatic neuro-critically ill patients and may also have a role in the prognostication of a subset of patients.

Biomechanical analysis of optic nerve injury treated by compound light granules and ciliary neurotrophic factor

In this study, rabbit models of optic nerve injury were reproduced by the clamp method. After modeling, rabbit models were given one injection of 50 ng recombinant human ciliary neurotrophic factor into the vitreous body and/or intragastric injection of 4 g/kg compound light granules containing Radix Angelicae Sinensis and Raidix Paeoniae Alba at 4 days after modeling, once per day for 30 consecutive days. After administration, the animals were sacrificed and the intraorbital optic nerve was harvested. Hematoxylin-eosin staining revealed that the injured optic nerve was thinner and optic nerve fibers were irregular. After treatment with recombinant human ciliary neurotrophic factor, the arrangement of optic nerve fibers was disordered but they were not markedly thinner. After treatment with compound light granules, the arrangement of optic nerve fibers was slightly disordered and their structure was intact. After combined treatment with recombinant human ciliary neurotrophic factor and compound light granules, the arrangement of optic nerve fibers was slightly disordered and the degree of injury was less than after either treatment alone. Results of tensile mechanical testing of the optic nerve showed that the tensile elastic limit strain, elastic limit stress, maximum stress and maximum strain of the injured optic nerve were significantly lower than the normal optic nerve. After treatment with recombinant human ciliary neurotrophic factor and/or compound light granules, the tensile elastic limit strain, elastic limit stress, maximum stress and maximum strain of the injured optic nerve were significantly increased, especially after the combined treatment. These experimental findings indicate that compound light granules and ciliary neurotrophic factor can alleviate optic nerve injury at the histological and biochemical levels, and the combined treatment is more effective than either treatment alone.

Research progress of stem cells on glaucomatous optic nerve injury

Glaucoma, the second leading cause of blindness, is an irreversible optic neuropathy. The mechanism of optic nerve injury caused by glaucoma is undefined at present. There is no effective treatment method for the injury. Stem cells have the capacity of self-renewal and differentiation. These two features have made them become the research focus on improving the injury at present. This paper reviews the application progress on different types of stem cells therapy for optic nerve injury caused by glaucoma.

Human umbilical cord blood stem cells and brainderived neurotrophic factor for optic nerve injury: a biomechanical evaluation

Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 10^6 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.

The role of monocytes in optic nerve injury

Monocytes,including monocyte-derived macrophages and resident microglia,mediate many phases of optic nerve injury pathogenesis.Resident microglia respond first,followed by infiltrating macrophages which regulate neuronal inflammation,cell proliferation and differentiation,scar formation and tissue remodeling following optic nerve injury.However,microglia and macrophages have distinct functions which can be either beneficial or detrimental to the optic nerve depending on the spatial context and temporal sequence of their activity.These divergent effects are attributed to pro-and anti-inflammatory cytokines expressed by monocytes,crosstalk between monocyte and glial cells and even microglia-macrophage communication.In this review,we describe the dynamics and functions of microglia and macrophages in neuronal inflammation and regeneration following optic nerve injury,and their possible role as therapeutic targets for axonal regeneration.

Selective deletion of zinc transporter 3 in amacrine cells promotes retinal ganglion cell survival and optic nerve regeneration after injury

Vision depends on accurate signal conduction from the retina to the brain through the optic nerve,an important part of the central nervous system that consists of bundles of axons originating from retinal ganglion cells.The mammalian optic nerve,an important part of the central nervous system,cannot regenerate once it is injured,leading to permanent vision loss.To date,there is no clinical treatment that can regenerate the optic nerve and restore vision.Our previous study found that the mobile zinc(Zn^(2+))level increased rapidly after optic nerve injury in the retina,specifically in the vesicles of the inner plexiform layer.Furthermore,chelating Zn^(2+)significantly promoted axonal regeneration with a long-term effect.In this study,we conditionally knocked out zinc transporter 3(ZnT3)in amacrine cells or retinal ganglion cells to construct two transgenic mouse lines(VGAT^(Cre)ZnT3^(fl/fl)and VGLUT2^(Cre)ZnT3^(fl/fl),respectively).We obtained direct evidence that the rapidly increased mobile Zn^(2+)in response to injury was from amacrine cells.We also found that selective deletion of ZnT3 in amacrine cells promoted retinal ganglion cell survival and axonal regeneration after optic nerve crush injury,improved retinal ganglion cell function,and promoted vision recovery.Sequencing analysis of reginal ganglion cells revealed that inhibiting the release of presynaptic Zn^(2+)affected the transcription of key genes related to the survival of retinal ganglion cells in postsynaptic neurons,regulated the synaptic connection between amacrine cells and retinal ganglion cells,and affected the fate of retinal ganglion cells.These results suggest that amacrine cells release Zn^(2+)to trigger transcriptomic changes related to neuronal growth and survival in reginal ganglion cells,thereby influencing the synaptic plasticity of retinal networks.These results make the theory of zinc-dependent retinal ganglion cell death more accurate and complete and provide new insights into the complex interactions between retinal cell networks.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist inhibits apoptosis of retinal ganglion cells in a rabbit model of optic nerve injury

A rabbit model of traumatic optic nerve injury, established by occlusion of the optic nerve using a vascular clamp, was used to investigate the effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist GYKI 52466 on apoptosis of retinal ganglion cells following nerve injury. Hematoxylin-eosin staining and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that retinal ganglion cells gradually decreased with increasing time of optic nerve injury, while GYKI 52466 could inhibit this process. The results demonstrate that following acute optic nerve injury, apoptosis of retinal ganglion cells is a programmed process, which can be inhibited by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist.

Neurolin expression in the optic nerve and immunoreactivity of Pax6-positive niches in the brain of rainbow trout(Oncorhynchus mykiss) after unilateral eye injury

In contrast to astrocytes in mammals, fish astrocytes promote axon regeneration after brain injury and actively participate in the regeneration process. Neurolin, a regeneration-associated, Zn8-1abeled protein, is involved in the repair of damaged optic nerve in goldfish. At 1 week after unilateral eye injury, the ex- pression of neurolin in the optic nerve and chiasm, and the expression of Pax6 that influences nervous system development in various brain regions in the rainbow trout （Oncorhynchus mykiss） were detected. Immunohistochemical staining revealed that the number of Zn8~ cells in the optic nerve head and intraor- bital segment was obviously increased, and the increase in Zn8~ cells was also observed in the proximal and distal parts of injured optic nerve. This suggests that Zn8＊ astrocytes participate in optic nerve regeneration. ELISA results revealed that Pax6 protein increased obviously at 1 week post-injury. Immunohistochemi- cal staining revealed the appearance of Pax6＋ neurogenic niches and a larger number of neural precursor cells, which are mainly from Pax6＋ radial glia cells, in the nuclei of the diencephalon and optic tectum of rainbow trout （Oncorhynchus mykiss）. Taken together, unilateral eye injury can cause optic nerve reaction, and the formation of neurogenic niches is likely a compensation phenomenon during the repair process of optic nerve injury in rainbow trout （Oncorhynchus mykiss）.

The clinical value of dynamic ultrasound measurement of optic nerve sheath diameter in the treatment of patients with moderate and severe craniocerebral injury

Objective:To explore the clinical value of dynamic ultrasound monitoring of optic nerve sheath diameter(ONSD)in the treatment of patients with moderate and severe head injury(CI).Methods:A total of 160 patients with moderate and severe CI admitted to the First Affiliated Hospital of Hainan Medical University from January 2018 to January 2020 were selected and divided into observation group(80 cases)and control group(80 cases)a Januaryccording to the random number table.Patients in control group and observation group were dehydrated to reduce intracranial pressure(ICP)according to clinical symptoms/brain CT and ONSD monitoring guidance.National Institutes of Health Stroke Scale(NIHSS),Acute Physiology and Chronic Health EvaluationⅡ(APACHEⅡ),Glasgow Coma Scale(GCS),complications,prognosis,ICU stay time and mechanical ventilation time were compared between the two groups.Results:NIHSS score[control group:(19.58±3.19)points vs(37.98±5.75)points,observation group:(10.33±2.42)points vs(38.05±5.83)points]and APACHE II score[control group:(14.55±2.17)points vs(19.87±3.50)points,observation group:(8.71±2.03)points vs(20.12±3.56)points]of the two groups at 1 month after injury were significantly lower than those at admission(P<0.05),GCS score[control group:(10.78±1.66)points vs(8.03±1.34)points,observation group:(13.10±1.72)points vs(7.99±1.32)points]were significantly higher than that at admission(P<0.05).At 1 month after injury,NIHSS score[(10.33±2.42)points vs(19.58±3.19)points],APACHE II score[(8.71±2.03)points vs(14.55±2.17)points]in the observation group were significantly lower than those in the control group(P<0.05),and GCS score[(13.10±1.72)points vs(10.78±1.66)points]in the observation group was significantly higher than that in the control group(P<0.05).The proportion of hydrocephalus(2.50%vs 12.50%),total complication rate(5.00%vs 21.25%),proportion of severe disability(5.00%vs 17.50%),proportion of survival in plant man(3.75%vs 15.00%),mortality rate(2.50%vs 12.50%),ICU stay time[(5.01±1.25)d vs(8.38±2.29)D],mechanical ventilation time[(2.18±0.75)D]in observation group were lower than those in the control group,and the good rate(56.25%vs 32.50%)and the total effective rate(93.75%vs 72.50%)in the control group were significantly higher than those in the control group(P<0.05).Conclusion:Dynamic ultrasound monitoring ONSD is effective in guiding dehydration treatment of patients with moderate and severe CI,it can significantly reduce ICP and complications,improve prognosis,which is worthy of promotion and application.

Establishing a cat model of chronic optic nerve compression injury

BACKGROUND： An animal model of chronic optic nerve injury is necessary to further understand the pathological mechanisms involved. OBJECTIVE： To establish a stabilized, chronic, optic nerve crush model, which is similar to the clinical situation to explore histopathological and optic electrophysiological changes involved in this injury. DESIGN, TIME AND SETTING： A randomized and controlled animal trial was performed at Shanghai Institute of Neurosurgery from May to October 2004, MATERIALS： A BAL3XRAY undetachable balloon and Magic-BD catheter were provided by BLAT, France; JX-2000 biological signal processing system by Second Military Medical University of Chinese PLA, China; inverted phase contrast microscopy by Olympus, Japan. METHODS： A total of twenty normal adult cats were randomly assigned to control （n = 5） and model （n = 15） groups, according to different doses of contrast agent injected through balloons as follows： 0.2 mL injection, 0.25 mL injection, and 0.35 mL injection, with each group containing 5 animals. Imitating the clinical pterion approach, the optic nerves were exposed using micro-surgical methods. An engorged undetachable balloon was implanted beneath the nerve and connected to a catheter. Balloon size was controlled with a contrast agent injection （0.1 mL/10 min） to form an occupying lesion model similar to sellar tumors. MAIN OUTCOME MEASURES： The visually evoked potential examination was used to study optical electrophysiology changes in pre-post chronic optical nerve injury. Ultrastructural pathological changes to the optic nerve were analyzed by electron microscopy. RESULTS： During the early period （day 11 after modeling）, visually evoked potential demonstrated no significant changes. In the late period （day 51 after modeling）, recorded VEP demonstrated that P1 wave latency was prolonged and P1 wave amplitude was obviously reduced. Following injury, the endoneurium, myelin sheath, lamella, axolemma, and axon appeared disordered. CONCLUSION： Results demonstrated that the chronic, intracranial, optical nerve crush model was stable and could simulate optic nerve lesions induced by sellar tumors. Under the condition of chronic optical nerve crush, visually evoked potentials were aggravated.

AB006.Longitudinal effects of an optic nerve injury on behavioural measures of visual functions

Background:Visual deficits,caused by ocular disease or trauma,can cause lasting damage.However,recent research has focused on neural plasticity as a means to regain visual functions.In order to better understand the involvement of neural plasticity and reorganization in partial vision restoration,we aim to evaluate the partial recovery of a visual deficit over time using two behavioural tests.In our study,a partial optic nerve crush(pONC)serves as an induced visual deficit,allowing for residual vision from surviving cells.Methods:Visual functions in C57BL/6 mice was measured using two behavioural tests prior to a bilateral pONC,then at various time points after the pONC.In this study,two injury intensities were used:a high intensity pONC with the full force of self-closing forceps,and a low intensity pONC,in which a calibrated space was left between the forceps at the closed position.The two behavioural tests consisted of the optomotor reflex(OMR)and the visual cliff(VC)tests.The OMR test measures the mouse’s tracking reflex in response to moving sinusoidal gratings.The VC test,on the other hand,evaluates exploratory behaviour,by simulating a cliff to observe the animal’s sense of depth perception.After the behavioural evaluation,surviving retinal ganglion cells were counted.Results:The high intensity pONC resulted in a total loss of visual acuity as measured by the OMR test,with no improvement in the following 4 weeks.However,the light intensity pONC showed the same initial loss,but recovery was observed as of day 3,and results in 40-60%recovery after 4 weeks.With the VC test,mice with intact vision will avoid the deep end,opting to spend more time in the shallow end.However,after both high and low intensity pONCs,this preference is no longer observed.Both groups show a return to the shallow end preference at day 14,though the low intensity pONC group showed a stronger preference similar to baseline performance.The percentage of surviving retinal ganglion cells was higher with the low intensity(68%)than with the high intensity(17%)pONC.Conclusions:There is evidence of visual recovery at the behavioural level following a pONC,though very little recovery was observed following a high intensity pONC,and only with the VC test.Therefore,a certain amount of residual retinal input may be required for recovery at the behavioural level.

Ultrastructural changes in the optic nerve and capillary vessels during early stages of optic nerve injury

BACKGROUND： Capillaries are the only blood supply for optic nerves, which makes the system more vulnerable to impaired blood circulation. OBJECTIVE： To observe the ultrastructural changes in the optic nerves and capillaries in rabbits following intracanalicular segment injury to the optic nerve. DESIGN, TIME AND SETTING： Comparative, observational, pathological morphology was performed at the Department of Anatomy, Weifang Medical College from September to November 2007. MATERIALS： Models of intracanalicular segment injury to the optic nerve were induced in the right eye of thirty healthy, adult rabbits by a free-falling metal cylinder. The H-7500 transmission electron microscope was provided by Hitachi, Japan. METHODS： All rabbits were randomly assigned into experimental （n = 25） and control （n = 5） groups. Optic nerve specimens were obtained from the experimental group at 0.5, 6, 12, 48, and 96 hours, respectively, following injury. Ultrastructural changes to the optic nerves and their capillaries were observed by electron microscopy. Optic nerve injury was not established in the control group, but optic nerve specimens were collected similarly to the experimental group. MAIN OUTCOME MEASURES： Ultrastructural changes in the injured optic nerves and their capillaries. RESULTS： Thirty rabbits were included in the final analysis. In the control group, cross-sections of the optic nerves exhibited varied thicknesses with regularly arranged fibers. The axons appeared to be smooth with condensed myelin sheaths and oval mitochondria. The microtubules and microfilaments were clearly seen. The lumens of the capillaries were regular with densely arranged endothelial cells and visible mitochondria. In the experimental group, 30 minutes after injury to the optic nerves, swollen axons, sparse myelin sheath, disordered microtubules and microfilaments, swollen mitochondria, and a decreased number of pinocytosis vesicles and microfilaments in endothelial cells of the capillaries were observed. At 6 hours, medullary and vacuolar degeneration in the mitochondria, and swollen endothelial cells in the capillary, were visible. At 12 hours, these changes were more obvious. At 48 hours, granular dissolution of microtubules, microfilaments, and mitochondria, as well as diffuse degeneration of mitochondria in the endothelial cells, were observed. At 96 hours, axonal disintegration, vacuolar degeneration, and dilated capillaries were observed. CONCLUSION： During early stages, the injured intracanalicular optic nerve exhibited swollen axons with vacuolar degeneration, swollen and degenerated mitochondria, decreased number of microtubules and microfilaments, and dilated capillaries with increased permeability.

Pathological changes in the retina and growth associated protein-43 expression following treatment of intracanalicular optic nerve injury via optic canal decompression,dexamethasone or their combination

BACKGROUND： The main clinical treatments for optic nerve injury are optic canal decompression and systemic administration of hormones, but both treatments have disadvantages. OBJECTIVE： To observe the pathological changes in the retina and growth associated protein-43 （GAP-43） expression, to compare the treatment of optic canal decompression, hormones, and their combination with the intracanalicular optic nerve injury.DESIGN, TIME AND SETTING： A randomized, controlled animal study was performed at the Department of Anatomy, Weifang Medical University, China, from September 2007 to November 2008.MATERIALS： Dexamethasone （Shandong Huaxin Pharmaceutical, China） and rabbit anti-GAP-43 polyclonal antibody （Boster, China） were used.METHODS： All 36 healthy adult rabbits were randomly assigned to control group （n = 4）, simple injury group （n = 20）, and treatment group （n = 12）. Intracanalicular optic nerve injury models were established using the metal cylinder free-fall impact method. The control group was left intact. The treatment group （four rabbits in each subgroup） was treated by optic nerve decompression, dexamethasone treatment （1 mg/kg daily via two intravenous infusions, 1/5 total dose reduction every 3 days, for 14 days）, and simultaneously giving surgery and hormone treatment.MAIN OUTCOME MEASURES： Pathological changes in the retina were determined using hematoxylin-eosin staining. GAP-43 expression was detected using immunohistochemistry in the retina.RESULTS： Retina injury induced obvious pathological changes in the retina. With prolonged time after optic nerve injury, the number of retinal ganglion cells was gradually decreased, and reached the minimum on day 14 （P〈0.01）. All three treatments increased the number of retinal ganglion cells （P〈0.01）, but surgery ＋ hormone treatment was most effective. No GAP-43 cells were present in the normal retinal, but they appeared 3 days after injury, peaked 7 days after injury, and then began to decline.CONCLUSION： Intracanalicular optic nerve injury induced obvious pathological changes in the retina, including increased GAP-43 expression. Optic canal decompression and hormones improved nerve repair after injury, and their combination produced better outcomes.

Establishing a cat model of acute optic nerve injury

BACKGROUND： In order to investigate the progress in optic nerve injury and the following regeneration and repair, many kinds of animal models of optic nerve injury have been established, such as models of acute and chronic ocular hypertension, compression, amputating wound, ischemia reperfusion or hypoxia, intravitreal injection of excitatory amino acids, etc. However, most of these models are established by squeezing intraorbital optic nerve, and suitable for ophthalmology, and there are fewer models suitable for the acute cranial contusion in neurosurgery. OBJECTIVE： To observe the changes of optic nerve after acute injury, and the characteristics of methods for establishing model of acute optic nerve injury in cats. DESIGN： A complete randomized grouping and controlled animal trial. SETTING： Department of Neurosurgery, General Hospital of Ji＇nan Military Area Command of Chinese PLA. MATERIALS： Twenty-eight healthy adult cats, common degree, either sex, weighing 2.0-3.5 kg, were provided by the animal experimental center of Fudan University. The cats were randomly divided into control group （n =3） and model group （n =25）, and 5 cats in the model group were observed at 6 hours and 1, 3, 7 and 14 days after injury respectively. JX-2000 biological signal processing system （Department of Physiology, Second Military Medical University of Chinese PLA, Shanghai）; Inverted phase contrast microscope （Olympus）; Axioplan 2 imaging microgram analytical system （Labsystems）. METHODS： The experiments were carried out in the Department of Neurosurgery, General Hospital of Jinan Military Area Command of Chinese PLA from June 2004 to June 2005. The cats in the model groups were made into models of acute optic nerve injury： The cats were anesthetized, then the limbs were fixed in a lateral recumbent position. Pterion approach in human was imitated, the operative incision was made along the line between lateral canthus and tragus, and it could be seen deep along the skull base that white optic nerve （about 3 mm） went through optic foramen and entered into brain tissue. It was squeezed with noninvasive vascular clip for 20 seconds, then the clip was removed, and then the skull was closed after it was examined to be no bleeding. The size of bilateral pupils, direct and indirect light reflexes were observed postoperatively. Successfully established models were judged by larger operated pupil than controlateral one, disappearance of direct light reflex and the existence of indirect light reflex. No model establishment was performed in the control group. Each cat was tested with flash visual evoked potential （F-VEP） to observe the electrophysiological changes before and after experiment. All the cats in the control group and model groups were killed under anesthesia before model establishment and at 6 hours, 1, 3, 7 and 14 days after model establishment respectively, and the pathological changes of the optic nerve after injury were observed under electron microscope and light microscope. MAIN OUTCOME MEASURES： VEP and the ultrastructural changes of optic nerve after acute optic nerve injury in both groups. RESULTS： All the 28 cats were involved in the analysis of results. ① VEP results： The VEP latencies were obviously different between the control group and model group at each time point （P 〈 0.05）, whereas there were no obvious differences among different time points in the model group （P 〉 0.05）. The VEP amplitudes were obviously different between the control group and model group at each time point （P 〈 0.05）, whereas there were no obvious differences among different time points in the model group （P 〉 0.05）. ② Ultrastructural changes of the optic nerve： Under electron microscope, normal optic nerve myelin sheath had complete structure, tramal plates were clear and arranged tightly, axolemma was complete, whereas the structures of endoneurium, myelin sheath, tramal plates, axolemma and axon were in disorders after optic nerve injury. CONCLUSION： Models of acute optic nerve injury established by squeezing intracranial optic nerve with noninvasive vascular clip can be applied in studying intracranial acute optic nerve injury.

Optic nerve injury-induced regeneration in the adult zebrafish is accompanied by spatiotemporal changes in mitochondrial dynamics

Axonal regeneration in the central nervous system is an energy-intensive process.In contrast to mammals,adult zebrafish can functionally recover from neuronal injury.This raises the question of how zebrafish can cope with this high energy demand.We previously showed that in adult zebrafish,subjected to an optic nerve crush,an antagonistic axon-dendrite interplay exists wherein the retraction of retinal ganglion cell dendrites is a prerequisite for effective axonal repair.We postulate a‘dendrites for regeneration’paradigm that might be linked to intraneuronal mitochondrial reshuffling,as ganglion cells likely have insufficient resources to maintain dendrites and restore axons simultaneously.Here,we characterized both mitochondrial distribution and mitochondrial dynamics within the different ganglion cell compartments(dendrites,somas,and axons)during the regenerative process.Optic nerve crush resulted in a reduction of mitochondria in the dendrites during dendritic retraction,whereafter enlarged mitochondria appeared in the optic nerve/tract during axonal regrowth.Upon dendritic regrowth in the retina,mitochondrial density inside the retinal dendrites returned to baseline levels.Moreover,a transient increase in mitochondrial fission and biogenesis was observed in retinal ganglion cell somas after optic nerve damage.Taken together,these findings suggest that during optic nerve injury-induced regeneration,mitochondria shift from the dendrites to the axons and back again and that temporary changes in mitochondrial dynamics support axonal and dendritic regrowth after optic nerve crush.

Cell proliferation and apoptosis in optic nerve and brain integration centers of adult trout Oncorhynchus mykiss after optic nerve injury

Fishes have remarkable ability to effectively rebuild the structure of nerve cells and nerve fibers after central nervous system injury.However,the underlying mechanism is poorly understood.In order to address this issue,we investigated the proliferation and apoptosis of cells in contralateral and ipsilateral optic nerves,after stab wound injury to the eye of an adult trout Oncorhynchus mykiss.Heterogenous population of proliferating cells was investigated at 1 week after injury.TUNEL labeling gave a qualitative and quantitative assessment of apoptosis in the cells of optic nerve of trout 2 days after injury.After optic nerve injury,apoptotic response was investigated,and mass patterns of cell migration were found.The maximal concentration of apoptotic bodies was detected in the areas of mass clumps of cells.It is probably indicative of massive cell death in the area of high phagocytic activity of macrophages/microglia.At 1 week after optic nerve injury,we observed nerve cell proliferation in the trout brain integration centers：the cerebellum and the optic tectum.In the optic tectum,proliferating cell nuclear antigen（PCNA）-immunopositive radial glia-like cells were identified.Proliferative activity of nerve cells was detected in the dorsal proliferative（matrix） area of the cerebellum and in parenchymal cells of the molecular and granular layers whereas local clusters of undifferentiated cells which formed neurogenic niches were observed in both the optic tectum and cerebellum after optic nerve injury.In vitro analysis of brain cells of trout showed that suspension cells compared with monolayer cells retain higher proliferative activity,as evidenced by PCNA immunolabeling.Phase contrast observation showed mitosis in individual cells and the formation of neurospheres which gradually increased during 1–4 days of culture.The present findings suggest that trout can be used as a novel model for studying neuronal regeneration.

Phosphorylated S6K1 and 4E-BP1 play different roles in constitutively active Rheb-mediated retinal ganglion cell survival and axon regeneration after optic nerve injury

Ras homolog enriched in brain(Rheb) is a small GTPase that activates mammalian target of rapamycin complex 1(mTORC1).Previous studies have shown that constitutively active Rheb can enhance the regeneration of sensory axons after spinal cord injury by activating downstream effectors of mTOR.S6K1 and4E-BP1 are important downstream effectors of mTORC1.In this study,we investigated the role of Rheb/mTOR and its downstream effectors S6K1 and 4E-BP1in the protection of retinal ganglion cells.We transfected an optic nerve crush mouse model with adeno-associated viral 2-mediated constitutively active Rheb and observed the effects on retinal ganglion cell survival and axon regeneration.We found that overexpression of constitutively active Rheb promoted survival of retinal ganglion cells in the acute(14 days) and chronic(21 and 42 days) stages of injury.We also found that either co-expression of the dominant-negative S6K1mutant or the constitutively active 4E-BP1 mutant together with constitutively active Rheb markedly inhibited axon regeneration of retinal ganglion cells.This suggests that mTORC1-mediated S6K1 activation and 4E-BP1 inhibition were necessary components for constitutively active Rheb-induced axon regeneration.However,only S6K1 activation,but not 4E-BP1 knockdown,induced axon regeneration when applied alone.Furthermore,S6K1 activation promoted the survival of retinal ganglion cells at 14 days post-injury,whereas 4E-BP1 knockdown unexpectedly slightly decreased the survival of retinal ganglion cells at 14 days postinjury.Ove rexpression of constitutively active 4E-BP1 increased the survival of retinal ganglion cells at 14 days post-injury.Likewise,co-expressing constitutively active Rheb and constitutively active 4E-BP1 markedly increased the survival of retinal ganglion cells compared with overexpression of constitutively active Rheb alone at 14 days post-injury.These findings indicate that functional 4E-BP1 and S6K1 are neuroprotective and that 4E-BP1 may exert protective effects through a pathway at least partially independent of Rhe b/mTOR.Together,our results show that constitutively active Rheb promotes the survival of retinal ganglion cells and axon regeneration through modulating S6K1 and 4E-BP1 activity.Phosphorylated S6K1 and 4E-BP1 promote axon regeneration but play an antagonistic role in the survival of retinal ganglion cells.

The mechanism of Chuanxiong Rhizoma on glaucomatous optic nerve injury based on network pharmacology and molecular docking

Based on network pharmacology,this study predicted the potential molecular mechanism and related pathways of the protective effect of traditional Chuanxiong Rhizoma,a traditional Chinese herb,on glaucomatous optic nerve injury,and conducted in vitro experimental verification of the predicted results of network analysis.We analyzed the molecular mechanism of Chuanxiong Rhizoma in the potential treatment of glaucoma by revealing its main active ingredients and predicting its targets,so as to provide reference for subsequent basic research.Network pharmacological research results showed that the potential hub targets and key signaling pathways of Chuanxiong Rhizoma in the treatment of glaucoma were closely related to biological processes such as apoptosis,autophagy,inflammation,oxidative stress and angiogenesis.Molecular docking showed that many active ingredients,such as chrysophanol(CHR),myricanone and retinol,could combine well with their target proteins by intermolecular forces,especially CHR had strong binding ability with each target.We speculated that the main active component of Chuanxiong Rhizoma might be involved in the regulation of PI3K-Akt,Nod-like receptor,IL-4 and IL-13,MAPK,AGE-RAGE and neurotrophin signaling pathway by regulating of PI3K,Akt,TLR4,RAGE,NTRK2 and other key targets.Furthermore,it may achieve multi-directional intervention on apoptosis/autophagy,inflammation/immunity,oxidative stress and nutrient metabolism of axoplasma flow,and then delay the degeneration of optic nerve injury.In vitro experiments showed that the active component CHR of Chuanxiong Rhizoma could reverse the M1-type polarization and autophagy/apoptosis of mouse microglia(BV2)induced by lipopolysaccharide(LPS)at the transcriptional level.Meanwhile,the expression of inflammatory mediators IL-1βand TNF-αwas inhibited,and the mRNA level of anti-inflammatory factor IL-10 was significantly increased.In addition,CHR down-regulates activation of the RAGE-NOX4 pathway mediated by LPS in reducing oxidative stress.In this study,network pharmacology and molecular docking technology were integrated for the first time to explore the potential molecular mechanism of traditional Chinese herb“Chuanxiong Rhizoma”in the treatment on glaucoma,and CHR was innovatively proposed as an important ingredient in Chuanxiong Rhizoma that plays a protective role in the damage of optic nerve.Preliminary verification was conducted through in vitro experiments.The results suggest that Chuanxiong Rhizoma may interfere with autophagy and apoptosis,inhibit immune inflammation,as well as reduce oxidative stress in the treatment of glaucoma through the active components represented by CHR,so as to resist progressive optic nerve injury.Our study provides theoretical basis for the clinical use of Chinese herbal medicine or its extract in glaucoma,and also lays a solid foundation for the research of Chinese medicine in the field of optic nerve protection.

Activation of autophagy in the retina after optic nerve crush injury in rats

AIM: To investigate the activation of autophagy in rat retina after optic nerve crush(ONC) and evaluate its relationship with apoptosis of retinal ganglion cells(RGCs).METHODS: The ONC model was established. Western blots were performed to investigate expression of p62, LC3 and Beclin-1. Transmission electron microscopy was performed to discover the autophagosomes in the retina after ONC. Immunohistochemistry was used to confirm the distribution of LC3. TUNEL was performed to confirm the relationship between autophagy and RGC apoptosis. RESULTS: p62/Beclin-1 ratio was declined shortly after ONC until to day 7 after ONC and then restored to a normal level at day 21. There was an opposite change in the LC3-II/LC3 I ratio in the retina compared to the p62/Beclin-1 ratio. Increased autophagosomes were found after ONC using transmission electron microscopy, and most of the LC3-stained cells were colocalized with RGCs and Müller cells. More LC3-immunoreactive cells and apoptotic RGCs were found on day 7 following ONC. CONCLUSION: Possible activation of autophagy in RGCs after ONC;autophagy mainly occurred in RGCs and Müller cells, and the apoptosis of RGCs after ONC may be partly associated with autophagic activation.