Spontaneous axonal regeneration after optic nerve injury in adult rat

Optic nerves of adult rats were crushed 2 mm behind the eye to examine the ability of retinal ganglion cells (RGCs) to regenerate their axons. Some animals were treated with the immunophilin ligands FK 506 or GPI 1046 for up to 4 weeks. After 10 days to 16 months, regenerating RGC axons were visualized using anterograde tracing and/or electron microscopy. A small proportion of RGC axons regenerated across the lesion site and grew very slowly along the entire optic nerve. Immunophilin ligands had no obvious effect. The regenerating axons were about 0.2 microm in diameter, and usually in clusters surrounded by astrocyte processes. Thus, some CNS axons can spontaneously regenerate long distances within degenerate white matter and this slow regeneration is not accelerated by immunophilin ligands.     

Radiation-induced impairment of optic nerve axonal transport in tree shrews and rats monitored by longitudinal manganese-enhanced MRI

PurposeRadiation-induced optic neuropathy (RION) is a serious complication that occurs after radiation therapy of tumors in the vicinity of the optic nerve, yet its mechanism and imaging features are poorly understood. In this study, we employed manganese-enhanced MRI (MEMRI) to assess optic nerve axonal transport in tree shrews and rats after irradiation.Materials and methodsA comparison of normal visual projections in tree shrews and rats was conducted by intravitreal MnCl₂ injection followed by MRI. Adult male tree shrews and rats received a total dose of 20 Gy delivered in two fractions (10 Gy per fraction) within 5 days. Longitudinal MEMRI was conducted 5, 10, 20 and 30 weeks after radiation. At the end of observation, motor proteins involved in axonal transport were detected by western blotting, and the axon cytoskeleton was assessed by immunofluorescence.ResultsThe eyeballs, lens sizes, vitreous volumes, optic nerves and superior colliculi of tree shrews were significantly larger than those of rats on MEMRI (P < 0.05). The Mn²⁺-enhancement of the optic nerve showed no significant changes at 5 and 10 weeks (P > 0.05) but decreased gradually from 20 to 30 weeks postirradiation (P < 0.05). The enhancement of the superior colliculus gradually decreased from 5 weeks to 30 weeks, and the decrease was most significant at 30 weeks (P < 0.05). The levels of the motor proteins cytoplasmic dynein-1, kinesin-1 and kinesin-2 in the experimental group were significantly decreased (P < 0.05). The immunofluorescence results showed that the α-tubulin, β-tubulin and SMI 31 levels in the experimental groups and control groups were not significantly different (P > 0.05).ConclusionTree shrews show great advantages in visual neuroscience research involving MEMRI. The main cause of the decline in axonal transport in RION is an insufficient level of motor protein rather than damage to the axonal cytoskeletal structure. Longitudinal MEMRI can be used to detect changes in axonal transport function and to observe the relatively intact axon structure from the early to late stages after radiation administration.     

Endothelin-1 impairs retrograde axonal transport and leads to axonal injury in rat optic nerve

The purpose of this study was to examine the effects of endothelin-1 (ET-1) on retrograde axonal transport in the rat optic nerve. Vehicle or ET-1 (0.2, 1, or 5 pmol/eye) were injected into the vitreous body in Sprague-Dawley rats. Retinal vessels were observed, using a fundus camera, before, and at 10 min, 3 days and 7 days after a single intravitreous injection. Two days after the injection, a neuronal tracer, fluoro gold, was administered via the superior colliculi to retrogradely label active retinal ganglion cells (RGCs). Five days after the tracer administration, retrogradely labeled RGCs were evaluated in the flat-mounted retina, and cross sections from each optic nerve were graded for injury by four independent, masked observers. ET-1 at 5 pmol/eye caused a significant constriction of retinal vessels (versus the vehicle-treated group) at 10 min after the injection. Intravitreous injection of ET-1 caused a dose-related decrease in the number of retrogradely labeled RGCs. Injection of 5 pmol/eye ET-1 led to a statistically significant decrease in the number of retrogradely labeled RGCs (versus the vehicle-treated group). ET-1 at 1 and 5 pmol/eye caused histological optic nerve damage (evaluated using a graded scale). The histological optic nerve damage correlated with the number of retrogradely labeled RGCs. In conclusion, a single intravitreous injection of ET-1 impaired retrograde axonal transport in the rat optic nerve and this impairment correlated with the histological optic nerve damage.     

Retrograde axonal transport in rat sciatic nerve after nerve crush injury

We investigated the quantitative alterations in retrograde transport of proteins following a nerve crush injury using the 3H N-succinimidyl propionate (3H NSP) method in rat sciatic nerve. After subepineurial injection of 3H NSP into the nerve the amount of radioactively labeled proteins accumulating in the cell bodies of the motor and sensory neurons was determined 1 day or 7 days later in nerves which had been crushed distal to the injection site 1, 3, 5, 7, or 33 days prior to 3H NSP labeling. One day accumulation in the DRG and spinal cord was not altered by nerve crush. Seven day accumulation in the DRG was initially slightly increased, then fell to 73% of control by 7 days, remaining reduced 33 days after crush. Seven day accumulation in the spinal cord was reduced to 25% of control 1 day after crush and remained at that low level except for 5 days post-crush when a normal amount of labeled protein was transported to the spinal cord. The time course of these changes suggests that quantitative alterations in retrograde transport may be involved in the long-term trophic interactions between the cell body and periphery, but are too slow to account for the earliest perikaryal responses to injury. In addition, the difference between the alterations of retrograde transport in motor and sensory neurons may reflect fundamental differences in the composition of retrograde transport in those different systems.     

Xylazine promotes axonal regeneration in the crushed optic nerve of adult rats

Xylazine, an alpha-2 adrenergic agonist, activates the endogenous trophic factors and neuronal survival signaling. Here, we tested the regenerative effect of xylazine on damaged optic nerve axons in adult rats. After optic nerve crush, xylazine was intraperitoneally injected into three groups of rats: a single administration immediately after the crush, intermittent administration, and daily administration. On day 14, the regenerated axons were quantitatively evaluated by anterograde labeling. Everyday administration but neither single nor intermittent administration markedly increased the number of labeled axons beyond the crush site, with upregulation of growth-associated protein-43 in the ganglion cell layer and the regenerated axons. It was concluded that xylazine promotes axonal regeneration in damaged optic nerves of adult rats.     

Bidirectional axonal transport of glycoproteins in goldfish optic nerve

The goldfish visual system was used to study the relationships between anterograde and retrograde transport of axonal glycoproteins. After intraocular injection of radioactive glucosamine, determinations were made of the normal time course of appearance of labeled glycoproteins in the optic nerve and tectum and of their time course of accumulation on both sides of an optic nerve crush. The labeled glycoproteins, transported at a maximum velocity of about 80 mm/day, continued to pass through the optic nerve in significant amounts for as long as 24 h after the injection, with a maximum at about 6 to 14 h. Retrograde transport of labeled materials back from the optic tectum to the same point in the nerve began about 5 h later, indicating a minimum possible retrograde velocity of about 36 mm/day and a maximum possible lag time in the axon terminals (with the assumption of equal retrograde and anterograde velocities) of 1 to 2 h. When delivery of glycoproteins from the retina to the optic tectum was interrupted by a nerve crush 8 h after injection, a component with rapid turnover in the tectum was revealed having a half-life of not more than 6 h. At least 40% of this turnover could be attributed to retrograde transport. The amount of labeled glycoprotein transported in a retrograde direction 8 to 10 h after injection was greatly elevated in regenerating axons 2 weeks after the optic tract was cut.     

依托咪酯促进大鼠视神经损伤后再生的研究

目的探讨依托咪酯对成年大鼠视神经损伤后再生的影响。方法选取25只成年SD大鼠,按随机数字表法随机分为依托咪酯治疗组（腹腔注射依托咪酯脂肪乳注射液,15只）、治疗对照组f腹腔注射脂肪乳,5只）和空白对照组（5只）;治疗组又分为低（2mg／kg）、中（4mg／kg）和高（6mg／kg）剂量3个亚组,每亚组5只。采用自体坐骨神经移植模型和荧光金逆行标记再生视网膜神经节细胞（RGCs）。自体坐骨神经移植术后4周,采用视网膜平铺技术计数再生RGCs。结果空白对照组每张视网膜中再生RGCs数量平均为（1032±147）个,治疗对照组为（1114±179）个,两者之间无明显差异fP〉0．05）。低、中和高剂量依托咪酯治疗组每张视网膜中再生RGCs数量分别为（2054±349）个、（2853±498）个和（4118±615）个,与空白对照组和治疗对照组相比均显著增高（P〈0．01）,而且不同剂量之间均差异显著（P〈0．01）。结论依托咪酯能显著促进大鼠视神经损伤后轴突再生,且具有剂量依赖性。     

Enhanced axonal transport of glycosaminoglycans in regenerating goldfish optic nerve

Glycosaminoglycans (GAGs) derived from axonally transported proteoglycans were evaluated in regenerating goldfish optic tracts. Fish were administered unilateral optic nerve crushes and stored for 21 days at 21 degrees C. Retinas were labeled by bilateral injection of 35SO4 and sulfated macromolecules axonally transported to the optic tracts were analyzed 8 h postinjection. Results show regenerating tracts contain 6.7-fold more transported 35SO4 in GAGs than their contralateral intact counterparts. Further analysis revealed that regenerating tract GAGs were comprised of 60% chondroitin (4 or 6) sulfate (CS) and 40% heparan sulfate (HS), while unoperated tract GAGs contained 26% CS and 74% HS. These results indicate that there is a large regeneration related increase in the axonal transport of proteoglycans and particular enrichment of transported molecules containing CS chains. The findings can be viewed in the context of recent implication of axonal proteoglycans in processes of fiber outgrowth, adhesion and induction of glial mitosis.     

Aluminum effect on slow axonal transport: a novel impairment of neurofilament transport

Administration of aluminum (Al) produces accumulation of neurofilaments (NF), called neurofibrillary tangles (NFT), in neuronal cell bodies and proximal axonal segments. This study was undertaken to investigate whether these changes are associated with impairment of the slow axonal transport. Local administration of AlCl3 induced the formation of NFT in 90 to 100% of the rabbit hypoglossal neurons. [35S]Methionine was then administered to the hypoglossal nerve nuclei. The hypoglossal nerves were processed 18 or 28 days later for one- and two-dimensional SDS-polyacrylamide gel electrophoresis and fluorography. Labeled NF polypeptides and a polypeptide of 57 kilodaltons (Kd) were not detectable beyond the proximal 9-mm segment of the hypoglossal nerve in Al-treated rabbits 18 days after labeling, whereas they were present up to 27 mm from the medulla in controls. Tubulin and polypeptides migrating with slow component b were not significantly affected. In rabbits sacrificed 28 days after labeling, accumulation of NF subunits within the proximal 9 mm of hypoglossal nerve was less dramatic, and labeled NF were present up to 30 mm from the medulla whereas they were detectable up to 45 mm in controls. Morphological studies demonstrated the presence of enlarged axons filled with NF in the proximal 9 mm of the hypoglossal nerve. In nerve segments immediately distal, axons were markedly reduced in size and contained no NF but an apparently normal number of microtubules and other organelles. Transport of NF and of a 57-Kd polypeptide is markedly but reversibly slowed down or blocked within the proximal 9-mm segments of the hypoglossal nerve following Al administration to the hypoglossal nucleus. It is suggested that NF transport is maintained distally, resulting in lack of NF in axonal segments immediately distal to the block. Local Al intoxication provides a novel model of impairment of NF transport.     

Methylprednisolone treatment does not influence axonal regeneration or degeneration following optic nerve injury in the adult rat.

BACKGROUND: Methylprednisolone (MP) is often used to treat optic nerve injury. However, its effects in experimental crush injury have not been extensively evaluated. METHODS: Adult Sprague-Dawley rats were subjected to a standardized optic nerve crush injury. Animals were treated either with 30 mg/kg MP intravenous bolus followed by subcutaneous injections every 6 hours for 48 hours, or with a drug vehicle alone. RESULTS: The injury resulted in a partial loss of neuronal nuclei-labeled retinal neurons and a corresponding degeneration of axons distal to the injury. EDI-labeled macrophages accumulated at the site of lesion, phagocyting FJ-labeled axonal debris. Regenerative fibers expressing growth associated protein-43 were seen proximal to the lesion, but did not traverse the glial scar. Analysis of optic nerve function using visual evoked potentials showed typical signals in intact animals, which were abolished after injury in MP-treated and untreated animals. CONCLUSIONS: We did not detect any effects of MP on retinal cell survival, macrophage activity at the site of injury, axonal degeneration/regeneration, or visual function. These experimental results provide a physiologic underpinning for the lack of efficacy demonstrated in a large trial of MP treatment of clinical optic nerve injury.     

Y-P30 confers neuroprotection after optic nerve crush in adult rats

The survival-promoting peptide, Y-P30, has been shown to be neuroprotective and stimulates neurite outgrowth in vitro. In this study, we examined whether the peptide increases survival and induces axon outgrowth of retinal ganglion cells after an incomplete optic nerve crush. A single intraocular injection of the peptide directly after optic nerve crush increased the number of retinal ganglion cells that preserved an axonal connection with the superior colliculus in the adult rat by more than 50%. However, administration of Y-P30 into the vitreous or optic nerve had no effect on the number of axons growing into the crush site after optic nerve crush. These findings suggest that the peptide is a neuroprotective agent after optic nerve damage, but does not stimulate the axon outgrowth.     

依托咪酯对成年大鼠视网膜神经节细胞的差异性保护作用

目的 探讨依托咪酯对成年大鼠视神经损伤后视网膜不同部位视网膜神经节细胞(RGCs)存活的影响.方法 取55只成年雌性SD大鼠,于双侧上丘和顶盖前区及外侧膝状荧光金逆行标记RGCs,然后随机分为6组:正常组(n=5),模型组(n=10),脂肪乳组(n= 10;视神经损伤后腹腔注射等体积脂肪乳,1次/d),以及低、中、高剂...     

Decreased axonal calibres without axonal loss in optic nerve following chronic alcohol feeding in adult rats: a morphometric study

Summary The effects of chronic ethanol exposure on number and calibres of optic nerve axons (and number of retinal ganglion cells) were investigated in a rat model. Male Sprague-Dawley rats were fed a liquid, ethanol-containing diet for 5, 10 and 17 weeks with littermates given isocaloric amounts of ethanol-free diet serving as controls. After fixation by perfusion, the optic nerves were imbedded in epoxy resin and sectioned for electron microscopy. Systematic random sampling was made from a cross-shaped area over the nerve. Axons within a counting frame were counted and morphometrically categorized with regard to mean diameter and the total number of axons estimated from number per area and the cross-sectional area of the nerve, which was measured using a digitizer table. According to non-parametric statistical analysis, ethanol exposure resulted in a significant reduction in mean cross-sectional area of the optic nerve and in mean axonal calibre but not in total axonal number in the ethanol-treated rats but there was no significant effect of duration of the exposure. The mean cross-sectional area of the nerve was reduced by 9%, 10% and 18% after 5, 10 and 17 weeks of exposure, respectively. The reduction in cross-sectional area appeared to be related to a proportional reduction in axonal and myelin area fractions. The findings indicate that chronic ethanol exposure results in decreased axonal calibres without axonal loss. This also implies that there is no reduction in the number of retinal ganglion cells.Supported by the Bank of Sweden Tercentenary Foundation (Proj. No. 86/80), the Swedish Medical Research Council (Proj. No. 07121) and Swedish Alcohol Monopoly Foundation for Alcohol Research (89/11)     

Methylprednisolone exacerbates axonal loss following optic nerve trauma in rats

Purpose: This study investigates the clinical dogma that very high doses of methylprodnisolone helpful in spinal cord injury are also helpful in optic nerve trauma. Methods: The right optic nerve of 29 male rats received a 5 second traumatic crush followed 30 minutes later by one of five intravenous treatments (methylprednisolone 30 mg/kg, 60 mg/kg, 90 mg/kg, 120 mg/kg, or saline). Treatment was continued for three additional administrations at 6 hour intervals. Untreated sham controls (n=7) were also prepared. Six weeks after injury, animals were sacrificed, perfused and optic nerves systematically counted. Results: Axon counts (means +/- s.e.m.) were as follows: Saline = 16,670+/-8,900 (n=5); Methylprednisolone: 30 mg/kg = 8,098+/-4,741 (n=5); 60 mg/kg=6,925+/-6,517 (n=4); 90 mg/kg=2,663+/-2,653 (n=4); 120 mg/kg=6,149+/-3,487 (n=6). Consequently, the data revealed that saline treated animals retained more axons than those that were administered methylprednisolone (p<0.02). Conclusions: We conclude that methylprednisolone exacerbates axonal loss following crush injury in the rat optic nerve. Based on the results of this study, clinical studies of traumatic optic neuropathy in the future should also examine the possibility that corticosteroid treatment may have an adverse effect on visual outcome following optic nerve trauma.     

Methylprednisolone exacerbates axonal loss following optic nerve trauma in rats

The excitability of human axons can be studied reliably using the technique of threshold tracking, which allows the strength of a test stimulus to be adjusted by computer to activate a defined fraction of the maximal nerve or muscle action potential. The stimulus current that just evokes the target response is considered the 'threshold' for that response. More useful than the resting threshold are other indices of axonal excitability derived from pairs of threshold measurements, such as refractoriness, supernormality, strength-duration time constant and 'threshold electrotonus' (i.e. the changes in threshold produced by long-lasting depolarizing or hyperpolarizing current pulses). Each of these measurements depends on membrane potential and on other biophysical properties of the axons. Together they can provide new information about the pathophysiology underlying abnormalities in excitability in neuropathy.     

Impaired slow axonal transport in diabetic peripheral nerve is independent of RAGE

Diabetic peripheral nerve dysfunction is a common complication occurring in 30–50% of long‐term diabetic patients. The pathogenesis of this dysfunction remains unclear but growing evidence suggests that it might be attributed, in part, to alteration in axonal transport. Our previous studies demonstrated that RAGE (Receptor for Advanced Glycation Endproducts) contributes to the pathogenesis of diabetic peripheral neuropathy and impairs nerve regeneration consequent to sciatic nerve crush, particularly in diabetes. We hypothesize that RAGE plays a role in axonal transport impairment via the interaction of its cytoplasmic domain with mammalian Diaphanous 1 (mDia1) – actin interacting molecule. Studies showed that mDia1–RAGE interaction is necessary for RAGE‐ligand‐dependent cellular migration, AKT phosphorylation, macrophage inflammatory response and smooth muscle migration. Here, we studied RAGE, mDia1 and markers of axonal transport rates in the peripheral nerves of wild‐type C57BL/6 and RAGE null control and streptozotocin‐injected diabetic mice at 1, 3 and 6 h after sciatic nerve crush. The results show that in both control and diabetic nerves, the amount of RAGE accumulated at the proximal and distal side of the crush area is similar, indicating that the recycling rate for RAGE is very high and that it is evenly transported from and towards the neuronal cell body. Furthermore, we show that slow axonal transport of proteins such as Neurofilament is affected by diabetes in a RAGE‐independent manner. Finally, our study demonstrates that mDia1 axonal transport is impaired in diabetes, suggesting that diabetes‐related changes affecting actin binding proteins occur early in the course of the disease.     

Cell proliferation and apoptosis in optic nerve and brain integration centers of adult troutOncorhynchus 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.     

Calcium channel inhibition-mediated axonal stabilization improves axonal regeneration after optic nerve crush

正Axonal projections are specialized neuronal compartments and the longest parts of neurons.Axonal degeneration is a common pathological feature in many neurodegenerative disorders,such as Parkinson’s disease,amyotrophic lateral sclerosis,glaucoma,as well as in traumatic lesions of the central nervous system(CNS),such as spinal cord injury.In many neurological disorders,the axon is     

Cognitive impairment and optic nerve axonal loss in patients with clinically isolated syndrome

Objective

To investigate cognitive impairment, to assess optical nerve axonal loss, and to determinate whether there is correlation between optical nerve axonal loss and cognition impairment in Clinically Isolated Syndrome (CIS).

Methods

Fifteen CIS patients and 15 controls were submitted to Wechsler memory scale, Rey Auditory Verbal Learning, Rey Complex Figure, Paced Auditory Serial Addition, Digit Span, verbal fluency, stroop color, D2, and Digit Symbol tests. CIS patients were evaluated by optical coherence tomography (OCT) (23 eyes).

Results

CIS patients had worse performance in Paced Auditory Serial Addition Test (PASAT) 2 seconds (P = 0.009) and fluency tests (P = 0.0038). Optical nerve axonal loss was found more frequently in eyes with previous optic neuritis (ON) (85.7%) than in those without previous ON (21.7%) (P = 0.0146). There were no significant correlations between optical nerve axonal loss and cognitive findings.

Conclusions

CIS patients had worse cognitive performance than controls. OCT can detect axonal loss resulting from optical neuritis and subclinical axonal loss in eyes without previous optical neuritis. Optical nerve axonal loss was not correlated with cognition.     

Node-like axonal undercoating in the optic nerve of heterozygous myelin-deficient rats

Focal regions of node-like axonal undercoating occur occasionally in the myelin-deficient rat spinal cord. In the optic nerve of heterozygous myelin-deficient rats, areas of amyelination are present, within which many axons show regions of node-like undercoating. These structures were most easily seen when fixation resulted in round axonal profiles. In one-year-old rats, these structures were common. The ubiquitous association of patches of axonal undercoating with regions of chronic amyelination and demyelination indicates that chronically demyelinated axons differ structurally from recently demyelinated axons. It is possible that such axons may have lost their ability to recruit myelin-forming cells.