Rat Optic Nerve Research Articles

Glia as transmitter sources and sensors in health and disease

Glial cells express a bewildering array of neurotransmitter receptors. To illustrate the complexity of expression, we have assayed non-glutamatergic neurotransmitter receptor mRNA in isolated rat optic nerve, a preparation devoid of neurons and neuronal synapses and from which relatively pure “glial” RNA can be isolated. Of the 44 receptor subunits examined which span the GABA-A, nicotinic, adreno- and glycine receptor families, over three quarters were robustly expressed in this mixed population of white matter glial cells, with several expressed at higher levels than found in control whole brain RNA. In addition to the complexity of glial receptor expression, numerous neurotransmitter release mechanisms have been identified. We have focused on glutamate release from astrocytes, which can occur via at least seven distinct pathways and which is implicated in excitotoxic injury and are neurons and glia. Recent findings suggest that non-glutamatergic receptors can also mediate acute glial injury are also discussed.

Comparative morphology of the rat optic nerve using light and electron microscopy

Optic nerve fibers are the axons of retinal ganglion cells, and as such they can provide information about the visual acuity and sensitivity capabilities of an animal. Consistent optic nerve fiber counts are not currently available for most animals. In this study, using light microscopy, we counted optic nerve fibers for 7 nerves from 4 adult Sprague‐Dawley rats. Using unbiased stereology we counted 4 adjacent sections of each nerve at 1000X, each using a different staining technique, including Bielschowsky's silver impregnation, Bodian's protagarol, Luxol Fast Blue stain, and a combination Bodian's protagarol and Luxol Fast Blue. We obtained counts of 75,000 to 90,000 fibers, with a fiber size range of 0.1μm to 5μm. We also examined preliminary electron micrographs of one Sprague‐Dawley rat optic nerve at magnifications up to 10,000X. Published counts of the pigmented rat optic nerve fibers using electron microscopy are 120,000, with a size range of 0.4μm to 5μm. This is significantly higher than our light microscopy counts. However, using EM at 10,000X we observe myelinated fibers as small as 0.02.μm. This discrepancy in fiber numbers calls into question which methodology is most suitable for counting myelinated fibers within the optic nerve.Grant Funding Source: Kenneth A. Suarez fellowship

Reduced Expression of Aquaporin-9 in Rat Optic Nerve Head and Retina following Elevated Intraocular Pressure

To investigate the effect of chronically elevated intraocular pressure (IOP) on the expression of water channel aquaporins (AQPs) 1, 4, and 9 in the optic nerve and retina in rats. Three episcleral veins were cauterized to elevate IOP in the left eyes of Sprague-Dawley rats. IOPs were monitored with a rebound tonometer. At 2 and 4 weeks after surgery, eyeballs with the attached optic nerve were enucleated for cryosectioning with immunohistochemistry, or dissected retinas and desheathed optic nerves were subjected to gene expression analyses. IOP was significantly increased after surgery up to 4 weeks (P=0.0008). In the control optic nerve, the unmyelinated portion showed only AQP9 immunoreactivity, whereas the myelinated portion expressed both AQP4 and AQP9 immunoreactivities colabeled for glial fibrillary acidic protein but not for neurofilament. In the control retina, AQP1 was expressed in the outer nuclear layer and photoreceptors, AQP4 was expressed in Müller cell endfeet, and AQP9 was expressed primarily in NeuN-positive cells in the ganglion cell layer (GCL). Elevated IOPs substantially reduced AQP9 expression in the optic nerve head (ONH) and the GCL and decreased the retinal gene expression, but not immunoreactivity, of AQP1. AQP9 was the only water channel expressed in the unmyelinated portion of the ONH and in the GCL whose expression was reduced after IOP elevation. Given that AQP9 presumably acts as a channel for metabolites to pass from astrocytes to neurons, the reduced expression of AQP9 at these specific sites may be implicated in the pathogenesis of glaucomatous optic neuropathy.

Mechanisms of acute axonal degeneration in the optic nerve in vivo

Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.

Exogenous modulation of intrinsic optic nerve neuroprotective activity

To characterize the molecular and functional status of the rat retina and optic nerve after acute elevation of intraocular pressure (IOP). Retinal ischemia was induced in rats by increasing the IOP (110 mmHg/60 minutes). Microarray analysis, quantitative RT-PCR (qRT-PCR) and immunohistochemistry were used to characterize retinal tissue. PLGA microspheres containing neurotrophic factors (BDNF, GDNF, or CNTF) or empty microspheres were injected into the vitreous of operated animals 1 day after elevation of IOP. Pupil light reflex (PLR) parameters and electroretinograms (ERG) were monitored at multiple time points during the 60-day postoperative recovery period. Molecular analysis showed a significant intrinsic up-regulation of CNTF at 10 and 25 days after induction of the acute ocular hypertension (p = 0.0067). Molecular tissue analysis of GDNF and its receptors (GDNFR1, GDNFR2), and BDNF and its receptor (trkB) showed no change in expression. Animals that received CNTF microspheres had no significant functional recovery compared to animals which received blank microspheres (p > 0.05). Animals that received GDNF or BDNF microspheres showed significant PLR recovery (p < 0.05 and p < 0.001 respectively) compared to non-treated animals. Continuous release of neurotrophic growth factors (NGFs) significantly protects optic nerve function in the experimental model of retinal ischemia observed by PLR analysis.

Ультраструктурные изменения элементов зрительного нерва крыс в динамике после действия метанола

Ультраструктурные изменения элементов зрительного нерва крыс в динамике после действия метанола

Hydrostatic Pressure–Induced Release of Stored Calcium in Cultured Rat Optic Nerve Head Astrocytes

Elevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90(RSK) and NHE1 phosphorylation after 2 hours. Here they examine calcium responses at the onset of HP to determine what precedes ERK1/2 phosphorylation. Cytoplasmic calcium concentration ([Ca(2+)](i)) was measured in cultured rat optic nerve astrocytes loaded with fura-2. The cells were placed in a closed imaging chamber and subjected to an HP increase of 15 mm Hg. Protein phosphorylation was detected by Western blot analysis. The increase of HP caused an immediate slow increase in [Ca(2+)](i). The response persisted in calcium-free solution and when nickel chloride (4 mM) was added to suppress channel-mediated calcium entry. Previous depletion of the ER calcium stores by cyclopiazonic acid abolished the HP-induced calcium level increase. The HP-induced increase persisted in cells exposed to xestospongin C, an inhibitor of IP3R-mediated calcium release. In contrast, ryanodine receptor (RyR) antagonist ruthenium red (10 microM) or dantrolene (25 microM) inhibited the HP-induced calcium increase. The HP-induced calcium increase was abolished when ryanodine-sensitive calcium stores were pre-depleted with caffeine (3 mM). HP caused ERK1/2 phosphorylation. The magnitude of the ERK1/2 phosphorylation response was reduced by ruthenium red and dantrolene. Increasing HP causes calcium release from a ryanodine-sensitive cytoplasmic store and subsequent ERK1/2 activation. Calcium store release appears to be a required early step in the initial astrocyte response to an HP increase.

Differential Patterns of Local Gene Regulation in Crush Lesions of the Rat Optic and Sciatic Nerve: Relevance to Posttraumatic Regeneration

Axon regrowth after nerve injury can occur in the peripheral but fails in the central nervous system. Cellular reactions at the lesion site affect axonal regrowth. We compared gene regulation in optic nerve (ON) and sciatic nerve (SN) crush lesions in adult rats by cDNA array analysis, quantitative RT-PCR and immunohistochemistry, focusing on the primary lesion site rather than the proximal or distal nerve stump. Four days after injury, identical gene regulation in ON and SN lesions was found for 19/1185 genes (15 up, 4 down). In contrast, tissue-specific regulations were identified for 48 genes in ON and 50 genes in SN crush lesions. Among these, in the ON many genes were downregulated (23 up, 25 down) whereas upregulation predominated in SN lesions (43 up, 7 down), especially for signaling, metabolism, and translation/transcription-related genes. In ON lesions aquaporin 4 downregulation corresponded to a transient loss of astrocytes. Tissue-type plasminogen activator was upregulated in the lesion and distal stump of SN while the urokinase-type plasminogen activator was upregulated only in the ON lesion indicating differences in local proteolysis between the systems. Typical neuronal genes were regulated at the crush site comprising neurotransmitter genes in ON and actin cytoskeleton-related genes in the SN. The differential orchestration of local gene regulation has implications for axonal regeneration in central nervous system lesions.

Degeneration of Retinal Ganglion Cells After Optic Nerve Sheath Fenestration in an Experimental Rat Model

Optic nerve sheath fenestration (ONSF) is a surgical procedure that is sometimes performed in patients with optic disc edema from increased intracranial pressure. The objective of this study was to assess the consequence of ONSF on optic nerve axons, retinal ganglion cells (RGCs), and retinal amacrine cells. The optic nerves of 22 male Wistar rats were assigned to one of three groups. In Group 1 (n = 12), the rats underwent unilateral ONSF. In Group 2 (n = 10), the rat optic nerves were unilaterally exposed but were not operated on. Group 3 (n = 22) consisted of the optic nerves of Group 1 and Group 2 rats that were neither operated on nor exposed. Thirty days later, a cresyl violet staining method was used to assess the number and sizes of RGCs and amacrine cells. Optic nerve axons were assessed by means of glial fibrillary acidic protein (GFAP) immunoreactivity. ONSF was associated with a significant reduction (P < 0.05) in the number and size of RGCs and amacrine cells. Optic nerve axons were undisturbed. Although ONSF does not apparently injure the optic nerve axons, loss and shrinkage of RGCs is a caution when considering ONSF as a treatment.

Glutamate receptor‐mediated ischemic injury of premyelinated central axons

Ischemic injury of axons is a feature of periventricular leukomalacia, a pathological correlate of cerebral palsy. Recent evidence suggests that axons are damaged before they receive the first layer of compact myelin. Here we examine the cellular mechanisms underlying ischemic-type injury of premyelinated central axons. Two-thirds of axons in the postnatal day 10 (P10) rat optic nerve are small premyelinated axons (<0.4microm in diameter), and one-third have undergone radial expansion in preparation for glial contact and the onset of myelination. Compound action potential recording and quantitative electron microscopy were used to examine the effect of modeled ischemia (oxygen-glucose deprivation) upon these two axon populations. Glutamate receptor (GluR) expression was investigated using polymerase chain reaction (PCR) and immunostaining approaches at the confocal light and ultrastructural levels. Oxygen-glucose deprivation produced action potential failure and focal breakdown of the axolemma of small premyelinated axons at sites of contact with oligodendrocyte processes, which were also disrupted. The resulting axon loss was Ca(2+)-dependent, Na(+)- and Cl(-)-independent, and required activation of N-methyl-D-aspartic acid (NMDA) and non-NMDA GluRs. NMDA receptor expression was localized to oligodendrocyte processes at sites of contact with premyelinated axons, in addition to expression within compact myelin. No periaxonal NMDA receptor expression was observed on oligodendrocyte processes ensheathing large premyelinated axons and no protective effect of GluR block was observed in these axons. NMDA receptor-mediated injury to oligodendrocyte processes navigating along small premyelinated axons precedes damage to the underlying axon, a phenomena that is lost following radial expansion and subsequent oligodendrocyte ensheathment.

Elevated hydrostatic pressure activates sodium/hydrogen exchanger-1 in rat optic nerve head astrocytes

Optic nerve head astrocytes become abnormal in eyes that have elevated intraocular pressure, and cultured astrocytes display altered protein expression after being subjected for > or = 1 days to elevated hydrostatic pressure. Here we show that 2-h elevated hydrostatic pressure (15 or 30 mmHg) causes phosphorylation of ERK1/2, ribosomal S6 protein kinase (p90(RSK)), and Na/H exchanger (NHE)1 in cultured rat optic nerve head astrocytes as judged by Western blot analysis. The MEK/ERK inhibitor U0126 abolished phosphorylation of NHE1 and p90(RSK) as well as ERK1/2. To examine NHE1 activity, cytoplasmic pH (pH(i)) was measured with BCECF and, in some experiments, cells were acidified by 5-min exposure to 20 mM ammonium chloride. Although baseline pH(i) was unaltered, the rate of pH(i) recovery from acidification was fourfold higher in pressure-treated astrocytes. In the presence of either U0126 or dimethylamiloride (DMA), an NHE inhibitor, hydrostatic pressure did not change the rate of pH(i) recovery. The findings are consistent with NHE1 activation due to phosphorylation of ERK1/2, p90(RSK), and NHE1 that occurs in response to hydrostatic pressure. These responses may precede long-term changes of protein expression known to occur in pressure-stressed astrocytes.

Elevated Hydrostatic Pressure Activates Sodium‐Hydrogen Exchanger‐1 in Rat Optic Nerve Head Astrocytes

ObjectiveOptic nerve head (ONH) astrocytes are thought to involve in the degeneration of ganglion cells and remodeling of surrounding extracellular matrix in glaucoma. Elevated intraocular pressure (IOP) is the main glaucoma risk factor. Here we report altered sodium hydrogen exchange (NHE) activity in cells exposed to elevated hydrostatic pressure .MethodsHydorstatic pressure (HP) of 15 mmHg was applied to the cells in a 20 cm glass cylinder filled with the culture medium equilibrated with 5% CO2/95% air for 2h. Proteins were analyzed by Western blot and cytoplasmic pH (pHi) was measured by BCECF ratiometry.ResultsElevated HP causes phosphorylation of ERK1/2, ribosomal S6 kinase (p90RSK), and Na‐H exchanger‐1 (NHE1). The MEK/ERK inhibitor UO126 abolished phosphorylation of NHE1 and p90RSK as well as ERK1/2. Although baseline pHi was unaltered in pressure treated cells, the rate of pHi recovery from ammonium chloride acidification was four fold higher. Dimethylamiloride (DMA), an NHE inhibitor, abolished and U0126 partially inhibited the increased rate of pHi recovery in pressure treated astrocytes.ConclusionsThe findings suggest elevated HP caused NHE‐1 activation through phosphorylation following activation of ERK1/2 and p90RSK. These events may lead to the long term changes of protein expression known to occur in pressure‐stressed astrocytes.FundingNIH Grant EY014069

Neuronal and glial cell expression of angiotensin II type 1 (AT 1) and type 2 (AT 2) receptors in the rat retina

The bio-active peptide, angiotensin II (Ang II), has been suggested to exert a neuromodulatory effect on inner retinal neurons. In this study, we examined the distribution of angiotensin receptors (ATRs) in the developing and mature rat retina and optic nerve using immunofluorescence immunocytochemistry. Double-labeling experiments were performed with established markers to identify different retinal cell populations. In adult retinae, ATRs were observed on neurons involved in “ON” pathways of neurotransmission. Angiotensin II type 1 receptors (AT 1Rs) were expressed by a sub-population of “ON” cone bipolar cells that also labeled for Gα 0 and islet-1. Extra-neuronal expression of AT 1Rs was evident on retinal astrocytes, Müller cells and blood vessels. Immunoreactivity for the angiotensin II type 2 receptor (AT 2R) was observed on conventional and displaced GABAergic amacrine cells. Co-localization studies showed that AT 2R-expressing amacrine cells constituted at least two separate sub-populations. Cell counts revealed that all wide-field amacrine cells expressing protein kinase C-alpha were also AT 2R-positive; a further subset of amacrine cells expressing AT 2Rs and stratifying in sublamina “b” of the inner plexiform layer (IPL) was identified. Developmental expression of AT 1Rs was dynamic, involving multiple inner neuronal classes. At postnatal day 8 (P8), AT 1R immunoreactivity was observed on putative ganglion cells. The characteristic bipolar cell labeling observed in adults was not evident until P13. In contrast, AT 2Rs were detected as early as P2 and localized specifically to amacrine cells from this age onward. These data provide further evidence for the potential role of angiotensin II in the modulation of retinal neurons and glia. The differential pattern of expression of these receptors across these cell types is similar to that observed in the brain and suggests that a similar functional role for Ang II may also exist within the retina.

The Role of αA- and αB-Crystallins in the Survival of Retinal Ganglion Cells after Optic Nerve Axotomy

Stress-induced crystallin expression is commonly viewed as activation of the cell survival mechanism. The authors analyzed the expression of alphaA- and alphaB-crystallins in a rat optic nerve transection (ONT) model characterized by specific retinal ganglion cell (RGC) degeneration and determined their role in RGC survival. ONT was performed on adult Wistar rats. Quantitative and spatial expression were examined with Western blot analysis and immunohistochemistry, respectively. Electroporation was used to deliver alphaA and alphaB expression constructs to RGCs. Cell-protective effects of alphaA and alphaB overexpression after ONT were determined by RGC density analysis. Expression of alphaA and alphaB in the retina was observed predominantly in the ganglion cell layer, where most crystallin-positive cells were colocalized with RGCs. Levels of alphaA and alphaB proteins after ONT were decreased 1.6-fold. The effect of alphaA and alphaB overexpression on RGC survival was evaluated 7 and 14 days after axotomy. At day 7 after ONT, 1426 +/- 70 and 1418 +/- 81 RGCs/mm(2) were present in retinas electroporated with alphaA and alphaB expression constructs, respectively, compared with 1010 +/- 121 RGCs/mm(2) in sham-transfected or 1016 +/- 88 RGCs/mm(2) in nontransfected retinas. Numbers of surviving RGCs at 14 days were 389 +/- 57 and 353.57 +/- 60 cells/mm(2) after alphaA and alphaB transfection, respectively, compared with 198 +/- 29 cells/mm(2) after transfection with the vector alone or 206 +/- 60 cells/mm(2) in nontransfected retinas. Increases of approximately 95% and 75% in RGC survival mediated by alphaA and alphaB overexpression, respectively, were observed 14 days after ONT. At day 7, the RGC protective effect of alphaA and alphaB overexpression was approximately 40%.

Effects of the Noradrenergic System in Rat White Matter Exposed to Oxygen–Glucose DeprivationIn Vitro

Norepinephrine (NE) is released in excess into the extracellular space during oxygen-glucose deprivation (OGD) in brain, increasing neuronal metabolism and aggravating glutamate excitoxicity. We used isolated rat optic nerve and spinal cord dorsal columns to determine whether the noradrenergic system influences axonal damage in white matter. Tissue was studied electrophysiologically by recording the compound action potential (CAP) before and after exposure to 60 min of OGD at 36 degrees C. Depleting catecholamine stores with reserpine was protective and improved CAP recovery after 1 h of reperfusion from 17% (control) to 35%. Adding NE during OGD decreased CAP recovery to 8%, and adding NE to reserpine during OGD eliminated the protective effect of the latter. Selective inhibitors of Na(+)-dependent norepinephrine transport desipramine and nisoxetine improved recovery to 58% and 44%, respectively. alpha2 adrenergic receptor agonists UK14,304 and medetomidine improved CAP recovery to 41% and 46% after 1 h of OGD. Curiously, alpha2 antagonists alone were also highly protective (e.g., atipamezole: 86% CAP recovery), at concentrations that did not affect baseline excitability. The protective effect of alpha2 receptor modulation was corroborated by imaging fluorescent Ca(2+) and Na(+) indicators within axons during OGD. Both agonists and antagonists significantly reduced axonal Ca(2+) and Na(+) accumulation in injured axons. These data suggest that the noradrenergic system plays an active role in the pathophysiology of axonal ischemia and that alpha2 receptor modulation may be useful against white matter injury.

Intraperitoneal administration of Ginkgo biloba extract (GBE 50) promotes the survival of retinal ganglion cells after crush of intraorbital optic nerve in rats

目的 探讨银杏叶提取物GBE 50(Ginkgo biloba extract 50)对大鼠视神经钳夹伤后视网膜神经节细胞(RGCs)的保护作用.方法 65只SD大鼠随机等分为正常对照组、假手术组、模型组、模型+生理盐水(NS)组、模型+GBE 50组,每只鼠的右眼用于实验.正常对照组不作任何处理;假手术组仅分离暴露视神经;其余3个组分离暴露视神经并进行钳夹;模型+NS组和模型+GBE 50组分别于实验前1周每日腹腔注射相应体积NS和0.35%GBE 50(100 mg/kg),术后继续给药4周;术后4 d,各组随机处死3只大鼠作凋亡RGCs的TUNEL荧光标记;术后4周后处死所有大鼠作光镜检查并计数视网膜垂直经线RGCs.结果 正常对照组和假手术组未见TUNEL阳性RGCs;其余3组均见TUNEL阳性RGCs,但模型+GBE 50组较前两组少.术后4周RGCs数目:模型组(131±10个)、模型+NS组(137±13个)、模型+GBE 50组(198±15个)均少于正常对照组和假手术组(P 0.05);但模型+GBE 50组显著多于模型组和模型+NS组(P<0.05).结论 腹腔注射银杏叶提取物GBE 50能部分抑制大鼠视神经钳夹后RGCs凋亡,具有一定的RGCs保护作用。

Conduction block and glial injury induced in developing central white matter by glycine, GABA, noradrenalin, or nicotine, studied in isolated neonatal rat optic nerve

The damaging effects of excessive glutamate receptor activation have been highlighted recently during injury in developing central white matter. We have examined the effects of acute exposure to four other neurotransmitters that have known actions on white matter. Eighty minutes of Glycine or GABA-A receptor activation produced a significant fall in the compound action potential recorded from isolated post-natal day 10 rat optic nerve. This effect was largely reversed upon washout. Nicotinic acetylcholine receptor (nAChR) or adrenoreceptor activation with noradrenalin resulted in an approximately 35% block of the action potential that did not reverse during a 30-min washout period. While the effect of nAChR activation was blocked by a nAChR antagonist, the effect of noradrenalin was not ablated by alpha- or beta-adrenoreceptor blockers applied alone or in combination. In the absence of noradrenalin, co-perfusion with alpha- and beta-adrenoreceptor blockers resulted in nonreversible nerve failure indicating that tonic adrenoreceptor activation is required for nerve viability, while overactivation of these receptors is also damaging. Nerves exposed to nAChR + adrenoreceptor activation showed no axon pathology but had extensive glial injury revealed by ultrastructural analysis. Oligodendroglia exhibited regions of membrane vacuolization while profound changes were evident in astrocytes and included the presence of swollen and expanded mitochondria, vacuolization, cell processes disintegration, and membrane breakdown. Blinded assessment revealed higher levels of astrocyte injury than oligodendroglial injury. The findings show that overactivation of neurotransmitter receptors other than those for glutamate can produce extensive injury to developing white matter, a phenomenon that may be clinically significant.

Effects of glutamate receptor activation on NG2-glia in the rat optic nerve.

NG2-glia are a substantial population of cells in the central nervous system (CNS) that can be identified by their specific expression of the NG2 chondroitin sulphate (CSPG). NG2-glia can generate oligodendrocytes, but it is unlikely this is their only function; indeed, they may be multipotent neural stem cells. Moreover, NG2-glia are a highly reactive cell type and a major function is to help form the axon growth inhibitory glial scar in response to CNS injury. The factors that regulate these diverse behaviours of NG2-glia are not fully resolved, but NG2-glia express receptors to the neurotransmitter glutamate, which has known potent effects on other glia. Here, we have examined the actions of glutamate receptor activation on NG2-glia in the rat optic nerve, a typical CNS white matter tract that does not contain neuronal cell bodies. Glutamate induces an increase in [Ca(2+)](i) in immuno-identified NG2-glia in situ and in vitro. In addition, we examined the effects of glutamate receptor activation in vivo by focal injection of the glutamate receptor agonist kainate into the optic nerve; saline was injected in controls. Changes in glial and axonal function were determined at 7 days post injection (dpi), by immunohistochemistry and electrophysiological measurement of the compound action potential (CAP). Injection of kainate resulted in a highly localized 'injury response' in NG2-glia, marked by dense labelling for NG2 at the lesion site, as compared to astrocytes, which displayed a more extensive reactive astrogliosis. Furthermore, injection of kainate resulted in an axonal conduction block. These glial and axonal changes were not observed following injection of saline vehicle. In addition, we provide evidence that endogenous glutamate induces calcium-dependent phosphorylation of extracellular signal-regulated kinases (ERK1/2), which may provide a potential mechanism by which glutamate-mediated changes in raised intracellular calcium could regulate the observed gliosis. The results provide evidence that activation of AMPA-kainate type ionotropic glutamate receptors evoke raised calcium in NG2-glia and induces an injury response in NG2-glia.

Combination of Mn2+‐enhanced and diffusion tensor MR imaging gives complementary information about injury and regeneration in the adult rat optic nerve

To evaluate manganese (Mn(2+))-enhanced MRI (MEMRI) and diffusion tensor imaging (DTI) as tools for detection of axonal injury and regeneration after intravitreal peripheral nerve graft (PNG) implantation in the rat optic nerve (ON). In adult Fischer rats, retinal ganglion cell (RGC) survival was evaluated in Flurogold (FG) back-filled retinal whole mounts after ON crush (ONC), intravitreal PNG, and intravitreal MnCl(2) injection (150 nmol) at 0 and 20 days post lesion (dpl). MEMRI and echo-planar DTI (DTI-EPI) was obtained of noninjured ON one day after intravitreal MnCl(2) injection, and at 1 and 21 dpl after ONC, intravitreal PNG, and intravitreal MnCl(2) injections given at 0 and 20 dpl. GAP-43 immunohistochemistry was performed after the last MRI. ONC reduced RGC density in retina by 94% at 21 dpl compared to noninjured ON without MnCl(2) injections. Both intravitreal PNG and intravitreal MnCl(2) injections improved RGC survival in retina, which was reduced by 90% (ONC+MnCl(2)), 82% (ONC+PNG), and 74% (ONC+PNG+MnCl(2)) compared to noninjured ON. DTI-derived parameters (fractional anisotropy [FA], mean diffusivity, axial diffusivity lambda( parallel), and radial diffusivity lambda( perpendicular)) were unaffected by the presence of Mn(2+) in the ON. At 1 dpl, CNR(MEMRI) and lambda( parallel) were reduced at the injury site, while at 21 dpl they were increased at the injury site compared to values measured at 1 dpl. GAP-43 immunoreactive axons were present in the ON distal to the ONC injury site. MEMRI and DTI enabled detection of functional and structural degradation after rat ON injury, and there was correlation between the MRI-derived and immunohistochemical measures of axon regeneration.

Reflections

CNS white matter from higher vertebrates and cultured differentiated oligodendrocytes are nonpermissive substrates for neurite growth and fibroblast spreading. Membrane proteins of 35 kd and 250 kd with highly nonpermissive substrate properties could be extracted from CNS myelin fractions. Monoclonal antibodies were raised against these proteins: IN-1 and IN-2 bound both to the 35 kd and 250 kd inhibitors and to the surface to differentiated cultured oligodendrocytes. Adsorption of nonpermissive CNS myelin or nonpermissive oligodendrocytes with either antibody markedly improved their substrate properties. Optic nerve explants injected with IN-1 or IN-2 allowed axon ingrowth of cocultured sensory and sympathetic neurons. We conclude that the nonpermissive substrate properties of CNS white matter are due to these membrane proteins on the surface of differentiated oligodendrocytes and to their in vivo product, myelin.