Intraorbital Optic Nerve Crush Research Articles

A hypothermia mimetic molecule (zr17-2) reduces ganglion cell death and electroretinogram distortion in a rat model of intraorbital optic nerve crush (IONC).

Introduction: Ocular and periocular traumatisms may result in loss of vision. Our previous work showed that therapeutic hypothermia prevents retinal damage caused by traumatic neuropathy. We also generated and characterized small molecules that elicit the beneficial effects of hypothermia at normal body temperature. Here we investigate whether one of these mimetic molecules, zr17-2, is able to preserve the function of eyes exposed to trauma. Methods: Intraorbital optic nerve crush (IONC) or sham manipulation was applied to Sprague-Dawley rats. One hour after surgery, 5.0µl of 330nmol/L zr17-2 or PBS, as vehicle, were injected in the vitreum of treated animals. Electroretinograms were performed 21days after surgery and a- and b-wave amplitude, as well as oscillatory potentials (OP), were calculated. Some animals were sacrificed 6days after surgery for TUNEL analysis. All animal experiments were approved by the local ethics board. Results: Our previous studies showed that zr17-2 does not cross the blood-ocular barrier, thus preventing systemic treatment. Here we show that intravitreal injection of zr17-2 results in a very significant prevention of retinal damage, providing preclinical support for its pharmacological use in ocular conditions. As previously reported, IONC resulted in a drastic reduction in the amplitude of the b-wave (p < 0.0001) and OPs (p < 0.05), a large decrease in the number of RGCs (p < 0.0001), and a large increase in the number of apoptotic cells in the GCL and the INL (p < 0.0001). Interestingly, injection of zr17-2 largely prevented all these parameters, in a very similar pattern to that elicited by therapeutic hypothermia. The small molecule was also able to reduce oxidative stress-induced retinal cell death in vitro. Discussion: In summary, we have shown that intravitreal injection of the hypothermia mimetic, zr17-2, significantly reduces the morphological and electrophysiological consequences of ocular traumatism and may represent a new treatment option for this cause of visual loss.

Early phosphoproteomic changes in the retina following optic nerve crush

Retinal ganglion cell (RGC) death causes irreversible blindness in adult mammals. Death of RGC occurs in diseases including glaucoma or injuries to the optic nerve (ON). To investigate mechanisms involved in RGC degeneration, we evaluated the phosphoproteomic changes in the retina induced by ON injury. Intraorbital optic nerve crush (ONC) was performed in adult C57BL/6J mice. Retinas were collected at 0, 6, and 12 h following ONC. Retinal proteins labeled with CyDye-C2 were subject to 2D-PAGE, followed by phosphoprotein staining and in-gel/cross-gel image analysis. Proteins with significant changes in phosphorylation (ratios ≥1.2) in retinas of the injured eyes compared to the control eyes were spot-picked, tryptic digested, and peptide fragments were analyzed by MALDI-TOF (MS) and TOF/TOF (tandem MS/MS). Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 29 significantly phosphorylated proteins were identified. PANTHER analysis showed that these proteins are associated with a variety of protein classes, cellular components, biological processes and signaling pathways. One of the identified proteins, phosphoprotein enriched in astrocytes 15 (PEA15), was further validated by western blotting and immunofluorescence staining. Functions of PEA15 were determined in cultured astrocytes. PEA15 knockdown reduced astrocyte phagocytic activity but promoted cell migration. Long term PEA15 knockdown also decreased astrocyte ATP level. This study provides new insights into mechanisms of RGC degeneration after ON injury, as well as central nervous system (CNS) neurodegeneration, since the retina is an extension of the CNS. These new insights will lead to novel therapeutic targets for retinal and CNS neurodegeneration.

Systemic and Intravitreal Antagonism of the TNFR1 Signaling Pathway Delays Axotomy-Induced Retinal Ganglion Cell Loss.

Here, we have blocked the signaling pathway of tumor necrosis factor α (TNFα) in a mouse model of traumatic neuropathy using a small cell permeable molecule (R7050) that inhibits TNFα/TNF receptor 1 (TNFR1) complex internalization. Adult pigmented mice were subjected to intraorbital optic nerve crush (ONC). Animals received daily intraperitoneal injections of R7050, and/or a single intravitreal administration the day of the surgery. Some animals received a combinatorial treatment with R7050 (systemic or local) and a single intravitreal injection of brain derived neurotrophic factor (BDNF). As controls, untreated animals were used. Retinas were analyzed for RGC survival 5 and 14 days after the lesion i.e., during the quick and slow phase of axotomy-induced RGC death. qPCR analyses were done to verify that Tnfr1 and TNFα were up-regulated after ONC. At 5 days post-lesion, R7050 intravitreal or systemic treatment neuroprotected RGCs as much as BDNF alone. At 14 days, RGC rescue by systemic or intravitreal administration of R7050 was similar. At this time point, intravitreal treatment with BDNF was significantly better than intravitreal R7050. Combinatory treatment was not better than BDNF alone, although at both time points, the mean number of surviving RGCs was higher. In conclusion, antagonism of the extrinsic pathway of apoptosis rescues axotomized RGCs as it does the activation of survival pathways by BDNF. However, manipulation of both pathways at the same time, does not improve RGC survival.

Tryptophan and Kynurenine Pathway Metabolites in Animal Models of Retinal and Optic Nerve Damage: Different Dynamics of Changes.

Kynurenines, products of tryptophan (TRP) metabolism, display neurotoxic (e.g., 3-hydroxykynurenine; 3-HK), or neuroprotective (e.g., kynurenic acid; KYNA) properties. Imbalance between the enzymes constituting the kynurenine pathway (KP) plays a role in several disease, including neurodegeneration. In this study, we track changes in concentrations of tryptophan and its selected metabolites after damage to retinal ganglion cells and link this data with expression of KP enzymes. Brown-Norway rats were subjected to intravitreal N-methyl-D-aspartate (NMDA) injection or partial optic nerve crush (PONC). Retinas were collected 2 and 7 days after the completion of PONC or NMDA injection. Concentrations of TRP, kynurenine (KYN), and KYNA were determined by high performance liquid chromatography (HPLC). Data on gene expression in the rat retina were extracted from GEO, public microarray experiments database. Two days after NMDA injection concentration of TRP decreased, while KYN and KYNA increased. At day 7 compared to day 2 decrease of KYN, KYNA and further reduction of TRP concentration were observed, but on day 7 KYN concentration was still elevated when compared to controls. At day 2 and 7 after NMDA injection no statistically significant alterations of 3-HK were observed. TRP and 3-HK concentration was higher in PONC group than in controls. However, both KYN and KYNA were lower. At day seven concentration of TRP, 3-HK, and KYN was higher, whereas concentration of KYNA declined. In vivo experiments showed that retinal damage or optic nerve lesion affect TRP metabolism via KP. However, the pattern of changes in metabolite concentrations was different depending on the model. In particular, in PONC KYNA and KYN levels were decreased and 3-HK elevated. These observations correspond with data on expression of genes encoding KP enzymes assessed after optic nerve crush or transection. After intraorbital optic nerve crush downregulation of KyatI and KyatIII between 24 h and 3 days after procedure was observed. Kmo expression was transiently upregulated (12 h after the procedures). After intraorbital optic nerve transsection (IONT) Kmo expression was upregulated after 48 h and 7 days, KyatI and KyatIII were downregulated after 12, 48 h, 7 days and upregulated after 15 days. Collected data point to the conclusion that development of therapeutic strategies targeting the KP could be beneficial in diseases involving retinal neurodegeneration.

Hypothermia Prevents Retinal Damage Generated by Optic Nerve Trauma in the Rat

Ocular and periocular traumatisms may result in loss of vision. Hypothermia provides a beneficial intervention for brain and heart conditions and, here, we study whether hypothermia can prevent retinal damage caused by traumatic neuropathy. Intraorbital optic nerve crush (IONC) or sham manipulation was applied to male rats. Some animals were subjected to hypothermia (8 °C) for 3 h following surgery. Thirty days later, animals were subjected to electroretinography and behavioral tests. IONC treatment resulted in amplitude reduction of the b-wave and oscillatory potentials of the electroretinogram, whereas the hypothermic treatment significantly (p < 0.05) reversed this process. Using a descending method of limits in a two-choice visual task apparatus, we demonstrated that hypothermia significantly (p < 0.001) preserved visual acuity. Furthermore, IONC-treated rats had a lower (p < 0.0001) number of retinal ganglion cells and a higher (p < 0.0001) number of TUNEL-positive cells than sham-operated controls. These numbers were significantly (p < 0.0001) corrected by hypothermic treatment. There was a significant (p < 0.001) increase of RNA-binding motif protein 3 (RBM3) and of BCL2 (p < 0.01) mRNA expression in the eyes exposed to hypothermia. In conclusion, hypothermia constitutes an efficacious treatment for traumatic vision-impairing conditions, and the cold-shock protein pathway may be involved in mediating the beneficial effects shown in the retina.

Low intensity repetitive transcranial magnetic stimulation does not induce cell survival or regeneration in a mouse optic nerve crush model.

Low intensity repetitive Transcranial Magnetic Stimulation (LI-rTMS), a non-invasive form of brain stimulation, has been shown to induce structural and functional brain plasticity, including short distance axonal sprouting. However, the potential for LI-rTMS to promote axonal regeneration following neurotrauma has not been investigated. This study examined the effect of LI-rTMS on retinal ganglion cell (RGC) survival, axon regeneration and levels of BDNF in an optic nerve crush neurotrauma model. Adult C57Bl/6J mice received a unilateral intraorbital optic nerve crush. Mice received 10 minutes of sham (handling control without stimulation) (n=6) or LI-rTMS (n = 8) daily stimulation for 14 days to the operated eye. Immunohistochemistry was used to assess RGC survival (β-3 Tubulin) and axon regeneration across the injury (GAP43). Additionally, BDNF expression was quantified in a separate cohort by ELISA in the retina and optic nerve of injured (optic nerve crush) (sham n = 5, LI-rTMS n = 5) and non-injured mice (sham n = 5, LI-rTMS n = 5) that received daily stimulation as above for 7 days. Following 14 days of LI-rTMS there was no significant difference in mean RGC survival between sham and treated animals (p>0.05). Also, neither sham nor LI-rTMS animals showed GAP43 positive labelling in the optic nerve, indicating that regeneration did not occur. At 1 week, there was no significant difference in BDNF levels in the retina or optic nerves between sham and LI-rTMS in injured or non-injured mice (p>0.05). Although LI-rTMS has been shown to induce structural and molecular plasticity in the visual system and cerebellum, our results suggest LI-rTMS does not induce neuroprotection or regeneration following a complete optic nerve crush. These results help define the therapeutic capacity and limitations of LI-rTMS in the treatment of neurotrauma.

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve.

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre(+/-)xRtn4/Nogo-A(flox/flox)) and neuron-specific (Thy1-Cre(tg+)xRtn4(flox/flox)) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4(flox/flox) animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.

Effect of intrathecal FK506 administration on intraorbital optic nerve crush: an ultrastructural study

Objective: Trauma to the optic nerve caused by fractures of the midface and (or) skull base has been simulated by an optic nerve crush injury model. Because the intraorbital segment of the optic nerve is surrounded by subarachnoidal cerebrospinal fluid and dura mater, we aimed to study the influence of intrathecal tacrolimus (FK506) administration after optic nerve crush injury and to determine its role in optic nerve protection or sparing after injury. Study Design: Experimental study. Participants: All optic nerves of the animals were included in the study. Methods: A total of 48 female Wistar rats were randomly divided into 4 groups (control, sham operated, FK506 treated, and vehicle treated). In vehicle- and FK506-treated groups, intrathecal catheter implantation and crush injury to the intraorbital part of the optic nerve were performed and then the animals were treated intrathecally. The optic nerve samples were harvested on the 30th postoperative day. Optic nerve appearances were analyzed qualitatively. Results: Light and electron microscopic evaluations revealed that numerous damaged myelin residues were present in the vehicle-treated group, whereas fibres of the optic nerve showed a well-shaped appearance in the FK506-treated group. Conclusion: We propose that such an intrathecal administration route and small-dose regimen should be used to obtain lesser immunosuppression and neurotoxicity and higher protection or sparing after injury.

Effects of different neurotrophic factors on the survival of retinal ganglion cells after a complete intraorbital nerve crush injury: A quantitative in vivo study

We examined in adult Sprague Dawley rats the loss of retinal ganglion cells (RGCs) induced by complete intraorbital optic nerve crush (IONC) as well as the effects of several neurotrophic factors to prevent IONC-induced RGC loss. Completeness of the IONC lesion was assessed by investigating the orthograde and retrograde transport of neuronal tracers applied to the origin and termination of the retinotectal pathway. RGC survival after IONC alone or combined with intraocular injection of the neurotrophic factors NT-4, BDNF or CNTF was quantified at survival intervals ranging from 5 to 12 days post-lesion (dpl) by identifying RGCs that had been pre-labelled with fluorogold (FG). RGC loss first appeared at 7 dpl and by 12 dpl only 32% of the RGC population remained in the retina. Intraocular administration of NT-4, BDNF or CNTF resulted in almost a complete protection against IONC-induced RGC loss by 7 dpl, and the protection remained significant by 12 dpl only for NT-4 and BDNF. We have analyzed these results taking into account our previous studies on the loss of RGCs induced by intraorbital optic nerve transection (IONT) and concluded that RGC loss induced by IONC is slower and less severe than that following IONT. Moreover, as for IONT-induced RGC loss, IONC-induced RGC loss may also be prevented with administration of NT-4, BDNF or CNTF, though for NT-4 and CNTF their neuroprotective effects differ depending on the injury type. Overall this data underscore the importance of the type of ON injury on the pattern of RGC degeneration as well as in their response to neuroprotective treatments.

Immediate Upregulation of Proteins Belonging to Different Branches of the Apoptotic Cascade in the Retina after Optic Nerve Transection and Optic Nerve Crush

To further investigate the molecular signals underlying optic nerve (ON) injury, the authors analyzed in adult control, ON-transected, and ON-crushed retinas the expression pattern and time-course regulation of the following proteins, all of which are linked to apoptosis through different pathways: Stat 1, caspase 11 (inflammation and death), cathepsins C and B (lysosomal death pathway), calpain 1 (endoplasmic reticulum stress), calreticulin (apoptosis marker), Jun (early response), and aryl hydrocarbon receptor (cell cycle arrest). Adult female rats were subjected to intraorbital optic nerve transection (IONT) or intraorbital optic nerve crush (IONC). Protein from naive and ON-injured adult rat retinas was extracted at different times postlesion, and Western blotting experiments were performed. For immunohistofluorescence analyses, retinal ganglion cells (RGCs) were retrogradely identified with fluorogold applied to the superior colliculi 1 week before injury. Western blotting analyses revealed upregulation of all the analyzed proteins as early as 12 hours postlesion (hpl), peaking at 48 hpl, in agreement with our previous RNA study findings. Furthermore, immunohistofluorescence to radial sections showed that all but Stat 1 were expressed by the primarily injured neurons, the RGCs, as seen by colocalization with fluorogold. All analyzed proteins were upregulated in the retina after IONT or IONC as early as 12 hpl, indicating that ON injury regulates several branches of the apoptotic cascade and suggesting that commitment to death might be an earlier event than previously anticipated.

FK506 blocks activation of the intrinsic caspase cascade after optic nerve crush

Retinal ganglion cells die by apoptosis after optic nerve crush. FK506 has been shown to be neuroprotective in this model but the mechanism(s) by which it exerts these actions remains unknown. We and others have shown that caspase 9 is cleaved in the retina in other injury models and we hypothesized that the neuroprotection observed with FK506 was mediated by interference with caspase 9 activation. The present study examined the cellular localization of caspase 9 cleavage after intraorbital optic nerve crush in rats, the time course of caspase 9 cleavage after optic nerve crush and the ability of orally administered FK506 to block caspase 9 cleavage after optic nerve crush. We show by immunohistochemistry that cleaved caspase 9 is present in retinal ganglion cells (identified by prior backlabelling) after optic nerve crush. Immunoblot analysis showed that caspase 9 cleavage is significantly elevated 5 and 8 days after optic nerve crush. We show that orally administered FK506 reaches the retina and is pharmacologically active in retinal tissue. Furthermore, the oral administration of FK506 5 mg kg −1 day −1 blocks the cleavage of caspase 9 at both time points. These data suggest that caspase 9 activation may play an important role in retinal ganglion cell death following optic nerve crush and that the neuroprotection seen with FK506 may be mediated by interfering with the activation of caspase 9.

Synergistic effect of Nogo-neutralizing antibody IN-1 and ciliary neurotrophic factor on axonal regeneration in adult rodent visual systems.

The presence of Nogo axon regeneration inhibitory molecules in the central nervous system (CNS) and the counteracting effect of IN-1 antibodies have been widely reported. In this study, we examined the effect of IN-1-producing hybridoma cells on axon regeneration in adult rodent retinal ganglion cells (RGCs) after various types of optic nerve (ON) injury, evaluating therein whether ciliary neurotrophic factor (CNTF) potentiated the effect of IN-1. We found that application of IN-1 alone failed to enhance regeneration of intracranially or intraorbitally transected RGC axons in a peripheral nerve (PN) graft. IN-1 hybridoma cells also failed to significantly promote intraorbitally crushed ON axons to reenter the distal part of the ON. However, a combined application of IN-1 and CNTF had a synergistic effect in both intracranial PN and intraorbital ON crush paradigms. This study suggests that the action of IN-1 antibodies in promoting axon regeneration in the CNS could be more effective when coupled with other appropriate factors.

Axonal regrowth in the amyelinated optic nerve of the myelin‐deficient rat: Ultrastructural observations and effects of ganglioside administration

It has been postulated that myelin degradation products may inhibit regrowth of mammalian central axons and that central nervous system (CNS) myelin and oligodendrocytes may constitute a "nonpermissive substrate" for axonal growth. To address these issues, we utilized an X-linked rat mutant, myelin-deficient or md. In the optic nerve of this mutant, 40 days and more postnatally, normal myelin is absent and oligodendrocytes are few (Dentinger et al. Brain Res. 344:255-266, 1985). Twenty-eight days before sacrifice, we operated on four groups of 50-day-old md rats and age-matched normal littermates according to the following protocols: 1) unilateral intraorbital optic nerve crush; 2) beginning within 1 hour of nerve crush, daily intraperitoneal injection of GM1 ganglioside (20 mg/kg) dissolved in phosphate-buffered saline (PBS); 3) daily intraperitoneal injection of PBS alone, also begun within 1 hour of nerve crush; 4) severance of the optic nerve immediately behind the papilla 16 or 21 days after the primary crush lesions. Additionally, normal and md rats were killed 4 and 14 days after unilateral optic nerve injury. Nerves of unoperated md rats and their normal littermates were also processed. In the operated animals that did not receive GM1, ultrastructural analysis 4, 14, and 28 days after lesioning revealed that md optic nerves contained significantly greater numbers of regenerating axons, including growth cones and varicosities, than nerves of normal rats. Notably, 28 days postoperatively, (group 1), regenerating axons were still abundant in md nerve, whereas, in nerves of normally myelinated littermates, axonal numbers were diminished markedly. Regenerating optic axons of both md and normally myelinated rats were oriented by linear astrocytic arrays and often were enclosed by astrocytic cytoplasm. In normal littermates, GM1 administration (group 2) induced a significant increase in the number of axons within the operative lesion. Paradoxically, GM1 inhibited the ordinarily robust regeneration of md axons. PBS-injected md and normal rats (group 3) showed no significant differences from noninjected, operated animals. Severance of the nerve at the papilla (group 4) 7-12 days before sacrifice confirmed the origination of axonal regrowth by retinal ganglion cells. The data provide in vivo support for a role of myelin breakdown products or the secretory products of oligodendroglia in the inhibition of regenerative axonal sprouting within mammalian CNS.

Studies of the early stages of optic axon regeneration in the goldfish

We have studied the early stages (4-14 days) of axonal regeneration following intraorbital optic nerve crush in the goldfish. We used 3H-proline autoradiography to anterogradely label and visualize the growing axons and wheat germ agglutinin-conjugated horseradish peroxidase (WGA:HRP) for retrograde labeling to determine the cells of origin of the earliest projections. The first retinal ganglion cells (RGCs) that could be retrogradely filled from the optic tract, following optic nerve crush, were in the central retina and were seen at 8 days postoperative. More peripheral cells were only labeled with longer postcrush survival periods. Thus, the first axons to regenerate past the lesion were from central RGCs. The axons of these cells extended into the cranial nerve stump between 4 and 5 days postcrush and entered the nerve as a fascicle, which travelled just beneath its surface. Studies of nerve cross sections from animals at 5-8 days postoperative demonstrated that initial outgrowth was not confined to any particular locale within the nerve although the early fibers appeared to avoid its temporal aspect. When the regenerating axons reached the optic tract they remained in fascicles but left the surface to run along the medial, deep portion of the tract, immediately adjacent to the diencephalon and pretectum. The positions occupied by the earliest-regenerating axons in the optic nerve were variable and not always appropriate for their central retinal origin. However, the abrupt change in growth trajectory as the fibers entered the optic tract brought them into the areas of the visual paths that are occupied by central axons in intact animals. We suggest that this change in position is related to both changes in the structural organization of the intracranial visual paths and to possible axon guidance signals in the region of the nerve-tract juncture.

Growth of retinal ganglion cell axons following optic nerve crush in adult hamsters

Regrowth of retinal ganglion cell axons was examined 2 to 60 days after intraorbital optic nerve crush lesions in adult hamsters. Anterograde axonal transport of intraocularly injected wheat germ agglutinin-horseradish peroxidase conjugate was used to label the axons after specific postinjury time periods. Labeled axons were present in the region of the optic nerve lying between the eye and the crush site at all times, but their numbers appeared to decrease with increasing survival time. Labeled axons were first detected in the segment of optic nerve lying distal to the crush site 1 week after injury and had extended as far as 2.3 mm beyond the crush site by 60 days postinjury, growing at a rate similar to that at which the collateral branches of developing ganglion cell axons extend into their targets. Although most axons failed to regrow after these lesions, the slow reextention exhibited by members of a small population of axons indicates that the degenerating adult mammalian optic nerve provides an adequate environment for a particular mode of regrowth by injured axons of the central nervous system.

Qualitative and quantitative ultrastructural observations on retinal ganglion cell layer of rat after intraorbital optic nerve crush.

Rat retinal ganglion cell layer (GCL) was examined ultrastructurally 1-180 days after intraorbital crushing of one optic nerve. It was confirmed quantitatively that axotomized ganglion cells lost cisternal membranes of the rough endoplasmic reticulum (RER) and showed disintegration of Nissl bodies and ribosomal rosettes 3 days postoperatively. Between 60 and 180 days after neurotomy there was partial reversion of the RER towards normal. At postoperative intervals of 14-60 days, chromatin aggregation became conspicuous and some nuclei were prominently furrowed and contained electron-dense inclusions. Concurrently, profiles of dead ganglion cells were encountered. Mean mitochondrial area increased in axotomized neurons but mitochondrial density declined, while the Golgi apparatus, lamellar specializations of the RER and the size of nuclei did not change significantly. Cytoplasmic atrophy was profound, however. Small nerve cells of the GCL appeared morphologically distinct from ganglion cells and did not undergo appreciable alteration. A decline in neuronal density, approximating 35%, occurred between the third and seventh postoperative day and progressed slowly thereafter. Neuronal density was 32% of normal 180 days postoperatively. A temporary increase in glial density 3-28 days after operation was due to microglial hyperplasia. Müller cell and astrocytic processes hypertrophied, infiltrated nerve fibre bundles, and surrounded and intruded into neuronal somata. Bundles of unmyelinated small axons, invested by astrocytes and basal lamina, were present within the necrotic cavity of the lesioned nerve 28-90 days postoperatively and had cytologic features of regenerative axonal sprouts. We conclude that intraorbital optic nerve crush is followed by a noteworthy degree of regenerative axonal sprouting which occurs and persists against a background of slow but relentless decline in the retinal ganglion cell population. This slow decline follows a rapidly-sustained loss of approximately one-third of the axotomized retinal ganglion cells during the first postoperative week. Intraorbital, as opposed to intracranial, injury of the optic nerve appears, paradoxically, to induce both a greater degree of ganglion cell death and a greater amount of regenerative axonal sprouting. Cytologic changes in axotomized retinal ganglion cells resemble those described for other populations of mammalian intrinsic neurons subjected to like injury.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased activity of ornithine decarboxylase in goldfish following optic nerve crush.

Ornithine decarboxylase (ODC) activity increased in goldfish retina, brain, and kidney several days following unilateral intraorbital optic nerve crush. Activity in both retinas followed a similar time course, an elevation being first detectable 3 days following crush, maximal on day 5 and returning to control levels by day 7. Increases were significantly higher in the retina on the lesioned side than in control retina. If one eye was surgically removed, a more prolonged elevation of ODC activity was seen in the remaining eye than was observed in control retinas following contralateral nerve crush. Sham operation or behavioral stress secondary to aversive electrical shock did not lead to significant increases in ODC activity in brain or retina. Explants of retinas whose optic nerve had been crushed 10--14 days prior to explantation, as well as those derived from the unlesioned side, showed increases in ODC activity following addition of beta-NGF to the medium. The response was greater in post-crush retinas than in control retinas. These results taken together suggest an increased responsiveness of the regenerating retina to stimuli that lead to increased ODC activity. While the nature of the in vivo signal(s) may be complex, it is inferred that degenerative changes in the distal stump play a significant role.