Optic nerve regeneration after intravitreal peripheral nerve implants: trajectories of axons regrowing through the optic chiasm into the optic tracts

We have studied axon regeneration through the optic chiasm of adult rats 30 days after prechiasmatic intracranial optic nerve crush and serial intravitreal sciatic nerve grafting on day 0 and 14 post-lesion. The experiments comprised three groups of treated rats and three groups of controls. All treated animals received intravitreal grafts either into the left eye after both left sided (unilateral) and bilateral optic nerve transection, or into both eyes after bilateral optic nerve transection. Control eyes were all sham grafted on day 0 and 14 post-lesion, and the optic nerves either unlesioned, or crushed unilaterally or bilaterally. No regeneration through the chiasm was seen in any of the lesioned control optic nerves. In all experimental groups, large numbers of axons regenerated across the optic nerve lesions ipsilateral to the grafted eyes, traversed the short distal segment of the optic nerve and invaded the chiasm without deflection. Regeneration was correlated with the absence of the mesodermal components in the scar. In all cases, axon regrowth through the chiasm appeared to establish a major crossed and a minor uncrossed projection into both optic tracts, with some aberrant growth into the contralateral optic nerve. Axons preferentially regenerated within the degenerating trajectories from their own eye, through fragmented myelin and axonal debris, and reactive astrocytes, oligodendrocytes, microglia and macrophages. In bilaterally lesioned animals, no regeneration was detected in the optic nerve of the unimplanted eye. Although astrocytes became reactive and their processes proliferated, the architecture of their intrafascicular processes was little perturbed after optic nerve transection within either the distal optic nerve segment or the chiasm. The re-establishment of a comparatively normal pattern of passage through the chiasm by regenerating axons in the adult might therefore be organised by this relatively immutable scaffold of astrocyte processes. Binocular interactions between regenerating axons from both nerves (after bilateral optic nerve transection and intravitreal grafting), and between regenerating axons and the intact transchiasmatic projections from the unlesioned eye (after unilateral optic nerve lesions and after ipsilateral grafting) may not be important in establishing the divergent trajectories, since regenerating axons behave similarly in the presence and absence of an intact projection from the other eye.     

Biochemical studies of myelin in Wallerian degeneration of rat optic nerve

Abstract— Wallerian degeneration of the optic nerves of the rat was induced by removal of the eyes. After 54, 66, 76 or 90 days of degeneration a myelin fraction of the nerves was obtained by the procedure of Laatsch et al. (1962). The yield of myelin from the degenerated nerves was decreased, but the isolated myelin appeared to be morphologically normal. The proportion of cholesterol in the myelin lipids was slightly increased, whereas that of the ethanolamineglycerophosphatides was decreased and galactolipids were normal. After one‘cycle’of myelin purification, the high-molecular-weight fraction formed a much greater percentage of the total protein in myelin isolated from degenerated optic nerves. After 2–3‘cycles’of purification, the distribution of protein in myelin isolated from degenerated and normal optic nerves was similar, an observation suggesting that the high-molecular-weight fraction in‘1-cycle myelin’from degenerated optic nerves may have been partly attributable to contamination. With the possible exception of ethanolamineglycerophosphatides, our data suggest that there was no preferential breakdown of myelin lipid constituents nor of protein constituents during Wallerian degeneration of rat optic nerve. As assessed by SDS-gel electrophoresis of the water-insoluble particulate fraction, the percentage of myelin protein was markedly decreased after 76 days of degeneration. However, the major myelin protein constituents in this fraction (the two basic proteins and proteolipid protein) appeared to decrease in the same relative proportions.     

Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush

Retinal ganglion cells (RGCs) are central nervous system (CNS) neurons that transmit visual information from the retina to the brain. Apoptotic RGC degeneration causes visual impairment that can be modeled by optic nerve crush. Neuronal apoptosis is also a salient feature of CNS trauma, ischemia (stroke), and diseases of the CNS such as Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis. Optic nerve crush induces the apoptotic cell death of ～ 70% of RGCs within the first 14 days after injury. This model is particularly attractive for studying adult neuron apoptosis because the time-course of RGC death is well established and axon regeneration within the myelinated optic nerve can be concurrently evaluated. Here, we performed a large scale iTRAQ proteomic study to identify and quantify proteins of the rat retina at 1, 3, 4, 7, 14, and 21 days after optic nerve crush. In total, 337 proteins were identified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated (>1.3 ×), 46 were downregulated (<0.7 ×), and 6 showed both positive and negative regulation over 21 days, relative to normal retinas. Among the differentially expressed proteins, Thymosin-β4 showed an early upregulation at 3 days, the time-point that immediately precedes the induction of RGC apoptosis after injury. We examined the effect of exogenous Thymosin-β4 administration on RGC death after optic nerve injury. Intraocular injections of Thymosin-β4 significantly increased RGC survival by ～ 3-fold compared to controls and enhanced axon regeneration after crush, demonstrating therapeutic potential for CNS insults. Overall, our study identified numerous proteins that are differentially regulated at key time-points after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the adult CNS.     

Induction of rat E and chicken A-I apolipoproteins and mRNAs during optic nerve degeneration

Apolipoprotein synthesis was measured in control optic nerves and optic nerves undergoing Wallerian degeneration. After short term organ culture with radiolabeled amino acid, optic nerve extracts were reacted with antiserum to rat or chicken apolipoproteins. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the degenerating rat optic nerve, apo-E synthesis increased from 0.30 to 0.90% of newly synthesized protein and from 0.45 to 1.4% of secreted protein. A DNA-excess solution hybridization assay was constructed to measure the absolute amount of apo-E mRNA in control and degenerating optic nerves. Paralleling the increase in apo-E protein synthesis, the absolute amount of apo-E mRNA was elevated 3- to 4-fold after enucleation. Similar to rat apo-E, apo-A-I synthesis was increased in degenerating chicken optic nerve. Chicken apo-A-I represented 0.65 and 3.5% of newly synthesized protein from control and enucleated optic nerves, respectively. Apo-A-I increased from 0.85 to 5.5% of secreted protein following enucleation. Using in vitro translation to quantitate relative amounts of chicken apo-A-I mRNA, enucleated optic nerve apo-A-I mRNA content was increased 5-fold. These results suggest that local apolipoprotein synthesis may be involved in the mobilization of myelin cholesterol which occurs during Wallerian degeneration. The similar response of the rat and chicken to increase optic nerve apolipoprotein synthesis during degeneration supports the idea that avian peripheral apo-A-I and mammalian peripheral apo-E may be performing functions common to both classes of animals.     

Optic nerve histopathology in a case of Wolfram Syndrome: A mitochondrial pattern of axonal loss

Mitochondrial dysfunction in Wolfram Syndrome (WS) is controversial and optic neuropathy, a cardinal clinical manifestation, is poorly characterized. We here describe the histopathological features in postmortem retinas and optic nerves (ONs) from one patient with WS, testing the hypothesis that mitochondrial dysfunction underlies the pathology. Eyes and retrobulbar ONs were obtained at autopsy from a WS patient, and compared with those of a Leber hereditary optic neuropathy (LHON) patient and one healthy control. Retinas were stained with hematoxylin & eosin for general morphology and ONs were immunostained for myelin basic protein (MBP). Immunostained ONs were examined in four “quadrants”: superior, inferior, nasal, and temporal. The WS retinas displayed a severe loss of retinal ganglion cells in the macular region similar to the LHON retina, but not in the control. The WS ONs, immunostained for MBP, revealed a zone of degeneration in the temporal and inferior quadrants. This pattern was similar to that seen in the LHON ONs but not in the control. Thus, the WS patient displayed a distinct pattern of optic atrophy observed bilaterally in the temporal and inferior quadrants of the ONs. This arrangement of axonal degeneration, involving primarily the papillomacular bundle, closely resembled LHON and other mitochondrial optic neuropathies, supporting that mitochondrial dysfunction underlies its pathogenesis.     

Enzyme histochemistry of Wallerian degeneration in the immature optic nerve of rabbits.

A histochemical study was performed on the activity of several phosphatases, esterases and oxidoreductases in the immature optic nerve of rabbits undergoing Wallerian degeneration. Unilateral enucleations of the eye bulb were performed on 7 days old animals and the degenerated optic nerves were examined in rabbits, 5, 23, 63 and 173 days afterwards. The following results were obtained: 1. The reactive cells appearing in the immature optic nerve undergoing Wallerian degeneration exhibit distinctly increased activities of many hydrolytic and oxidoreductive enzymes. 2. The histoenzymic pattern of changes displayed by the reactive cells occurring in the immature, degenerating optic nerve is distinct from and bears no relation to that seen in the normally developing optic nerve. 3. The genetic formation contained in the oligodendroglial cells is not the sole factor safeguarding the transformation of immature and mature oligodendroglia into myelinating cells.     

Ultrastructural sequence of myelin breakdown during Wallerian degeneration in the rat optic nerve

Summary Adult albino rats were subjected to unilateral surgical removal of the eyeball. After survival times of 7–140 days, the numerical response of the neuroglial cells, and the progressive disintegration of the myelin sheaths in the optic nerves, were studied qualitatively and quantitatively in electron-microscopic montages. The distribution density of microglia and astroglia in degenerating optic nerve increased to peaks after 35 and 56 days respectively, whereas, the oligodendroglia gradually decreased. During the early stage of degeneration, microglial cells appeared and invaded the sheath at the intraperiod line, peeling off the outer lamellae, which were then engulfed by phagocytosis. Within the microglia, myelin sheath fragments were surrounded by a membrane curled to form a myelin ring. In the intermediate stage of degeneration, the paired electrondense lines of the ring, made up of myelin basic protein, decomposed and formed a homogenous or heterogenous osmiophilic layered structure, the myelin body, which, in the final stages, disintegrated and transformed into globoid lipid droplets and needle shaped cholesterol crystals.     

Quantitative characteristics of degeneration of the fibers of the optic nerve after section

The optic nerve of the dog has been histologically studied. In the intact nerve 160,000 axons have been revealed. In 3 weeks after the nerve section in the orbit and near the nerve disk 72% retinoencephal fibers, 28% encephaloretinal and 0.23% of centrofugal axons are found.     

Neuroglia of the adult rat optic nerve in the course of wallerian degeneration

The neurological reactions in Wallerian degeneration have been studied by electron microscopy in the optic nerve of adult albino rats from 7 to 120 days after unilateral enucleation. Reactive astrocytes contained abundant dense bodies, numerous microtubules and hyperplastic glial filaments. These astrocytes also assisted phagocytosis of degenerated myelin sheaths and in glial scar formation. Oligodendrocytes disconnected their cytoplasmic extensions, which were phagocytosed by microglial cells and astrocytes, by increased production of lysosomes. Microglial cells consisted of crinkled, long, rough endoplasmic reticula, several highly-active Golgi complexes, laminar inclusions and globoid lipid droplets. Microglia engulfed and lysed the disintegrated axons and myelin sheaths.     

Axonal protection by Nmnat3 overexpression with involvement of autophagy in optic nerve degeneration

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the crucial role of nicotinamide mononucleotide adenylyltransferase (Nmnat) 1, 2, and 3 in axonal protection. In this study, Nmnat3 immunoreactivity was observed inside axons in the optic nerve. Overexpression of Nmnat3 exerts axonal protection against tumor necrosis factor-induced and intraocular pressure (IOP) elevation-induced optic nerve degeneration. Immunoblot analysis showed that both p62 and microtubule-associated protein light chain 3 (LC3)-II were upregulated in the optic nerve after IOP elevation. Nmnat3 transfection decreased p62 and increased LC3-II in the optic nerve both with and without experimental glaucoma. Electron microscopy showed the existence of autophagic vacuoles in optic nerve axons in the glaucoma, glaucoma+Nmnat3 transfection, and glaucoma+rapamycin groups, although preserved myelin and microtubule structures were noted in the glaucoma+Nmnat3 transfection and glaucoma+rapamycin groups. The axonal-protective effect of Nmnat3 was inhibited by 3-methyladenine, whereas rapamycin exerted axonal protection after IOP elevation. We found that p62 was present in the mitochondria and confirmed substantial colocalization of mitochondrial Nmnat3 and p62 in starved retinal ganglion cell (RGC)-5 cells. Nmnat3 transfection decreased p62 and increased autophagic flux in RGC-5 cells. These results suggest that the axonal-protective effect of Nmnat3 may be involved in autophagy machinery, and that modulation of Nmnat3 and autophagy may lead to potential strategies against degenerative optic nerve disease.