Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.     

Comparative study of the lamina cribrosa and the pial septa in the vertebrate optic nerve and their relationship to the myelinated axons

The optic nerve contains the connective tissues, i.e. the lamina cribrosa and pial septa. This report presents a histological comparison of the lamina cribrosa and pial septa in the five classes (mammals, birds, reptiles, amphibians and teleosts) of vertebrates. Furthermore, the distribution of myelinated fibers was observed from the optic nerve through the retina in the same animals. The lamina cribrosa is found in mammals except for mice, and in birds. Structural complexity of the lamina was different in animals but generally dependent of the optic nerve thickness. The pial septa were present in the optic nerve proper of the mammals except for the mice, in birds and in a part of teleosts. Fasciculation of the optic nerve by the pial septa tended to be more prominent as the optic nerve become thicker. The optic nerve consisted of largely myelinated fibers in vertebrates. The retina contained some myelinated fibers in submammals but was thoroughly devoid of myelinated fibers in mammals. The borderline between myelinated and unmyelinated portions in the optic nerve of different species did not related to the lamina cribrosa. Amphibians had exceptionally only a few myelinated fibers in the optic nerve and no myelinated fibers in the retina.     

猫视神经年龄相关的形态学变化

对4只青年猫(1-3龄)和4只老年猫(10-13龄)视神经进行形态计量比较研究。取两个年龄组的颅内相应部分视神经进行横向连续切片,H.E染色于光镜下观察其基本结构;相邻切片进行结晶紫染色显示胶质细胞;神经丝蛋白(NF)免疫染色显示视神经纤维,胶质纤维酸性蛋白(GFAP)免疫染色显示星形胶质细胞(AS),对实验结果进行统计学分析并绘制纤维直径谱。与青年猫相比,老年猫视神经外膜厚度、直径、面积均显著增加,视神经纤维的密度和数量显著下降,且以视神经中央部纤维密度下降最显著;纤维直径谱分析结果显示,青、老年猫纤维直径分布范围相似,但老年猫的峰直径及纤维平均直径比青年猫的显著减小;另外,老年猫视神经束中的星形胶质细胞明显膨大,胶质细胞密度以及星形胶质细胞占胶质细胞总数的百分比均显著增加。结果表明:在衰老过程中视神经纤维出现明显的丢失现象,纤维平均直径显著减小使其对视觉信息的传导速度减慢,这可能是导致老年个体视觉分析速度下降的重要原因;老年个体视神经束内胶质细胞活动增强可能对维持视神经纤维形态、功能或延缓视神经进一步衰老起保护作用     

Promoting Optic Nerve Regeneration in Adult Mice with Pharmaceutical Approach

Our previous research has suggested that lack of Bcl-2-supported axonal growth mechanisms and the presence of glial scarring following injury are major impediments of optic nerve regeneration in postnatal mice. Mice overexpressing Bcl-2 and simultaneously carrying impairment in glial scar formation supported robust optic nerve regeneration in the postnatal stage. To develop a therapeutic strategy for optic nerve damage, the combined effects of chemicals that induce Bcl-2 expression and selectively eliminate mature astrocytes—scar forming cells—were examined in mice. Mood-stabilizer, lithium, has been shown to induce Bcl-2 expression and stimulate axonal outgrowth in retinal ganglion cells in culture and in vivo. Moreover, astrotoxin (alpha-aminoadipate), a glutamate analogue, selectively kills astrocytes while has minimal effects on surrounding neurons. In the present study, we sought to determine whether concurrent applications of lithium and astrotoxin were sufficient to induce optic nerve regeneration in mice. Induction of Bcl-2 expression was detected in the ganglion cell layer (GCL) of mice that received a lithium diet in compared with control-treated group. Moreover, efficient elimination of astrocytes and glial scarring was observed in the optic nerve of mice treated with astrotoxin. Simultaneous application of lithium and astrotoxin, but not any of the drugs alone, induced robust optic nerve regeneration in adult mice. These findings further support that a combinatorial approach of concurrent activation of Bcl-2-supported growth mechanism and suppression of glial scarring is required for successful regeneration of the severed optic nerve in adult mice. They suggest a potential therapeutic strategy for treating optic nerve and CNS damage. Special issue article in honor of Dr. Ji-Sheng Han.     

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.     

Myocilin expression in the astrocytes of the optic nerve head

We investigated the expression of myocilin in the optic nerve head of porcine eyes by Western blotting and immunohistochemical staining. Myocilin was localized in the nucleus, centrosome, glial filament, mitochondria, and some parts of the cell membranes of the astrocytes. Myocilin was also detected at the edge-feet portion of the processes of astrocytes adjacent to the inner limiting membrane and blood vessel wall. The astrocytes are the major cell population in the optic nerve head, contributing to the architecture of the nerve axon and blood vessels. Therefore, myocilin gene mutation and change of myocilin protein are likely to affect the architecture of the optic nerve head and induce various forms of glaucomatous optic nerve damage.     

The correlation of changes of the optic nerve diameter in the acute retrobulbar neuritis with the brain changes in multiple sclerosis

The aim of this paper is to compare diameter of healthy and affected optic nerve determined by ultrasound with brain lesions in acute retrobulbar neuritis in patients with multiple sclerosis. In this prospective study 20 patients with multiple sclerosis and acute retrobulbar neuritis were examined. Optic nerve diameter was measured by ultrasound. Brain lesions were detected by magnetic resonance. Correlation between demyelinating lesions of the brain in multiple sclerosis and optic nerve diameter was tested by Kruskal-Wallis test. Significant difference in diameter between healthy and affected optic nerve in acute retrobulbar neuritis was found. Demyelinating brain changes examined by magnetic resonance revealed periventricular lesions, subcortical lesions and lesions in corpus callosum. There is statistically significant correlation between optic nerve diameter and number of brain lesions in multiple sclerosis, p < 0.05. Diameter of optic nerve in retrobulbar neuritis measured by ultrasound correlates with brain lesions detected by magnetic resonance in multiple sclerosis.     

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.     

Origin of Oligodendrocytes in the Vertebrate Optic Nerve: A Review

One of the unsolved problems in the research field of oligodendrocyte (OL) development has been the site(s) of origin of optic nerve OLs and its precursor cells (OPCs). It is generally accepted that OLs in the optic nerve are derived from the brain, and thus optic nerve OLs are immigrant cells. We previously demonstrated the brain origin of optic nerve OPCs in chick embryos. However, the site of optic nerve OPC origin has not been examined experimentally in developing rodents for the past two decades. We have recently reported that optic nerve OPCs in mice arise in the preoptic area by E12.5 and gradually migrate caudally and enter the optic nerve. These OPCs give rise to myelinating OLs in the optic nerve in the postnatal or adult stages. Surprisingly, there are species differences with respect to the origin of optic nerve OPCs between chicks and mice. Here, we summarize the site of OPC origin in the optic nerve based on our own previous and recent results, and discuss possible mechanisms underlying these species differences.     

Na,K-ATPase alpha3 subunit in the goldfish retina during optic nerve regeneration

The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up-regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the Na,K-ATPase catalytic subunit alpha3 isoform by high- sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5-20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50-100 nm ) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up-regulation of the Na,K-ATPase alpha3 subunit is involved in the regrowth of ganglion cell axons after axotomy.     

Keratin 8 of simple epithelia is expressed in glia of the goldfish nervous system

The intermediate filament protein composition in glial cells of goldfish optic nerve differs from that found in glial cells of the goldfish spinal cord and brain. Brain and spinal cord glial cells contain glial fibrillary acidic protein (GFAP), whereas glial cells in the optic nerve contain ON3. The ON3 protein of the goldfish optic nerve was recently identified as the goldfish equivalent to the mammalian type II keratin 8 protein. In addition to the ON3 protein, the goldfish optic nerve also contains a 48-kDa protein. Immunoblotting experiments suggest that this protein is equivalent to the mammalian type I keratin 18 protein, which typically pairs with keratin 8 to form filaments. We show that these proteins are not specific to the optic nerve. The ON3 and 48-kDa proteins of the goldfish optic nerve share common antigenic properties with the predominant keratin pair expressed in the goldfish liver. These proteins are also expressed at low levels in the goldfish brain and spinal cord. In addition RNase protection assays and Northern blots indicate that the mRNA for the ON3 protein in optic nerve is identical to the message found in other goldfish tissues. The expression of ON3 was also examined in cultured glial cells from goldfish spinal cord and optic nerve and cultured fibroblast cells. Analysis of intermediate filament protein expression in cultured glial cells taken from goldfish spinal cord demonstrated the absence of GFAP in these cells and the expression of ON3. This protein was also the predominant intermediate filament protein of cultured optic nerve glial cells and fibroblasts. The differences in the expression of intermediate filament proteins in mammals and lower vertebrates are discussed. In addition, we discuss how the expression of a simple epithelial keratin pair in glial cells of the goldfish optic nerve may be associated with this system's capacity for continuous growth and regeneration.     

58,000 Dalton Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Visual Pathway

A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two-dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal-enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one-dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one-dimensional gel electrophoresis.     

Comparison of the glial investment of normal and regenerating fiber bundles in the optic nerve and optic tectum of the goldfish and the Crucian carp

Summary The architecture of normal and regenerating nerve fiber bundles in the optic nerve of the goldfish and the Crucian carp was compared to that of the axonal fascicles in the optic tectum of these teleost species with the use of ultrathin sections and freeze-fracture replicas. The fascicles in the optic nerve are clearly demarcated by astrocytic processes, in contrast to the fascicles in the tectum. No astrocytes could be identified in the tectum; in this region processes of astrocytes or of radial glial cells do not form channeling structures reminiscent of those in the optic nerve. Furthermore, tectal blood vessels lack complete investments of glial processes. It can be assumed that at least in lower vertebrates a framework of astrocytic processes might be important for growth of optic fibers over large distances, i.e., from the eye to the tectum, but may be dispensable in the target region itself.     

Dolphin peripheral visual pathway in chronic unilateral ocular atrophy: Complete decussation apparent

Components of the peripheral visual pathway were examined in two bottlenose dolphins, Tursiops truncatus, each with unilateral ocular degeneration and scarring of 3 or more years' duration. In both animals, the optic nerve associated with the blind eye right eye in Tg419 and left eye in Tt038 had a translucent, gel-like appearance upon gross examination. This translucency was also evident in the optic tract contralateral to the affected eye. In Tg419, myelinated axons of varying diameters were apparent in the left optic nerve, whereas the right optic nerve, serving the blind eye, appeared to be devoid of axons. In Tt038, myelinated axons were associated with the right optic nerve (serving the functional eye) and left optic tract but were essentially absent in the left optic nerve and right optic tract. Examined by light microscopy in serial horizontal sections, the optic chiasm of Tt038 was arranged along its central plane in segregated, alternating pathways for the decussation of right and left optic nerve fibers. Ventral to this plane, the chiasm was comprised of fibers from the left optic nerve, whereas dorsal to the central plane, fibers derived from the right optic nerve. Because of this architectural arrangement, the right and left optic nerves grossly appeared to overlap as they crossed the optic chiasm with the right optic nerve coursing dorsally to the left optic nerve. At the light and electron microscopic levels, the optic nerves and tracts lacking axons were well vascularized and dominated by glial cell bodies and glial processes, an expression of the marked glial scarring associated with postinjury axonal degeneration. The apparent absence of axons in one of the optic tract pairs (right in Tt038 and left in Tg419) supports the concept of complete decussation of right and left optic nerve fibers at the optic chiasm in the bottlenose dolphin. © 1994 Wiley-Liss, Inc.     

62K Proteins Constitute the Major Neurofilament Proteins in the Frog Optic Nerve

The frog optic nerve contains a major group of proteins at a molecular weight of 62K. These proteins are insoluble in nonionic detergents, reactive with a general antibody to intermediate filament proteins, and not labeled by ex vivo incubations of optic nerve. They were therefore considered neurofilament proteins. Axonal transport and enucleation studies were performed to characterize further the origin of these proteins. The results show that the 62K proteins are transported into the optic nerve at a very slow rate (0.1 mm/day). After enucleation, these proteins are substantially reduced in concentration to 20% of the control value at 13 weeks. The predominant neurofilament proteins of the frog optic nerve are 62K in molecular weight. These results are discussed in terms of the anatomy of the frog optic nerve and also contrasted to findings obtained for the goldfish optic nerve.     

Axonal Transport in the Garfish Optic Nerve: Comparison with the Olfactory System

Fast and slow axonal transports were studied in the optic nerve of the garfish and compared with previous studies on the olfactory nerve. The composition of fast-transport proteins was very similar in the two nerves. Although the velocity of fast transport was slightly lower in the optic nerve, there was a linear increase in velocity with temperature in both nerves. As in the olfactory nerve, only a single wave of slow-transport protein radioactivity moves along the nerve. The velocity of slow transport also increased linearly with temperature, but the coefficient was less than in the olfactory system. The composition of slow transport in the optic nerve was significantly different from that in the olfactory nerve, a finding reflecting the different cytoskeletal constituents of the two types of axons. The slow wave could be differentiated into several subcomponents, with the order of velocities being a 105-kilodalton protein and actin greater than tubulins and clathrin greater than fodrin much greater than neurofilaments. It can be concluded that the temperature dependence of fast and slow axonal transport in different nerves reflects the influence of temperature on the individual polypeptides constituting the various transport phases. The garfish optic nerve preparation may be advantageous for studies of axonal transport in retinal ganglion cell axons, because its great length avoids the complications of having to study transport in the optic tract or in material accumulating at the tectum.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Topotecan Delivery to the Optic Nerve after Ophthalmic Artery Chemosurgery

Extraocular retinoblastoma is a major challenge worldwide, especially in developing countries. Current treatment involves the administration of systemic chemotherapy combined with radiation, but there is a clear need for improvement of chemotherapy bioavailability in the optic nerve. Our aim was to study the ophthalmic artery chemosurgery (OAC) local route for drug delivery assessing ocular and optic nerve exposure to chemotherapy and to compare it to exposure after intravenous infusion (IV) of the same dose in an animal model. Topotecan was used as a prototype drug that is active in retinoblastoma and based on the extensive knowledge of its pharmacokinetics in preclinical and clinical settings. Five Landrace pigs received 4mg of topotecan via OAC as performed in retinoblastoma patients. At the end of the infusion, the eyes were enucleated, the optic nerve and retina were dissected, and the vitreous and plasma were separated. After recovery and a wash-out period, the animals received a 30-min IV infusion of topotecan (4 mg). The remaining eye was enucleated and tissues and fluids were separated. All samples were stored until quantitation using HPLC. A significantly higher concentration of topotecan in the optic nerve, vitreous, and retina was obtained in eyes after OAC compared to IV infusion (p<0.05). The median (range) ratio between topotecan concentration attained after OAC to IV infusion in the optic nerve, retina and vitreous was 84(54–668), 143(49–200) and 246(56–687), respectively. However, topotecan systemic exposure after OAC and IV infusion remained comparable (p>0.05). The median optic nerve-to-plasma ratio after OAC and IV was 44 and 0.35, respectively. Topotecan OAC delivery attained an 80-fold higher concentration in the optic nerve compared to the systemic infusion of the same dose with similar plasma concentrations in a swine model. Patients with retinoblastoma extension into the optic nerve may benefit from OAC for tumor burden by increased chemotherapy bioavailability in the optic nerve without increasing systemic exposure or toxicity.     

Guidance of glial precursor cell migration by secreted cues in the developing optic nerve

Oligodendrocyte precursors are produced in restricted foci of the germinative neuroepithelium in embryo brains and migrate to their sites of function, while astrocytes are produced in a wider area in the neuroepithelium. We investigated the guidance mechanisms of glial precursor (GP) cell migration in the optic nerve. GP cell migration in newborn rat optic nerve was monitored by the UV-thymine-dimer (TD) method. A double labeling study using NG2 and TD revealed that many of these in vivo migrating cells were NG2 positive, while some of them with large TD-positive nuclei were NG2 negative. An in vitro cell migration study using optic nerve with chiasma and/or eyeball tissue revealed that the GP cells migrated under the guidance of repulsive cues secreted from the optic chiasma. We detected the expression of netrin 1 and Sema3a in the optic chiasma, and that of Unc5h1 and neuropilin 1 in the optic nerve. Co-culture experiments of the optic nerve with cell clusters expressing guidance cues revealed that the migrating GP cells in the optic nerve were heterogeneous. Netrin 1 repelled a subtype of NG2-positive and PLP-positive GP cells with small nuclei. Sema3a repelled a subtype of GP cells with large nuclei.