Gene Therapy with Single-Subunit Yeast NADH-Ubiquinone Oxidoreductase (NDI1) Improves the Visual Function in Experimental Autoimmune Encephalomyelitis (EAE) Mice Model of Multiple Sclerosis (MS).

Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.

miR-219 Cooperates with miR-338 in Myelination and Promotes Myelin Repair in the CNS.

A lack of sufficient oligodendrocyte myelination contributes to remyelination failure in demyelinating disorders. miRNAs have been implicated in oligodendrogenesis; however, their functions in myelin regeneration remained elusive. Through developmentally regulated targeted mutagenesis, we demonstrate that miR-219 alleles are critical for CNS myelination and remyelination after injury. Further deletion of miR-338 exacerbates the miR-219 mutant hypomyelination phenotype. Conversely, miR-219 overexpression promotes precocious oligodendrocyte maturation and regeneration processes in transgenic mice. Integrated transcriptome profiling and biotin-affinity miRNA pull-down approaches reveal stage-specific miR-219 targets in oligodendrocytes and further uncover a novel network for miR-219 targeting of differentiation inhibitors including Lingo1 and Etv5. Inhibition of Lingo1 and Etv5 partially rescues differentiation defects of miR-219-deficient oligodendrocyte precursors. Furthermore, miR-219 mimics enhance myelin restoration following lysolecithin-induced demyelination as well as experimental autoimmune encephalomyelitis, principal animal models of multiple sclerosis. Together, our findings identify context-specific miRNA-regulated checkpoints that control myelinogenesis and a therapeutic role for miR-219 in CNS myelin repair.

The effect of prenatal exposure of a non-steroidal anti-inflammatory drug on the optic nerve of female rats: a stereological, histological, and electron microscopic study.

OBJECTIVE: Non-steroidal anti-inflammatory drugs (NSAIDs) can have adverse effects for in both mother and fetus following administration during the prenatal period. If given during pregnancy, diclofenac sodium (DS), an NSAID, is given during the pregnancy, may also affect the development of the central nervous system (CNS) or related structures. METHODS: Pregnant rats were separated into pure control (PG), saline (SG) and diclofenac groups (DG). A daily dose of 1 mg/kg of DS and 1 mL/kg saline was injected intraperitoneally to the DG and SG groups, respectively, from the 5th gestation day for a 15 day of period; the PG group received no treatment. After spontaneous delivery, female offspring were obtained from all groups. After the 20th week of postnatal life, the animals (n = 6 for each group) were perfused and the right optic nerves were resected. Sections were subjected to stereological and histological analysis. RESULTS: There were no significant differences (p > 0.05) between PG, SG and DG groups with respect to myelin thickness, axonal cross-sectional area, axon numerical density, total section area of optic nerve and axon number. CONCLUSIONS: Histological and stereological results indicated that treatment with DS or saline produced undesirable effects on female rat optic nerve development and myelinization with respect to morphology.

The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.

Dominant optic atrophy is a rare inherited optic nerve degeneration caused by mutations in the mitochondrial fusion gene OPA1. Recently, the clinical spectrum of dominant optic atrophy has been extended to frequent syndromic forms, exhibiting various degrees of neurological and muscle impairments frequently found in mitochondrial diseases. Although characterized by a specific loss of retinal ganglion cells, the pathophysiology of dominant optic atrophy is still poorly understood. We generated an Opa1 mouse model carrying the recurrent Opa1(delTTAG) mutation, which is found in 30% of all patients with dominant optic atrophy. We show that this mouse displays a multi-systemic poly-degenerative phenotype, with a presentation associating signs of visual failure, deafness, encephalomyopathy, peripheral neuropathy, ataxia and cardiomyopathy. Moreover, we found premature age-related axonal and myelin degenerations, increased autophagy and mitophagy and mitochondrial supercomplex instability preceding degeneration and cell death. Thus, these results support the concept that Opa1 protects against neuronal degeneration and opens new perspectives for the exploration and the treatment of mitochondrial diseases.

Preclinical retinal neurodegeneration in a model of multiple sclerosis.

Neurodegeneration plays a major role in multiple sclerosis (MS), in which it is thought to be the main determinant of permanent disability. However, the relationship between the immune response and the onset of neurodegeneration is still a matter of debate. Moreover, recent findings in MS patients raised the question of whether primary neurodegenerative changes can occur in the retina independent of optic nerve inflammation. Using a rat model of MS that frequently leads to optic neuritis, we have investigated the interconnection between neurodegenerative and inflammatory changes in the retina and the optic nerves with special focus on preclinical disease stages. We report that, before manifestation of optic neuritis, characterized by inflammatory infiltration and demyelination of the optic nerve, degeneration of retinal ganglion cell bodies had already begun and ultrastructural signs of axon degeneration could be detected. In addition, we observed an early activation of resident microglia in the retina. In the optic nerve, the highest density of activated microglia was found within the optic nerve head. In parallel, localized breakdown in the integrity of the blood-retinal barrier and aberrations in the organization of the blood-brain barrier marker aquaporin-4 in the optic nerves were observed during the preclinical phase, before onset of optic neuritis. From these findings, we conclude that early and subtle inflammatory changes in the retina and/or the optic nerve head reminiscent of those suggested for preclinical MS lesions may initiate the process of neurodegeneration in the retina before major histopathological signs of MS become manifest.

Evidence of compromised circulation in the pathogenesis of optic nerve damage in chronic glaucomatous rabbit.

The protective effect of improving the microcirculation on the retinal ganglion cells (RGC) and the ultrastructure of the optic nerve after intravenous (i.v.) use of Salviae miltiorrhiza (SMR) was studied in a chronic intraocular hypertension model in 36 rabbits. These rabbits were treated with topical 0.5% timolol, or i.v. SMR or i.v. 0.9% saline for 1, 3, 5 or 8 weeks. The intraocular pressure (IOP) was 19.6 +/- 2 mmHg and the RGC density was 1140 +/- 121.2/mm2 in control rabbits. After one week of intraocular hypertension (IOP between 30 and 40 mmHg), the RGC densities decreased to between 650 and 820/mm2. Following the use of timolol for 1 to 8 weeks, IOP returned to control level and the RGC density increased to 1015 +/- 7/mm2. After treatment with i.v. saline for 8 weeks, the RGC density decreased progressively from 651 +/- 1/mm2 at the beginning of treatment to 83 +/- 3/mm2. The RGC density of those rabbits treated with i.v. SMR for 8 weeks remained virtually unchanged (658 +/- 5/mm2) compared to the RGC density at the beginning of the treatment. Severe ultrastructural damage of optic nerve and collapsed capillaries were found in rabbits treated with saline while these findings were mild in rabbits treated with SMR. During these periods of treatment, the intraocular hypertension did not decrease in rabbits treated with either saline or SMR. From these findings and the fact that SMR improves local microcirculation, it is considered that the compromised microcirculation is one of the mechanisms in the pathogenesis of optic nerve damage in chronic glaucoma.

Visual evoked potentials and optic nerve histopathology in normal and diabetic rats and effect of ginkgo biloba extract.

The purpose of this study was to test the possible therapeutic role of ginkgo biloba extract on the impairment of visual function and pathological histology of the optic nerve caused by early diabetes. Ginkgo biloba extract entraps oxygenated free radicals and is also a strong inhibitor of the platelet activation factor (PAF). For this purpose, VEP recordings and optic nerve histopathology were studied on alloxan diabetic and normal Swiss albino rats in four experimental groups. The VEP recordings showed no statistical significance between diabetic and normal rats. However, the amplitudes were significantly increased in diabetic animals with ginkgo biloba extract compared with the diabetics, supposing an impression of axonal protection. But the amplitude values were decreased in normal rats treated with the same extract compared with normal animals, assuming a toxic activity. Optic nerve ultrastructural findings also confirmed these VEP changes. It was concluded that this extract could be encouraging for human clinical trials of diabetes.

[The effect of Inj. Salviae Miltiorrhizae Co. on the retrograde axoplasmic transport in the optic nerve of rabbits with chronic IOP elevation].

With the horseradish peroxidase histochemical technique and electron microscope, the effects of 0.5% timolol, Inj. Salviae Miltiorrhizae Co. and their combination on the retrograde axoplasmic transport in the optic nerve of 230 rabbit models of chronic IOP elevation were studied. The results showed that (1) the nerve damage in chronic glaucoma was due to multiple factors; (2) the use of a drug that improved microcirculation in combination with an IOP depressor better protected the optic nerve functions than did the latter alone; and (3) Inj. Salviae Miltiorrhizae Co. improved the optic nerve axoplasmic transport under chronic IOP elevation. The mechanism of the drug in protecting the optic nerve could be through its actions of improving the local microcirculation and the tolerance of nerve tissues to anoxia.

Regeneration of axons from the adult rat optic nerve: influence of fetal brain grafts, laminin, and artificial basement membrane.

After transection of the optic nerve of adult rats, most of the axons in the proximal stump die and the surviving ones are unable to regenerate into the distal optic nerve. Since the fetal brain has an inherent capacity to regenerate axons, we investigated whether fetal (E16) target regions of optic axons (thalamus and tectum) transplanted to the completely transected optic nerve of adult rats would promote axon regeneration. In control operated rats, axon growth beyond the site of transection was restricted to a few fibers that grew irregularly within the connective tissue scar. By contrast, in grafted animals directed outgrowth of optic axons toward the transplant started at 6 days postoperation (p.o.) and reached its maximum 15 days p.o. and later, when numerous single optic fibers and small axon fascicles had grown toward and into the graft, where they formed arborizations and terminal varicosities. Regenerating optic axons were further advanced than GFAP-positive strands of astroglia that emanated from the proximal optic nerve stump. Laminin immunoreactivity appeared at 6 days p.o. in the zone of reactive astroglia in the terminal part of the optic nerve stump. Later it showed a distribution complementary to the pattern of GFAP immunoreactivity, which it seemd to circumscribe. There was no unequivocal codistribution of laminin immunoreactivity with regenerating axons. In further experiments, target regions from different ontogenetic stages (E14 to neonate and adult) and nontarget regions (E16, cerebral cortex or spinal cord) were grafted to the optic nerve stump. With the exception of the adult grafts, all transplants had effects on axon regeneration comparable to those of E16 target regions. In order to test the effects of extracellular matrix molecules on axon regeneration, a basement membrane gel reconstituted from individual components of the Engelbreth-Holm-Sarcoma (EHS) sarcoma was implanted between proximal and distal optic nerve stumps. No axons were induced to regenerate by this matrix. Likewise, laminin adsorbed to nitrocellulose paper and implanted at the lesion site did not stimulate axon growth from the proximal optic nerve stump. These results indicate that fetal brain is able to induce and direct regrowth of axons from the optic nerve toward the graft across a substrate that is not composed of astroglia or basement membrane components like laminin. The directed growth of axons in the absence of a preformed substrate implies a chemotactic growth response along a concentration gradient mediated by neurotropic molecules released from the graft.

Enlargement of uncrossed retinal projections in the albino rat: additive effects of neonatal eye removal and thalamectomy.

The present study in the albino rat investigates the effects of neonatal unilateral eye removal and/or thalamectomy upon the number of ipsilaterally projecting retinal ganglion cells (IPRGCs), using retrograde fluorescent labelling and electron microscopy. The results show that neonatal unilateral eye removal and thalamectomy result in a significant increase in the number of aberrant IPRGCs as compared to that observed in normal rats, and the effects of the two different neonatal lesions appear to be additive. These findings strongly suggest that there are at least two subpopulations of IPRGCs, which normally do not exist in mature albino rats, but which can be preserved into adulthood by neonatal enucleation or thalamectomy. The data also suggest that about 70% of the entire population of IPRGCs, most of which normally exist only transiently in neonatal retinas, can be retained in rats which receive both unilateral eye removal and thalamectomy at the neonatal stage.

Brain and optic system pathology in hypocholesterolemic dogs treated with a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The cholesterol lowering compound lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34 HMG CoA reductase), was given in nine separate experiments to normocholesterolemic dogs at rates up to 180 times the maximum therapeutic dose in man (1 mg/kg/day). Mean serum total cholesterol concentrations were reduced as much as 88% below normal. Clinical evidence of neurotoxicity occurred in up to 37% of animals given 180 mg/kg/day lovastatin for 11 or more days, especially in one laboratory where the dosing regime resulted in higher concentrations of plasma drug levels. Dogs receiving 60 mg/kg/day or less never exhibited neurologic signs. The central nervous system (CNS) of affected dogs exhibited endothelial degeneration and hemorrhagic encephalopathy. Focal extravasation of horseradish peroxidase occurred frequently (6/8) in the retrolaminar optic nerve of asymptomatic or clinically affected dogs given 180 mg/kg/day lovastatin, with endothelial degeneration and discrete optic nerve degenerative lesions interpreted as ischemic. The association between the degree of hypocholesterolemia and occurrence of clinical signs was not exact. Total brain cholesterol was similar in treated and control dogs. Hypocholesterolemic dogs had proportionally lowered serum concentrations of alpha-tocopherol, but oral supplementation of this vitamin did not prevent the neurologic syndrome. Endothelial degeneration in the CNS and optic nerve may have reflected in vitro morphologic effects of HMG CoA reductase inhibitors due to extreme inhibition of nonsterol isoprene synthesis. Retinogeniculate axonal (Wallerian-like) degeneration occurred in greater than or equal to 12% of dogs given 60 mg/kg/day or more lovastatin, with central chromatolysis of occasional retinal ganglion cells. These neuroaxonal changes may have been secondary to vascular effects, but superimposed direct neurotoxic action at the high dosage levels of lovastatin could not be excluded. There was no evidence of drug induced adverse effects in the CNS of dogs given up to 30 mg/kg/day lovastatin for 2 years.

Visual system degeneration in the glaucomatous albino quail.

The peripheral (eye, retina, optic nerve) and central (primary optic tracti and centers, centrifugal visual tractus and nucleus) visual system of an imperfect albino quail mutant with a sex linked recessive gene was examined in 32 specimens ages 1 week - 16 months-hatch using various histological techniques. During the first weeks the visual system was normal and comparable in its overall organization to that found in the pigmented quail. However, the ipsilateral retinal projections were observed to be weaker in the young mutant, then completely disappeared two months after birth. Initial signs of the bupthalmos, a form of spontaneous glaucoma, appeared between the 3rd and 5th months. This was characterized by a distention of the eye linked to an increase in intraocular pressure. The pathological process was progressive and at 16 months the eye was very prominent, the anterior chamber deep and a large and globular cornea was noted. The glaucoma progressively induced different histopathological changes in the visual system including: cupping of the optic disc, degeneration of optic axons and their parent ganglion and centrifugal cells and cavernous degeneration. All of these phenomena were identifiable at about the 10th post-natal month and progressed in a relatively constant and orderly manner. The retinal projections to the nucleus ectomamillaris, ventral and lateral optic tectum and ventral pretectum were the first to degenerate. The degeneration of optic fibers attaining the dorsal pretectum and dorsal thalamus occurred later. Furthermore the retrograde degeneration in the centrifugal isthmo-optic nucleus progressed from the external to the internal pole. The mechanisms involved in the selective degeneration of centrifugal and centripetal optic fibers is discussed.

Early uncrossed component of the developing optic nerve with a short extracerebral course: a light and electron microscopic study of fetal ferrets.

During the study of the developing optic nerve described in the preceding paper (Guillery and Walsh, '87), small bundles of nerve fibers were seen passing between the optic nerve and the ipsilateral hypothalamus of 24-to 27-day-old prenatal ferrets. The bundles appear before any other fiber groups of the retinofugal pathway and are identifiable while the main portions of the retinofugal system are growing into the optic tracts. The bundles, made up of 50 or more axons, leave the optic nerve, emerge through the otherwise continuous layer of subpial glia and through the basal lamina of the nerve, run a short, naked, extracerebral course among collagen fibers and presumed fibroblasts, and then re-enter the central nervous system, passing rostrally and dorsally to the superficial parts of the ipsilateral hypothalamus away from the region of the chiasm. These fibers represent the earliest link between the optic nerve and the brain, but their course is not followed by the majority of retinofugal fibers developing later, which pass toward one or the other optic tract.

Laser-induced primate glaucoma. II. Histopathology.

A sustained, moderate pressure elevation was produced in 15 nonhuman primate eyes by application of laser energy to the trabecular meshwork. By light and electron microscopy, the trabecular beams were blunted, and scattered synechiae were present. Backward bowing of the lamina cribrosa, partial loss of the myelin sheath surrounding axonal segments just posterior to the lamina, and diffuse axonal loss involving the entire nerve cross section were noted. A quantitative analysis of this axonal loss revealed that eyes with moderate nerve head damage (cup-disc ratio, 0.6 to 0.8) had only 38% to 69% of the expected normal axonal count. The eyes with nearly total cupping (cup-disc ratio, 0.9 to 1.0) maintained between 10% and 36% of the normal axonal count. The disc changes in these experimental eyes are similar to those previously described in human eyes with glaucoma.

Plasticity in synaptic appositions of optic nerve afferents under different lighting conditions.

The influence of constant light and darkness (14 days) on the ultrastructure of synaptic appositions of optic nerve afferents has been studied in the suprachiasmatic nucleus (SCN) of hooded rats. Light exposure causes a general loss of postsynaptic density (PD) material, whereas after constant darkness the postsynaptic densities are thicker and the number of subjunctional bodies is higher. Accordingly, there are changes in the relative number of 'asymmetrical' (excitatory?) and 'symmetrical' (inhibitory?) synaptic appositions in optic and non-optic synapses. This phenomenon seems to reflect an adaptive response of the postsynaptic neurons to a long-term change of input activity.

Synapses of optic nerve afferents in the rat suprachiasmatic nucleus. I. Identification, qualitative description, development and distribution.

Synapses of optic nerve afferents (optic synapses) in the rat suprachiasmatic nucleus (SCN) have been identified ultrastructurally. They are easily distinguished from other types of synapses. The optic boutons are characterized by the presence of large mitochondria with a swollen electron lucent matrix and an interconnected tubular system formed by their inner membrane. Other, more variable features include: 1) a scattered pattern of synaptic vesicles which are found throughout the entire presynaptic element with relatively little accumulation near the active zones; 2) the occurrence of dense core vesicles and glycogen granules; 3) the active zones, the majority of which is Gray-type I, but a minority can obviously be classified as Gray's type II; 4) the innervation of smaller peripheral dendrites and dendritic spines. Boutons of this kind are exclusively filled with anterogradely transported horseradish peroxidase injected into both eyes. Very few neuronal elements containing the typical mitochondria have been observed in the SCN on the 6th day post partum, increasingly more on the 9th and 12th day, but considerably higher numbers after opening of the eyes on the 17th and the following days. The location of normal and degenerating optic boutons was examined light- and electron microscopically. In the rostral third of the SCN there are relatively few optic synapses which are found close to the optic chiasma. In the middle portion of the SCN optic synapses increase in number; they are found not only in the ventral part of the nucleus but also in lateral regions. This becomes particularly obvious in the caudal third of the SCN.

Axoplasmic flow during chronic experimental glaucoma. 1. Light and electron microscopic studies of the monkey optic nervehead during development of glaucomatous cupping.

The anterior optic nerve and the macular region of the retina of glaucomatous eyes of five rhesus monkeys (Macaca mulatta) have been examined by light and electron microscopy. The experimental glaucoma had been induced by argon laser treatment of the anterior chamber angle. The eyes were examined 3 to 11 weeks after the onset of sustained elevation of intraocular pressure above 20 mm Hg. Severe degenerative changes were seen in eyes with higher intraocular pressure and longer duration of glaucoma. Eyes with a lesser elevation of intraocular pressure and shorter duration of glaucoma showed changes sharply localized to the axon bundles in the scleral lamina cribrosa. Accumulation of mitochondria and dense bodies occurred anterior and posterior to collagenous septae. The location of these changes is in agreement with the localization of block of axoplasmic transport identified by autoradiographic studies. It is speculated that these cytologic changes reflect blockage of axoplasmic flow in the optic nerve of eyes with glaucoma.

Prenatal development of central optic pathways in albino rats.

The development of the central optic projections in albino rat fetuses has been studied using light and electron microscopic degeneration techniques and the horseradish peroxidase method for demonstrating axonal projections of neurons. The first optic axons to reach the region of the optic chiasm arrive at day 15. By day 16, a substantial optic chiasm is seen and the optic tract can be traced into the epithalamus, having first passed through the ventral lateral geniculate nucleus and a thin lamina of cells which is thought to correspond to part of the future dorsal geniculate nucleus. A growth rate of 80-100 mum per hour is estimated for the fastest growing axons. By day 16-1/3 the first axons have entered the anterior border of the superior colliculus and in the next day have grown across the entire rostrocaudal extent with the exception of the medial and lateral edges. The optic axons are recognized at day 17 as bundles lying just below the surface, but in older animals they come to lie deeper, as the whole layer of optic innervation broadens. The first synapses to be formed in the superior colliculus (some of them of optic origin) appear on day 17. Subsequently, there is a gradual increase in the number of contacts, the great majority being formed by optic axons. Compared with previous studies on Xenopus and chick, one of the most striking features of the development of the central visual connections in the rat is the relatively long time before the first optic axons reach the brain and the speed with which they innervate the central structures once they have arrived.