Retinal ganglion cell distribution and visual acuity in alpacas (Vicugna pacos).

OBJECTIVE: To investigate the distribution of retinal ganglion cells (RGCs) and visual acuity in alpacas (Vicugna pacos) through Brn-3a immunofluorescent labeling. PROCEDURES: Five eyes from four healthy alpacas with normal ophthalmic examination findings were included in the study. The axial length of the globes was measured before fixation. All five retinas were treated with Brn-3a antibodies to label RGCs. Images taken with a fluorescent microscope were used for RGC counting. RGC density maps were reconstructed by computer software. Visual acuity was estimated based on the results of peak RGC density and ocular anatomical parameters. RESULTS: The reconstructed retinal maps from Brn-3a labeling showed a horizontal streak across the retinal meridian superior to the optic nerve head with a temporal, upward extension. The highest RGC densities were in the temporal retinas. The maximal visual acuity was located in the temporal retina and was estimated to range between 12.5 and 13.4 cycles per degree. CONCLUSIONS: Alpacas have a horizontal streak across the retinal meridian superior to the optic disk with a temporal, upward extension based on the Brn-3a labeling of RGCs. The maximal visual acuity was located in the temporal retina. The reconstructed retinal maps indicate the RGC topography of alpacas is similar to that of other herbivores, but is different from that of dromedary camels.

Oxidative stress increases in retinas of dogs in acute glaucoma but not in chronic glaucoma.

OBJECTIVE: To test the hypothesis that oxidative stress occurs early in the pathogenesis of glaucoma in dogs. ANIMALS: Sections from eight control retinas and 25 retinas from dogs with primary glaucoma. METHODS: For retinas embedded in paraffin, sections were immunohistochemically stained for malondialdehyde (MDA) and 3-nitrotyrosine (NT). For retinas embedded in plastic, serial 0.5-µm sections were immunogold-stained for total glutathione, taurine, and glutamate. RESULTS: Increased immunostaining for MDA and NT, markers of oxidative stress, occurred in retinal ganglion cells (RGCs) and other neurons in acute glaucoma, but not in chronic glaucoma. In minimally damaged regions, immunostaining for the antioxidant glutathione was decreased in RGCs, neurons of the inner nuclear layer (INL), and Müller cell processes. The loss of glutathione immunostaining in RGCs occurred without a decrease in glutamate immunostaining. Neurons with nuclear damage in the INL had low levels of glutathione, taurine, and glutamate. In severely damaged regions, immunostaining for glutathione was increased in the remaining retinal tissue. CONCLUSIONS: Immunohistochemical staining revealed an increase in markers of oxidative stress and loss of glutathione in neurons with minimal damage during acute glaucoma. Oxidative changes were no longer present in chronic glaucomatous retinas, suggesting transient oxidative stress occurs early in glaucoma. The loss of glutathione in minimally damaged regions occurred without a significant redistribution of glutamate, suggesting oxidative stress may occur before glutamate redistribution. Alteration in markers of oxidative stress occurs early in canine glaucoma, suggesting oxidative stress may contribute to subsequent glutamate redistribution and other damaging processes.

Domoic acid-induced seizures in California sea lions (Zalophus californianus) are associated with neuroinflammatory brain injury.

California sea lions (CSLs) exposed to the marine biotoxin domoic acid (DA) develop an acute or chronic toxicosis marked by seizures and act as sentinels of the disease. Experimental evidence suggests that oxidative stress and neuroinflammation are important mechanisms underlying the seizurogenic potential of environmental toxicants but these pathways are relatively unstudied in CSLs. In the current study, we investigated the role of glutamate-glutamine changes and gliosis in DA-exposed CSLs to better understand the neurotoxic mechanisms occurring during DA toxicity. Sections from archived hippocampi from control and CSLs diagnosed with DA toxicosis were immunofluorescently stained for markers of gliosis, oxidative/nitrative stress and changes in glutamine synthetase (GS). Quantitative assessment revealed increasing loss of microtubule associated protein-2 positive neurons with elevations in 4-hydroxynonenal correlating with chronicity of exposure, whereas the pattern of activated glia expressing nitric oxide synthase 2 and tumor necrosis factor followed pathological severity. There was no significant change in the amount of GS positive cells but there was increased 3-nitrotyrosine in GS expressing cells and in neurons, particularly in animals with chronic DA toxicosis. These changes were consistently seen in the dentate gyrus and in the cornu ammonis (CA) sectors CA3, CA4, and CA1. The results of this study indicate that gliosis and resultant changes in GS are likely important mechanisms in DA-induced seizure that need to be further explored as potential therapies in treating exposed wildlife.

alpha-Luminol prevents decreases in glutamate, glutathione, and glutamine synthetase in the retinas of glaucomatous DBA/2J mice.

OBJECTIVE: To test the hypothesis that in DBA/2J mice, oxidative stress decreases glutamine synthetase (GS) levels resulting in a loss of neuronal glutamate and that the antioxidant alpha-luminol (GVT) decreases this stress and glutamate loss in some types of glaucoma. ANIMALS: DBA/2J mice were separated into two groups, of which one was not treated, and the other treated with GVT in the drinking water. At 7 months of age, retinas were examined from five untreated DBA/2J mice, seven GVT-treated mice, and five C57BL/6 mice (negative controls). METHODS: Serial 0.5 microm plastic sections were immunogold stained for glutamate, GS, and total glutathione, followed by image analysis for staining patterns and density. RESULTS: Focal decreases in glutamate immunostaining were common in the inner nuclear layer (INL) of DBA/2J retinas, but not in C57BL/6 or GVT-treated DBA/2J retinas. Decreases in glutathione and GS immunostaining were found in DBA/2J retinal regions where neuronal glutamate immunostaining was reduced. Retinas from GVT-treated DBA/2J had no significant decreases in INL levels of glutamate, glutathione, or GS. CONCLUSIONS: Retinas of dogs with primary glaucoma are reported to have focal depletion of neuronal glutamate. In DBA/2J mice, similar changes occur prior to the development of clinical disease. In these focal glutamate-depleted regions, levels of glutathione and GS are also reduced, consistent with the hypothesis that oxidative stress contributes to retinal changes in glaucoma. The ability of GVT, an antioxidant, to inhibit retinal abnormalities in DBA/2J mice provides further support for this hypothesis.

Insulin and IGF-I prevent brain atrophy and DNA loss in diabetes.

The aim of this study was to identify factors that regulate the bulk of adult brain mass, and test the hypothesis that concomitantly reduced insulin and insulin-like growth factor (IGF) levels are pathogenic for brain atrophy associated with impaired learning and memory in diabetes. Doses of insulin, or insulin plus IGF-I that were too small to prevent hyperglycemia were infused for 12 weeks into the brain lateral ventricles of streptozotocin-diabetic adult rats. Brain wet, water and dry weights were significantly decreased in diabetic rats; insulin prevented these decreases. The decrease in brain DNA and protein contents in diabetic rats was prevented by the combination treatment, but not by insulin alone. Levels of several glia- and neuron-associated proteins were reduced in diabetes; these reductions were also prevented by the combination treatment. Although hyperglycemia was not prevented in plasma or cerebrospinal fluid, insulin prevented brain atrophy but not bulk DNA loss in diabetes, whereas the combination prevented both. Insulin actively prevented the loss of brain water content as well. Brain atrophy is associated with concomitantly reduced levels of insulin and IGF in other disorders such as Alzheimer's disease.

Loss of glutamine synthetase immunoreactivity from the retina in canine primary glaucoma.

PURPOSE: Changes in retinal glutamate distribution occur in primary glaucoma (PG) in dogs. Although the redistribution resembles that induced by ischemia, decreases in glutamine synthetase (GS) activity may also induce a similar glutamate redistribution. We examined the distribution of GS, glutamate, and glial fibrillary acidic protein (GFAP), a marker for reactive glia, in PG retinas by immunohistochemistry to determine whether decreases in GS and formation of reactive glia are associated with glutamate redistribution and neuronal damage. ANIMALS: Sections from 14 control dog eyes and 22 eyes from dogs with PG. METHODS: Sections from 14 control and 22 glaucomatous globes were immunohistochemically stained for GS, glial fibrillary acidic protein or glutamate. RESULTS: In semiquantitative immunogold studies, decreases in GS staining density were strongly correlated with glutamate redistribution and neuronal damage. In less quantitative immunoperoxidase staining of acute (< or = 5 days after clinical signs) and chronic PG retinas, GS immunoreactivity was decreased in focal regions of some acute PG retinas, and there were widespread decreases in chronic PG retinas. GFAP immunoreactivity was increased in Müller cells primarily in severely damaged regions of chronic PG retinas. CONCLUSIONS: Decreases in GS immunoreactivity were associated with glutamate redistribution. These decreases in GS occurred even in mildly damaged regions of retina before retinal thinning. Reactive Müller cells were seen primarily in chronic PG in severely damaged regions. Decreases in GS may potentiate ischemia-induced early glutamate redistribution and neuronal damage in canine PG.

Microvessel loss, vascular damage and glutamate redistribution in the retinas of dogs with primary glaucoma.

OBJECTIVE: Vascular damage and ischemia-like changes in glutamate distribution occur in primary glaucoma (PG) in dogs. We measured the microvessel density in PG retinas to determine whether microvessel loss may induce ischemia and glutamate redistribution. ANIMALS STUDIED: Sections from 12 control and 33 glaucomatous dog retinas. PROCEDURES: Vessels in retinas were identified by staining with Griffonia simplicifolia isolectin B4 or immunohistochemical staining for laminin or glutamate. Damage to regions of the inner nuclear layer (INL) was classified as mild (< 10% damaged neurons), moderate (> or = 10% damaged neurons, INL > or = 2 cells thick) or severe (INL < 2 cells thick). RESULTS: Glutamate redistribution was found in some mildly damaged regions and increased as damage increased. Regions with increased glutamate redistribution and increased damage had lower densities of microvessels in plastic sections. However, neuronal damage and glutamate redistribution were seen even in areas adjacent to the remaining microvessels. Microvessel loss in damaged regions was confirmed in paraffin sections with lectin staining and immunohistochemical localization of laminin. The density of larger vessels was not decreased in PG, but larger vessels often had thickened walls, cuffing with leukocytes, and leakage of albumin. CONCLUSIONS: Microvessel loss may occur in regions of glutamate redistribution and neuronal damage in PG retinas. Larger vessels were often damaged. The redistribution of glutamate is associated with a loss of microvessels, even in mildly damaged regions. However, neuronal damage and glutamate redistribution may occur close to remaining microvessels, suggesting microvessel loss was not the sole factor inducing these changes.

Retinal pigment epithelial damage, breakdown of the blood-retinal barrier, and retinal inflammation in dogs with primary glaucoma.

OBJECTIVE: This paper aims to determine if abnormalities of the retinal pigment epithelium (RPE) and retinal inflammation occur in primary glaucoma. PROCEDURE: Twenty-three canine globes with primary glaucoma, goniodysgenesis, and elevated intraocular pressure were evaluated. Sections from 6 control and 23 glaucomatous canine globes were stained with hematoxylin and eosin, Griffonia simplicifolia isolectin B4, or immunohistochemically stained for CD3 or albumin. The retinal sections were evaluated with light microscopy for morphological and immunohistochemical evidence of pigmentary changes and inflammation. RESULTS: Abnormal pigmented cells including displaced RPE cells and macrophages (identified by lectin binding) were found in the neuroretinas and vitreous bodies of glaucomatous eyes. Other abnormalities included hypertrophy of RPE cells and loss of RPE continuity. Regions of neuroretina with more displaced pigment had fewer remaining neurons. Signs of retinal inflammation found in glaucomatous eyes included infiltration with leukocytes, retinal swelling, and albumin leakage from vessels. Accumulation of perivascular CD3-positive T lymphocytes also occurred in glaucomatous retinas. Chronic glaucomatous retinas had increased pigmentary changes, fewer neutrophils, and less swelling than acute glaucomatous retinas. CONCLUSIONS: Disruption of the RPE, increased permeability of the vascular endothelium, accumulation of inflammatory cells, and retinal swelling or thinning occur in canine primary glaucoma. The displacement of pigment and accumulation of inflammatory cells in the neuroretina suggests that inflammation may be an important contributor to retinal damage.

Manganese-induced neurotoxicity: the role of astroglial-derived nitric oxide in striatal interneuron degeneration.

Chronic exposure to excessive manganese (Mn) is the cause of a neurodegenerative movement disorder, termed manganism, resulting from degeneration of neurons within the basal ganglia. Pathogenic mechanisms underlying this disorder are not fully understood but involve inflammatory activation of glial cells within the basal ganglia. It was postulated in the present studies that reactive astrocytes are involved in neuronal injury from exposure to Mn through increased release of nitric oxide. C57Bl/6 mice subchronically exposed to Mn by intragastric gavage had increased levels of Mn in the striatum and displayed diminutions in both locomotor activity and striatal DA content. Mn exposure resulted in neuronal injury in the striatum and globus pallidus, particularly in regions proximal to the microvasculature, indicated by histochemical staining with fluorojade and cresyl fast violet. Neuropathological assessment revealed marked perivascular edema, with hypertrophic endothelial cells and diffusion of serum albumin into the perivascular space. Immunofluorescence studies employing terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (DUTP)-biotin nick-end labeling revealed the presence of apoptotic neurons expressing neuronal nitric oxide synthase (NOS), choline acetyltransferase, and enkephalin in both the striatum and globus pallidus. In contrast, soma and terminals of dopaminergic neurons were morphologically unaltered in either the substantia nigra or striatum, as indicated by immunohistochemical staining for tyrosine hydroxylase. Regions with evident neuronal injury also displayed increased numbers of reactive astrocytes that coexpressed inducible NOS2 and localized with areas of increased neuronal staining for 3-nitrotyrosine protein adducts, a marker of NO formation. These data suggest a role for astrocyte-derived NO in injury to striatal-pallidal interneurons from Mn intoxication.

Assessment of glutamate loss from the ganglion cell layer of young DBA/2J mice with glaucoma.

OBJECTIVE: To determine whether glutamate contents are decreased in the ganglion cell layer (GCL) of retinas of DBA/2J mice with glaucoma, compared with unaffected control mice. SAMPLE POPULATION: 20 eyes from DBA/2J mice (9-week-old mice [n = 8] and 4- [4], 6- [4], and 12-month-old [4] mice) and 17 eyes from control CD-1 (7) and C57/BL6 (10) mice of similar age. PROCEDURE: After euthanasia, the eyes were rapidly dissected and fixed. Serial 0.5-microm sections were prepared from eyecups and stained with toluidine blue (to identify damaged cells) or immunogold (to localize glutamate). Microscopic images were captured digitally for comparison; immunostaining densities were assessed via special software. RESULTS: In the GCL of control mice, few cells appeared damaged; large amounts of glutamate were detected in 83 +/- 8.3% of cells. In DBA/2J mice > or = 9 weeks of age, damaged neurons were observed in retinal sections; the level of glutamate immunoreactivity was high in a few cells near areas of damage (13 +/- 3.2%) and in many cells in less-damaged regions of the same sections (82 +/- 4.2%). Many neurons with low amounts of glutamate in damaged regions did not appear damaged histologically. CONCLUSIONS AND CLINICAL RELEVANCE: In retinas of young DBA/2J mice, damaged and undamaged GCL cells had decreased levels of immunostaining for glutamate, compared with less-damaged adjacent regions or retinas from control mice. The loss of neuronal glutamate in damaged retinal regions suggests that glutamate is contributing to early retinal damage prior to changes in intraocular pressure.

Depletion of taurine and glutamate from damaged photoreceptors in the retinas of dogs with primary glaucoma.

OBJECTIVE: To determine whether taurine and glutamate contents are reduced in damaged photoreceptors in dogs with primary glaucoma (PG) in a manner consistent with an ischemia-like release of both of these amino acids from damaged cells. SAMPLE POPULATION: Retinas from 6 dogs with PG and 3 control dogs. PROCEDURE: Serial, semithin sections of each canine retina were stained with toluidine blue to identify damaged photoreceptors or via immunogold techniques to quantify taurine and glutamate content in retinal cells. RESULTS: Regions with a thin outer nuclear layer and pathologic nuclear changes in photoreceptors were evident in retinas of dogs with PG. The density of immunostaining for taurine in damaged photoreceptors was significantly reduced to (mean +/- SEM) 37.5 +/- 2.6% of the density in adjacent undamaged photoreceptors. Photoreceptors with decreased taurine immunostaining also had decreased glutamate immunostaining, consistent with ischemia-like release of both of these amino acids from damaged cells. Immunostaining for glutamate, but not taurine, was increased in presumptive radial glial cells (i.e., Miller cells) in damaged regions, consistent with an ischemia-induced redistribution of amino acids in dogs with PG. CONCLUSIONS AND CLINICAL RELEVANCE: Retinal damage in dogs with PG includes ischemia-like losses of taurine and glutamate from photoreceptors and accumulation of glutamate, but not taurine, in nearby Müller cells. These changes are consistent with glutamate release and depletion of intracellular taurine in damaged regions, perhaps contributing to progressive damage in these areas.

Evaluation of glutamate loss from damaged retinal cells of dogs with primary glaucoma.

OBJECTIVE: To determine whether retinal damage in dogs with primary glaucoma (PG) is consistent with ischemia-induced glutamate toxicosis. SAMPLE POPULATION: Retinal tissue sections from 25 dogs with PG and 12 normotensive control dogs. PROCEDURE: Retinal sections from control and glaucomatous dogs were stained for morphometric and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analyses to determine whether retinal damage was consistent with glutamate toxicosis. Immunohistochemical analysis was performed to detect ischemia-like loss of glutamate from neurons in damaged areas. RESULTS: In severely damaged glaucomatous retinas, all neurosensory layers had focal regions that were thin or disrupted. There was less thinning of the outer nuclear layer (ONL) and inner nuclear layer (INL) in moderately damaged retinas than in severely damaged retinas. Acute signs of damage in the INL included cells with dark, condensed chromatin and lightly stained cytoplasm interspersed with a few TUNEL-positive cells, which was consistent with glutamate toxicosis. Glutamate immunoreactivity was reduced in thin areas and in damaged cells of the INL and ONL, which was consistent with glutamate release in damaged areas. Glutamate immunoreactivity was increased in putative Müller cells in damaged areas, which also was consistent with glutamate release. CONCLUSIONS AND CLINICAL RELEVANCE: Retinal damage in dogs with PG differs in intensity in focal areas. Damage in affected regions resembles damage induced by glutamate. Glutamate is lost from damaged neurons and accumulates in Müller cells, which is consistent with increased glutamate release contributing to the damage. Glutamate antagonists may protect INL cells in dogs with glaucoma.