Sleep Disorder Kleine-Levin Syndrome (KLS) Joins the List of Polygenic Brain Disorders Associated with Obstetric Complications.

Kleine-Levin Syndrome is a rare neurological disorder with onset typically during adolescence that is characterized by recurrent episodes of hypersomnia, behavioral changes, and cognitive abnormalities, in the absence of structural changes in neuroimaging. As for many functional brain disorders, the exact disease mechanism in Kleine-Levin Syndrome is presently unknown, preventing the development of specific treatment approaches or protective measures. Here we review the pathophysiology and genetics of this functional brain disorder and then present a specific working hypothesis. A neurodevelopmental mechanism has been suspected based on associations with obstetric complications. Recent studies have focused on genetic factors whereby the first genome-wide association study (GWAS) in Kleine-Levin Syndrome has defined a linkage at the TRANK1 locus. A Gene x Environment interaction model involving obstetric complications was proposed based on concepts developed for other functional brain disorders. To stimulate future research, we here performed annotations of the genes under consideration for Kleine-Levin Syndrome in relation to factors expected to be associated with obstetric complications. Annotations used data-mining of gene/protein lists related to for hypoxia, ischemia, and vascular factors and targeted literature searches. Tentative links for TRANK1, four additional genes in the TRANK1 locus, and LMOD3-LMO2 are described. Protein interaction data for TRANK1 indicate links to CBX2, CBX4, and KDM3A, that in turn can be tied to hypoxia. Taken together, the neurological sleep disorder, Kleine-Levin Syndrome, shows genetic and mechanistic overlap with well analyzed brain disorders such as schizophrenia, autism spectrum disorder and ADHD in which polygenic predisposition interacts with external events during brain development, including obstetric complications.

New aspects for the brain in Hartnup disease based on mining of high-resolution cellular mRNA expression data for SLC6A19.

Hartnup disease is an autosomal recessive, metabolic disorder caused by mutations of the neutral amino acid transporter, SLC6A19/B0AT1. Reduced absorption in the intestine and kidney results in deficiencies in neutral amino acids and their down-stream metabolites, including niacin, associated with skin lesions and neurological symptoms. The effects on the nervous system such as ataxia have been related to systemic deficiencies of tryptophan (and other neutral amino acids) as no expression of the B0AT1 transporter was found in the brain. In the intestine, SLC6A19 cooperates with ACE2 which has received major attention as the cellular receptor for SARS-CoV-2. When transcriptomics data for ACE2 and its partner proteins were examined, a previously unrecognized expression of Slc6a19 mRNA in the ependymal cells of the mouse brain was encountered that is set into the context of neurological manifestations of Hartnup disease with this communication. A novel role for SLC6A19/B0AT1 in amino acid transport from CSF into ependymal cells is proposed and a role of niacin in ependymal cells highlighted.

Analysis of GWAS-Derived Schizophrenia Genes for Links to Ischemia-Hypoxia Response of the Brain.

Obstetric complications (OCs) can induce major adverse conditions for early brain development and predispose to mental disorders, including schizophrenia (SCZ). We previously hypothesized that SCZ candidate genes respond to ischemia-hypoxia as part of OCs which impacts neurodevelopment. We here tested for an overlap between SCZ genes from genome-wide association study (GWAS) (n=458 genes from 145 loci of the most recent GWAS dataset in SCZ) and gene sets for ischemia-hypoxia response. Subsets of SCZ genes were related to (a) mutation-intolerant genes (LoF database), (b) role in monogenic disorders of the nervous system (OMIM, manual annotations), and (c) synaptic function (SynGO). Ischemia-hypoxia response genes of the brain (IHR genes, n=1,629), a gene set from RNAseq in focal brain ischemia (BH, n=2,449) and genes from HypoxiaDB (HDB, n=2,289) were overlapped with the subset of SCZ genes and tested for enrichment with Chi-square tests (p < 0.017). The SCZ GWAS dataset was enriched for LoF (n=112; p=0.0001), and the LoF subset was enriched for IHR genes (n=25; p=0.0002), BH genes (n=35; p=0.0001), and HDB genes (n=23; p=0.0005). N=96 genes of the SCZ GWAS dataset (21%) could be linked to a monogenic disorder of the nervous system whereby IHR genes (n=19, p=0.008) and BH genes (n=23; p=0.002) were found enriched. N=46 synaptic genes were found in the SCZ GWAS gene set (p=0.0095) whereby enrichments for IHR genes (n=20; p=0.0001) and BH genes (n=13; p=0.0064) were found. In parallel, detailed annotations of SCZ genes for a role of the hypoxia-inducible factors (HIFs) identified n=33 genes of high interest. Genes from SCZ GWAS were enriched for mutation-intolerant genes which in turn were strongly enriched for three sets of genes for the ischemia-hypoxia response that may be invoked by OCs. A subset of one fifth of SCZ genes has established roles in monogenic disorders of the nervous system which was enriched for two gene sets related to ischemia-hypoxia. SCZ genes related to synaptic functions were also related to ischemia-hypoxia. Variants of SCZ genes interacting with ischemia-hypoxia provide a specific starting point for functional and genomic studies related to OCs.

Methionine sulfoxide reductase (Msr) dysfunction in human brain disease.

Extensive research has shown that oxidative stress is strongly associated with aging, senescence and several diseases, including neurodegenerative and psychiatric disorders. Oxidative stress is caused by the overproduction of reactive oxygen species (ROS) that can be counteracted by both enzymatic and nonenzymatic antioxidants. One of these antioxidant mechanisms is the widely studied methionine sulfoxide reductase system (Msr). Methionine is one of the most easily oxidized amino acids and Msr can reverse this oxidation and restore protein function, with MsrA and MsrB reducing different stereoisomers. This article focuses on experimental and genetic research performed on Msr and its link to brain diseases. Studies on several model systems as well as genome-wide association studies are compiled to highlight the role of MSRA in schizophrenia, Alzheimer's disease, and Parkinson's disease. Genetic variation of MSRA may also contribute to the risk of psychosis, personality traits, and metabolic factors.

Involvement of hemoglobins in the pathophysiology of Alzheimer's disease.

Hemoglobins (Hbs) are heme-containing proteins binding oxygen, carbon monoxide, and nitric oxide. While erythrocytes are the most well-known location of Hbs, Hbs also exist in neurons, glia and oligodendroglia and they are primarily localized in the inner mitochondrial membrane of neurons with likely roles in cellular respiration and buffering protons. Recently, studies have suggested links between hypoxia and neurodegenerative disorders such as Alzheimer Disease (AD) and furthermore suggested involvement of Hbs in the pathogenesis of AD. While cellular immunohistochemical studies on AD brains have observed reduced levels of Hb in the cytoplasm of pre-tangle and tangle-bearing neurons, other studies on homogenates of AD brain samples observed increased Hb levels. This potential discrepancy may result from differential presence and function of intracellular versus extracellular Hbs. Intracellular Hbs may protect neurons against hypoxia and hyperoxia. On the other hand, extracellular free Hb and its degradation products may trigger inflammatory immune and oxidative reactions against neural macromolecules and/or damage the blood-brain barrier. Therefore, biological processes leading to reduction of Hb transcription (including clinically silent Hb mutations) may influence intra-erythrocytic and neural Hbs, and reduce the transport of oxygen, carbon monoxide and nitric oxide which may be involved in the (patho)physiology of neurodegenerative disorders such as AD. Agents such as erythropoietin, which stimulate both erythropoiesis, reduce eryptosis and induce intracellular neural Hbs may exert multiple beneficial effects on the onset and course of AD. Thus, evidence accumulates for a role of Hbs in the central nervous system while Hbs deserve more attention as possible candidate molecules involved in AD.

Pre- and Perinatal Ischemia-Hypoxia, the Ischemia-Hypoxia Response Pathway, and ADHD Risk.

This review focuses on how measured pre- and perinatal environmental and (epi)genetic risk factors are interrelated and potentially influence one, of many, common developmental pathway towards ADHD. Consistent with the Developmental Origins of Health and Disease hypothesis, lower birth weight is associated with increased ADHD risk. Prenatal ischemia-hypoxia (insufficient blood and oxygen supply in utero) is a primary pathway to lower birth weight and produces neurodevelopmental risk for ADHD. To promote tissue survival in the context of ischemia-hypoxia, ischemia-hypoxia response (IHR) pathway gene expression is altered in the developing brain and peripheral tissues. Although altered IHR gene expression is adaptive in the context of ischemia-hypoxia, lasting IHR epigenetic modifications may lead to increased ADHD risk. Taken together, IHR genetic vulnerability to ischemia-hypoxia and IHR epigenetic alterations following prenatal ischemia-hypoxia may result in neurodevelopmental vulnerability for ADHD. Limitations of the extant literature and future directions for genetically-informed research are discussed.

Genomic approach to selective vulnerability of the hippocampus in brain ischemia-hypoxia.

Transient global ischemia selectively damages neurons in specific brain areas. A reproducible pattern of selective vulnerability is observed in the dorsal hippocampus of rodents where ischemic damage typically affects neurons in the CA1 area while sparing neurons in CA3 and granule cells. The "neuronal factors" underlying the differential vulnerability of CA1 versus CA3 have been of great interest. This review first provides on overview of the histological pattern of ischemic-hypoxic damage, the phenomenon of delayed neuronal death, the necrosis-apoptosis discussion, and multiple molecular mechanisms studied in the hippocampus. Subsequently, genomic studies of basal gene expression in CA1 and CA3 are summarized and changes in gene expression in response to global brain ischemia are surveyed. A formal analysis is presented for the overlap between genes expressed under basal conditions in the hippocampus and genes responding to ischemia-hypoxia in general. A possible role of the elusive vascular factors in selective vulnerability is reviewed, and a gene set for angiogenesis is then shown to be enriched in the CA3 gene set. A survey of selective vulnerability in the human hippocampus in relation to genomic studies in ischemia-hypoxia is presented, and neurodegeneration genes with high expression in CA1 are highlighted (e.g. WFS1). It is concluded that neuronal factors dominate the selective vulnerability of CA1 but that vascular factors also deserve more systematic studies.

S100b counteracts neurodegeneration of rat cholinergic neurons in brain slices after oxygen-glucose deprivation.

Alzheimer's disease is a severe chronic neurodegenerative disorder characterized by beta-amyloid plaques, tau pathology, cerebrovascular damage, inflammation, reactive gliosis, and cell death of cholinergic neurons. The aim of the present study is to test whether the glia-derived molecule S100b can counteract neurodegeneration of cholinergic neurons after oxygen-glucose deprivation (OGD) in organotypic brain slices of basal nucleus of Meynert. Our data showed that 3 days of OGD induced a marked decrease of cholinergic neurons (60% of control), which could be counteracted by 50 mug/mL recombinant S100b. The effect was dose and time dependent. Application of nerve growth factor or fibroblast growth factor-2 was less protective. C-fos-like immunoreactivity was enhanced 3 hours after OGD indicating metabolic stress. We conclude that S100b is a potent neuroprotective factor for cholinergic neurons during ischemic events.

Expression of the diabetes risk gene wolframin (WFS1) in the human retina.

Wolfram syndrome 1 (WFS1, OMIM 222300), a rare genetic disorder characterized by optic nerve atrophy, deafness, diabetes insipidus and diabetes mellitus, is caused by mutations of WFS1, encoding WFS1/wolframin. Non-syndromic WFS1 variants are associated with the risk of diabetes mellitus due to altered function of wolframin in pancreatic islet cells, expanding the importance of wolframin. This study extends a previous report for the monkey retina, using immunohistochemistry to localize wolframin on cryostat and paraffin sections of human retina. In addition, the human retinal pigment epithelial (RPE) cell line termed ARPE-19 and retinas from both pigmented and albino mice were studied to assess wolframin localization. In the human retina, wolframin was expressed in retinal ganglion cells, optic axons and the proximal optic nerve. Wolframin expression in the human retinal pigment epithelium (RPE) was confirmed with intense cytoplasmic labeling in ARPE-19 cells. Strong labeling of the RPE was also found in the albino mouse retina. Cryostat sections of the mouse retina showed a more extended pattern of wolframin labeling, including the inner nuclear layer (INL) and photoreceptor inner segments, confirming the recent report of Kawano et al. [Kawano, J., Tanizawa, Y., Shinoda, K., 2008. Wolfram syndrome 1 (Wfs1) gene expression in the normal mouse visual system. J. Comp. Neurol. 510, 1-23]. Absence of these cells in the human specimens despite the use of human-specific antibodies to wolframin may be related to delayed fixation. Loss of wolframin function in RGCs and the unmyelinated portion of retinal axons could explain optic nerve atrophy in Wolfram Syndrome 1.

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain.

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.

Microvessel length density, total length, and length per neuron in five subcortical regions in schizophrenia.

Recent studies (Prabakaran et al. in Mol Psychiat 9:684-697, 2004; Hanson and Gottesman in BMC Med Genet 6:7, 2005; Harris et al. in PLoS ONE 3:e3964, 2008) have suggested that microvascular abnormalities occur in the brains of patients with schizophrenia. To assess the integrity of the microvasculature in subcortical brain regions in schizophrenia, we investigated the microvessel length density, total microvessel length, and microvessel length per neuron using design-based stereologic methods in the caudate nucleus, putamen, nucleus accumbens, mediodorsal nucleus of the thalamus, and lateral nucleus of the amygdala in both hemispheres of 13 postmortem brains from male patients with schizophrenia and 13 age-matched male controls. A general linear model multivariate analysis of variance with diagnosis and hemisphere as fixed factors and illness duration (patients with schizophrenia) or age (controls), postmortem interval and fixation time as covariates showed no statistically significant differences in the brains from the patients with schizophrenia compared to the controls. These data extend our earlier findings in prefrontal cortex area 9 and anterior cingulate cortex area 24 from the same brains (Kreczmanski et al. in Acta Neuropathol 109:510-518, 2005), that alterations in microvessel length density, total length, and particularly length per neuron cannot be considered characteristic features of schizophrenia. As such, compromised brain metabolism and occurrence of oxidative stress in the brains of patients with schizophrenia are likely caused by other mechanisms such as functional disruption in the coupling of cerebral blood flow to neuronal metabolic needs.

Hypoxia-regulated components of the U4/U6.U5 tri-small nuclear riboprotein complex: possible role in autosomal dominant retinitis pigmentosa.

PURPOSE: High oxygen consumption and cyclical changes related to dark-adaptation are characteristic of the outer retina. Oxygenation changes may contribute to the selective vulnerability of the retina in retinitis pigmentosa (RP) patients, especially for those forms involving genes with global cellular functions. Genes coding for components of the U4/U6.U5 tri small nuclear ribonucleoprotein (tri-snRNP) complex of the spliceosome stand out, because mutations in four genes cause RP, i.e., RP9 (PAP1), RP11 (PRPF31), RP13 (PRPF8), and RP18 (PRPF3), while there is no degeneration outside the retina despite global expression of these genes. With the assumption that variable oxygenation plays a role in RP forms related to pre-mRNA splicing and the retina and brain are similar, we searched a data collection of ischemia-hypoxia regulated genes of the brain for oxygen regulated genes of the U4/U6.U5 tri-snRNP complex. METHODS: A database of ischemia-hypoxia response (IHR) genes in the brain was generated from gene expression profiling studies [n=24]. Public databases (NCBI) were searched for RP genes with global function that are expressed in the brain. From the IHR gene list, we extracted genes that were directly related to retinal degeneration through a listed mutation (OMIM, Retnet, RISN). The database was then examined for indirect links to RP forms affecting the U4/U6.U5 tri-snRNP complex by searching for IHR genes contributing to this complex. Potential expression of matched genes in the retina was ascertained using NEIBank. Immunohistochemistry was used to localize a selected protein of the U4/U6.U5 tri-snRNP complex in cynomolgus monkey and human retina specimens. RESULTS: The approach identified genes that cause retinal degeneration (CNGB1, SEMA4A, RRG4) or developmental changes (SOX2) when mutated. One IHR gene, Pim1, is the immediate binding partner for PAP1 (RP9). Three IHR genes linked the U4/U6.U5 tri-snRNP complex to regulation by oxygenation: PRPF4; SART1, also known as 110 kDa SR-related protein of the U4/U6.U5 tri-snRNP or as hypoxia associated factor (HAF); and LSM8, U6 snRNA-associated Sm-like protein. The 110 kDa SR-related protein was localized in all retinal cells including photoreceptors. CONCLUSIONS: Regulation by changes in oxygenation within the U4/U6.U5 tri-snRP complex could be particularly important for photoreceptors where oxygen consumption follows a circadian rhythm. If the U4/U6.U5 tri-snRP complex is already impaired by mutations in any of the four genes causing RP, it may be unable to follow properly the physiological demands of oxygenation which are mediated by the four hypoxia-regulated proteins emerging in this study. Selective vulnerability may involve complex combinations of widely expressed genes, specific cellular functions and local energy availability.

Volume, neuron density and total neuron number in five subcortical regions in schizophrenia.

Several studies have pointed to alterations in mean volumes, neuron densities and total neuron numbers in the caudate nucleus (CN), putamen, nucleus accumbens (NA), mediodorsal nucleus of the thalamus (MDNT) and lateral nucleus of the amygdala (LNA) in schizophrenia. However, the results of these studies are conflicting and no clear pattern of alterations has yet been established in these subcortical regions, possibly due to differences in quantitative histological methods used as well as differences in the investigated case series. The present study investigates these subcortical regions in both hemispheres of the same post-mortem brains for volume, neuron density and total neuron number with high-precision design-based stereology. The analysed case series consisted of 13 post-mortem brains from male schizophrenic patients [age range: 22-64 years; mean age 51.5 +/- 3.3 years (mean +/- SEM)] and 13 age-matched male controls (age range: 25-65 years; mean age 51.9 +/- 3.1 years). A general linear model multivariate analysis of variance with diagnosis and hemisphere as fixed factors and illness duration (schizophrenic patients) or age (controls), post-mortem interval and fixation time as covariates showed a number of statistically significant alterations in the brains from schizophrenic patients compared with the controls. There was a reduced mean volume of the putamen [-5.0% on the left side (l) and -4.1% on the right side (r)] and the LNA (l: -12.1%, r: -17.6%), and a reduced mean total neuron number in the CN (l: -10.4%, r: -10.2%), putamen (l: -8.1%, r: -11.6%) and the LNA (l: -15.9%, r: -16.2%). These data show a previously unreported, distinct pattern of alterations in mean total neuron numbers in identified subcortical brain regions in a carefully selected sample of brains from schizophrenic patients. The rigorous quantitative analysis of several regions in brains from schizophrenic patients and matched controls is crucial to provide reliable information on the neuropathology of schizophrenia as well as insights about its pathogenesis.

Moderate loss of cerebellar Purkinje cells after chronic bilateral common carotid artery occlusion in rats.

Pathological effects of moderate ischemia (oligemia, hypoperfusion) are relevant in relation to vascular factors in dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult Wistar rats induces oligemia and leads to acute changes in gene expression, subacute changes in cortical astrocytes and prolonged changes in white matter tracts, while largely sparing neurons in the forebrain areas. Dilation and remodeling of the basilar artery ensures blood flow to the forebrain. The present study examined the hypoxia-sensitive Purkinje cells in the cerebellum after 6 months of BCCAO using conventional neuropathological analysis, immunohistochemistry and high-precision design-based stereologic methods. Purkinje cells in the vermis region revealed abnormally shaped nuclei. A stereologic analysis showed that the mean total number of Purkinje cells within the vermis was statistically significantly smaller in the BCCAO animals than in the control animals (d = 11.8%; P < 0.0001). BCCAO had no significant effect on the mean volumes of the molecular layer, granule cell layer and white matter in the vermis or the entire cerebellum. Remodeling of the basilar artery indicated that secondary vascular perturbations might be responsible for the effects of BCCAO on the cerebellar Purkinje cells.

Wolfram syndrome 1 (WFS1) protein expression in retinal ganglion cells and optic nerve glia of the cynomolgus monkey.

Wolfram syndrome (WFS1, OMIM 222300) is a rare genetic disorder associated with multiple organ abnormalities, most prominently optic nerve atrophy and diabetes. Mutations in the WFS1 gene coding for wolframin have been identified. The pathogenesis for optic nerve atrophy remains elusive. We here tested the hypothesis that wolframin is expressed in glial cells of the optic nerve and in retinal ganglion cells in the cynomolgus monkey. Paraffin sections through the retina and optic nerve were examined with immunohistochemistry using affinity-purified antibodies to wolframin. Retinal ganglion cells and optic nerve glial cells were found to be strongly labeled. Dual dysfunction of wolframin in optic nerve glial cells and retinal ganglion cells may explain the progressive optic nerve atrophy in Wolfram syndrome.

Neuroglobin mRNA expression after transient global brain ischemia and prolonged hypoxia in cell culture.

Neuroglobin is a nerve-specific respiratory protein that has been proposed to play an important role in the protection of brain neurons from ischemic and hypoxic injuries. Here, we investigated the regulation of neuroglobin expression after transient global ischemia in the rat brain using mRNA in situ hybridization and under hypoxic stress in cultured neuronal cell lines (PC12, HN33) by quantitative RT-PCR. While neuroglobin mRNA expression was significantly enhanced in cell culture after severe prolonged hypoxia (0-1% O2 for 24 h), we did not find any significant increases in neuroglobin mRNA levels in the rat brain after transient global ischemia. Vegf and Glut1 mRNAs showed increases in the hippocampus as expected. Therefore, it is unlikely that neuroglobin is instrumental in the acute response of neurons to hypoxic or ischemic insults, for which the mammalian brain is not adapted.

Gene regulation by hypoxia and the neurodevelopmental origin of schizophrenia.

Neurodevelopmental changes may underlie the brain dysfunction seen in schizophrenia. While advances have been made in our understanding of the genetics of schizophrenia, little is known about how non-genetic factors interact with genes for schizophrenia. The present analysis of genes potentially associated with schizophrenia is based on the observation that hypoxia prevails in the embryonic and fetal brain, and that interactions between neuronal genes, molecular regulators of hypoxia, such as hypoxia-inducible factor 1 (HIF-1), and intrinsic hypoxia occur in the developing brain and may create the conditions for complex changes in neurodevelopment. Consequently, we searched the literature for currently hypothesized candidate genes for susceptibility to schizophrenia that may be subject to ischemia-hypoxia regulation and/or associated with vascular expression. Genes were considered when at least two independent reports of a significant association with schizophrenia had appeared in the literature. The analysis showed that more than 50% of these genes, particularly AKT1, BDNF, CAPON, CCKAR, CHRNA7, CNR1, COMT, DNTBP1, GAD1, GRM3, IL10, MLC1, NOTCH4, NRG1, NR4A2/NURR1, PRODH, RELN, RGS4, RTN4/NOGO and TNF, are subject to regulation by hypoxia and/or are expressed in the vasculature. Future studies of genes proposed as candidates for susceptibility to schizophrenia should include their possible regulation by physiological or pathological hypoxia during development as well as their potential role in cerebral vascular function.

Complex neurodegeneration in retina following moderate ischemia induced by bilateral common carotid artery occlusion in Wistar rats.

Bilateral common carotid artery occlusion (BCCAO) produces moderate levels of ischemia in the retina of rats, which may simulate the inflow disturbances in severe carotid artery disease. ERG changes following acute BCCAO have been well described, but the effects of chronic BCCAO on the histopathology of the retina remain to be characterized in a reproducible model. Chronic BCCAO was induced in halothane-anaesthetized male Wistar rats and the retina fixed after 3, 6, or 24 hr, 1 week, and 2, 4, or 6 months. Cell counts and measurements of retinal layers were performed in H&E stained paraffin sections. Immunohistochemistry with a panel of fourteen antibodies served to examine the survival of different retinal cell class, astrocytic reactions and the expression of acute stress response proteins. A lectin method was used to label activated microglial cells. Microglial activation, heme oxygenase-1 upregulation and caspase-3 cleavage occurred during the first 24hr in the absence of overt cell death of retinal ganglion cells (RGC). Three waves of neurodegeneration followed. RGCs were affected after 1 week, followed by neurons in the inner nuclear layer at 2 months, and finally photoreceptors at 4 months. Immunomarkers indicated acute damage to horizontal cells and prolonged survival of amacrine cells. In conclusion, chronic BCCAO produced delayed neuronal death in the retina of adult male Wistar rats. The window of moderate changes of at least 1 day may facilitate molecular studies on retinal ganglion cell loss.

Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats.

The effects of oligemia (moderate ischemia) on the brain need to be explored because of the potential role of subtle microvascular changes in vascular cognitive impairment and dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult rats has been used to study effects of oligemia (hypoperfusion) using neuropathological and neurochemical analysis as well as behavioral tests. In this study, BCCAO was induced for 1 week, or 2, 4, and 6 months. Sensitive immunohistochemistry with marker proteins was used to study reactions of astrocytes (GFAP, nestin), and lectin binding to study microglial cells during BCCAO. Overt neuronal loss was visualized with NeuN antibodies. Astrocytes reacted to changes in the optic tract at all time points, and strong glial reactions also occurred in the target areas of retinal fibers, indicating damage to the retina and optic nerve. Astrocytes indicated a change in the corpus callosum from early to late time points. Diffuse increases in GFAP labeling occurred in parts of the neocortex after 1 week of BCCAO, in the absence of focal changes of neuronal marker proteins. No significant differences emerged in the cortex at longer time points. Nestin labeling was elevated in the optic tract. Reactions of microglia cells were seen in the cortex after 1 week. Measurements of the basilar artery indicated a considerable hypertrophy, indicative of macrovascular compensation in the chronic occlusion model. These results indicate that chronic BCCAO and, by inference, oligemia have a transient effect on the neocortex and a long-lasting effect on white matter structures.

Stereological studies of capillary length density in the frontal cortex of schizophrenics.

The presence of microvasculature abnormalities in the prefrontal cortex of schizophrenics was proposed in a recent study of molecular signatures of schizophrenia [Prabakaran et al (2004) Mol Psychiat 9:684-697]. To assess this possibility further, we investigated capillary length densities in prefrontal cortex area 9 and anterior cingulate cortex area 24 in postmortem brains from 13 schizophrenics and 13 age- and sex-matched controls. To check that our sample of brains shared cardinal neuropathological features of schizophrenia with previously reported case studies, we also measured cortical gray matter volumes and cortical thickness in areas 9 and 24. The mean cortical gray matter volume was significantly reduced in brains from schizophrenics compared to controls. Mean cortical thickness was significantly reduced in area 24, but not in area 9, in schizophrenics. There were no differences in mean capillary length densities in either area 9 or 24 between the two groups. Thus, alterations in capillary length density in the prefrontal cortex cannot be considered a general feature of schizophrenia. Compromised brain metabolism and occurrence of oxidative stress in the brain of schizophrenics are likely caused by other mechanisms.