Ian Everall

Neuronal loss in the frontal cortex in HIV infection.

In an attempt to elucidate the cause and mechanism of the dementia and other neurological disorders that can occur in HIV-1 infection, we have quantitatively assessed neuronal populations, by means of a stereological technique (the disector), in the frontal cortex of patients with HIV infection. Eleven of sixty-five brains in the Medical Research Council Central AIDS Brain Bank were selected for study. The selected patients died without opportunistic infection or neoplasm affecting the brain; they had HIV encephalitis or minimal changes. We compared their neuronal counts with those of eight control subjects (seven died of systemic illness, one of pontine haemorrhage which did not affect the cerebral hemispheres). The neuronal numerical density was significantly lower in the HIV group than in the control group (mean [SD] 307 [46] vs 499 [113] x 10(2) per mm3; p less than 0.001). This difference represents a loss of about 38%. There was no significant difference between the HIV subgroups, which suggests that neuronal loss occurs in cases of minor pathology as well as in HIV encephalitis. This finding contributes to the understanding of dementia in AIDS patients and has important implications for their future treatment.

Glial cell abnormalities in major psychiatric disorders: the evidence and implications

Recent quantitative post-mortem investigations of the cerebral cortex have convincingly demonstrated cortical glial cell loss in subjects with major depression. Evidence is also mounting that glial cell loss may also be a feature of schizophrenia. These findings coincide with a re-evaluation of the importance of glial cells in normal cortical function. In addition to their traditional roles in neuronal migration and inflammatory processes, glia are now accepted to have roles in providing trophic support to neurons, neuronal metabolism, and the formation of synapses and neurotransmission. Consequently, reduced cortical glial cell numbers could be responsible for some of the pathological changes in schizophrenia and depression, including reduced neuronal size, reduced levels of synaptic proteins, and abnormalities of cortical neurotransmission. Additionally, as astrocytes provide the energy requirements of neurons, deficient astrocyte function could account for aspects of the functional magnetic imaging abnormalities found in these disorders. We discuss the possible basis of glial cell loss in these disorders and suggest that elevated levels of glucocorticoids, due to illness-related stress or to hyperactivity of the hypothalamic-pituitary-adrenal may down-regulate glial activity and so predispose to, or exacerbate psychiatric illness through enhanced excitotoxicity. The potential therapeutic impact of agents which up-regulate glial activity or normalise glial cell numbers is also discussed.

Microglial activation and increased microglial density observed in the dorsolateral prefrontal cortex in autism

BACKGROUND In the neurodevelopmental disorder autism, several neuroimmune abnormalities have been reported. However, it is unknown whether microglial somal volume or density are altered in the cortex and whether any alteration is associated with age or other potential covariates. METHODS Microglia in sections from the dorsolateral prefrontal cortex of nonmacrencephalic male cases with autism (n = 13) and control cases (n = 9) were visualized via ionized calcium binding adapter molecule 1 immunohistochemistry. In addition to a neuropathological assessment, microglial cell density was stereologically estimated via optical fractionator and average somal volume was quantified via isotropic nucleator. RESULTS Microglia appeared markedly activated in 5 of 13 cases with autism, including 2 of 3 under age 6, and marginally activated in an additional 4 of 13 cases. Morphological alterations included somal enlargement, process retraction and thickening, and extension of filopodia from processes. Average microglial somal volume was significantly increased in white matter (p = .013), with a trend in gray matter (p = .098). Microglial cell density was increased in gray matter (p = .002). Seizure history did not influence any activation measure. CONCLUSIONS The activation profile described represents a neuropathological alteration in a sizeable fraction of cases with autism. Given its early presence, microglial activation may play a central role in the pathogenesis of autism in a substantial proportion of patients. Alternatively, activation may represent a response of the innate neuroimmune system to synaptic, neuronal, or neuronal network disturbances, or reflect genetic and/or environmental abnormalities impacting multiple cellular populations.

Comparative gene expression analysis of blood and brain provides concurrent validation of SELENBP1 up-regulation in schizophrenia

Microarray techniques hold great promise for identifying risk factors for schizophrenia (SZ) but have not yet generated widely reproducible results due to methodological differences between studies and the high risk of type I inferential errors. Here we established a protocol for conservative analysis and interpretation of gene expression data from the dorsolateral prefrontal cortex of SZ patients using statistical and bioinformatic methods that limit false positives. We also compared brain gene expression profiles with those from peripheral blood cells of a separate sample of SZ patients to identify disease-associated genes that generalize across tissues and populations and further substantiate the use of gene expression profiling of blood for detecting valid SZ biomarkers. Implementing this systematic approach, we: (i) discovered 177 putative SZ risk genes in brain, 28 of which map to linked chromosomal loci; (ii) delineated six biological processes and 12 molecular functions that may be particularly disrupted in the illness; (iii) identified 123 putative SZ biomarkers in blood, 6 of which (BTG1, GSK3A, HLA-DRB1, HNRPA3, SELENBP1, and SFRS1) had corresponding differential expression in brain; (iv) verified the differential expression of the strongest candidate SZ biomarker (SELENBP1) in blood; and (v) demonstrated neuronal and glial expression of SELENBP1 protein in brain. The continued application of this approach in other brain regions and populations should facilitate the discovery of highly reliable and reproducible candidate risk genes and biomarkers for SZ. The identification of valid peripheral biomarkers for SZ may ultimately facilitate early identification, intervention, and prevention efforts as well.

Cortical Synaptic Density is Reduced in Mild to Moderate Human Immunodeficiency Virus Neurocognitive Disorder

Dendritic and synaptic damage (without frank neuronal loss) may be seen in milder human immunodeficiency virus (HIV)‐related cognitive disorders. Synapse volume estimates, performed by stereological methods, could enhance the ability to detect subtle neuronal changes that may accompany cognitive impairment in HIV infection. For the present study, synaptic density and neuronal number were assessed by combined stereology/confocal microscopy and these measures were then correlated with ante‐mortem levels of cognitive performance in AIDS patients. Three‐dimensional stereological measures showed a significant correlation between reduced synaptic density and poor neuropsychological performance. To evaluate the specificity of any observed associations, additional variables including viral burden, astrogliosis and number of calbindin‐immunoreactive neurons were measured. Factor analysis of a set of neuropathological variables revealed two factors; one defined by synaptic density and volume fraction, calbindin pyramidal neuronal densities and viral burden; the second by astrocytosis and calbindin interneuron density. Only the first factor correlated significantly with neuropsychological functioning during life.

Neuropathology of early HIV-1 infection

Early HIV‐1 invasion of the central nervous system has been demonstrated by many cerebrospinal fluid studies; however, most HIV‐1 carriers remain neurologically unimpaired during the so called “asymptomatic” period lasting from seroconversion to symptomatic AIDS. Therefore, neuropathological studies in the early pre‐AIDS stages are very few, and the natural history of central nervous system changes in HIV‐1 infection remains poorly understood. Examination of brains of asymptomatic HIV‐1 positive individuals who died accidentally and of rare cases with acute fatal encephalopathy revealing HIV infection, and comparison with experimental simian immunodeficiency virus and feline immunodeficiency virus infections suggest that, invasion of the CNS by HIV‐1 occurs at the time of primary infection and induces an immunological process in the central nervous system. This includes an inflammatory T‐cell reaction with vasculitis and leptomeningitis, and immune activation of brain parenchyma with increased number of microglial cells, upregulation of major histocompatibility complex class II antigens and local production of cytokines. Myelin pallor and gliosis of the white matter are usually found and are likely to be the consequence of opening of the blood brain barrier due to vasculitis; direct damage to oligodendrocytes by cytokines may also interfere. These white matter changes may explain, at least partly, the early cerebral atrophy observed, by magnetic resonance imaging, in asymptomatic HIV‐1 carriers. In contrast, cortical damage seems to be a late event in the course of HIV‐1 infection. There is no significant neuronal loss at the early stages of the disease, no accompanying increase in glial fibrillary acid protein staining in the cortex, and only exceptional neuronal apoptosis. Although HIV‐1 proviral DNA may be demonstrated in a number of brains, viral replication remains very low during the asymptomatic stage of HIV‐1 infection. This makes it likely that, although opening of the blood brain barrier may facilitate viral entry into the brain, specific immune responses including both neutralising antibodies and cytotoxic T‐lymphocytes, continuously inhibits viral replication at that stage.

Two-dimensional assessment of cytoarchitecture in the anterior cingulate cortex in major depressive disorder, bipolar disorder, and schizophrenia: evidence for decreased neuronal somal size and increased neuronal density

BACKGROUND Abnormalities of cortical neuronal organization and reductions in neuronal somal size have been reported in schizophrenia. The purpose of this investigation was to assess patterns of neuronal and glial distribution in the anterior cingulate cortex (ACC) in major depressive disorder (MDD), schizophrenia, bipolar disorder (BPD), and normal control subjects (15 subjects per group). METHODS Estimates for neuronal somal and glial nuclear size and density were obtained. We employed two-dimensional morphometric analysis to examine the location of neurons and glia in a 1000-microm-wide strip of cortex. RESULTS A decreased clustering of neurons was seen in BPD (p =.001). No other group differences were observed in the clustering of neurons, glia, or of neurons about glia. Neuronal somal size was reduced in layer 5 in schizophrenia (18%, p =.001), BPD (16%, p <.001), and MDD (9%, p =.01). Neuronal density was increased in layer 6 in BPD (63%, p =.004) and schizophrenia (61%, p =.006) and in layer 5 in MDD (24%, p =.018) and schizophrenia (33%, p =.003). CONCLUSIONS The results of this study indicate that reduced neuronal somal size and increased neuronal density in cortical layers 5 and 6 of the ACC may be key features of schizophrenia, MDD, and BPD.

The density and spatial distribution of GABAergic neurons, labelled using calcium binding proteins, in the anterior cingulate cortex in major depressive disorder, bipolar disorder, and schizophrenia.

BACKGROUND There is strong evidence for the presence of a deficit in cortical gamma aminobutyric acid (GABA) neurotransmission in schizophrenia. In this investigation we have used the calcium binding proteins (CBPs) parvalbumin (PV), calretinin (CR), and calbindin-D28K (CB) as markers of these neuronal populations, and have characterized their pattern and density in schizophrenia, bipolar disorder (BPD), and major depressive disorder (MDD). METHODS We examined the anterior cingulate cortex (ACC) in four groups of 15 brains each from subjects with schizophrenia, MDD, and BPD, and from control subjects. Using immunocytochemistry to identify these distinct neuronal populations, we quantified their areal density and spatial pattern organization. RESULTS There were reductions in the density of CB-labeled neurons in layer 2 in schizophrenia (34%; p =.04) and BPD (33%; p =.05) compared with control subjects; however, after correction for multiple comparisons these findings no longer attained formal statistical significance. We observed no differences in the density of the neuronal populations labeled by CR or PV in any layer of the cortex in any disorder compared with control subjects. There was increased clustering among PV-labeled neurons in BPD compared with control subjects but no significant differences in the spatial organization of the other neuronal subpopulations in any disorder. CONCLUSIONS The study provides some support for the presence of a deficit in GABAergic neurons in schizophrenia and shows that these changes are not specific to schizophrenia. The findings indicate that there may be a pathophysiological condition, shared by subjects with schizophrenia and BPD, which operates to selectively reduce the number or protein expression of CB-immunoreactive neurons.

Detection of the human immunodeficiency virus regulatory protein tat in CNS tissues.

Neuropathologically, human immunodeficiency virus (HIV) is associated with a range of inflammatory disorders, extensive cortical neuronal loss, and dendritic and synaptic damage. Although the mechanisms resulting in these abnormalities are still unclear, the neurotoxic effects are thought to be due in part to viral products including the tat gene product. We have previously shown that Tat when presented to neurons extracellularly interacts with neuronal cell membranes to cause neuronal excitation and toxicity in fmole amounts. To determine the role of Tat in mediating HIV encephalitis (HIVE), we detected tat mRNA and protein in tissue extracts of nine patients with HIVE and seven patients without HIVE. Despite long autopsy times and significant degradation, tat mRNA was detected in 4/9 patients with HIVE but not in any of the seven patients without dementia. Similarly, the env mRNA was also detected in 5/9 patients with HIVE but not in the patients without HIVE. However, vif mRNA was detected in both groups of patients with (5/9) or without (2/7) HIVE. Using protein extracts from the brains of the same groups of patients we were unable to detect Tat by enzyme linked immunosorbant assay (ELISA) (sensitivity of 2 ng Tat/ml of brain tissue). However, Tat could be detected immunohistochemically and in protein extracts from the brains of rhesus macaques with encephalitis due to a chimeric strain of HIV and simian immunodeficiency virus (SHIV). Our observations support the role of Tat in the neuropathogenesis of HIV and SHIV encephalitis.

A review of neuronal damage in human immunodeficiency virus infection: its assessment, possible mechanism and relationship to dementia.

Over the past decade it has been realized that HIV affects the central nervous system, and various investigations have illuminated the spectrum of neuropathology in AIDS. One major advance has been the demonstration that there is substantial neuronal loss, which appears independent of the HIV-associated inflammatory lesions. Quantitative studies on neuronal populations, while fraught with methodological difficulties, are essential to the understanding of the mechanism of this neurotoxic damage. This article will review, firstly, the modern stereological procedures available for quantitative investigations; secondly, the pattern, degree and time scale of HIV-associated neuronal loss; thirdly, other morphological evidence of neuronal damage; and finally, the pathological and clinical implications of these findings.