My pathway to a career in neuropathology.

(No abstract).

Rescue of infralimbic mGluR2 deficit restores control over drug-seeking behavior in alcohol dependence.

A key deficit in alcohol dependence is disrupted prefrontal function leading to excessive alcohol seeking, but the molecular events underlying the emergence of addictive responses remain unknown. Here we show by convergent transcriptome analysis that the pyramidal neurons of the infralimbic cortex are particularly vulnerable for the long-term effects of chronic intermittent ethanol intoxication. These neurons exhibit a pronounced deficit in metabotropic glutamate receptor subtype 2 (mGluR(2)). Also, alcohol-dependent rats do not respond to mGluR(2/3) agonist treatment with reducing extracellular glutamate levels in the nucleus accumbens. Together these data imply a loss of autoreceptor feedback control. Alcohol-dependent rats show escalation of ethanol seeking, which was abolished by restoring mGluR(2) expression in the infralimbic cortex via viral-mediated gene transfer. Human anterior cingulate cortex from alcoholic patients shows a significant reduction in mGluR(2) transcripts compared to control subjects, suggesting that mGluR(2) loss in the rodent and human corticoaccumbal neurocircuitry may be a major consequence of alcohol dependence and a key pathophysiological mechanism mediating increased propensity to relapse. Normalization of mGluR(2) function within this brain circuit may be of therapeutic value.

Neuroanatomy and neuropathology associated with Korsakoff's syndrome.

Although the neuropathology of Korsakoff's syndrome (KS) was first described well over a century ago and the characteristic brain pathology does not pose a diagnostic challenge to pathologists, there is still controversy over the neuroanatomical substrate of the distinctive memory impairment in these patients. Cohort studies of KS suggest a central role for the mammillary bodies and mediodorsal thalamus, and quantitative studies suggest additional damage to the anterior thalamus is required. Rare cases of KS caused by pathologies other than those of nutritional origin provide support for the role of the anterior thalamus and mammillary bodies. Taken together the evidence to date shows that damage to the thalamus and hypothalamus is required, in particular the anterior thalamic nucleus and the medial mammillary nucleus of the hypothalamus. As these nuclei form part of wider memory circuits, damage to the inter-connecting white matter tracts can also result in a similar deficit as direct damage to the nuclei. Although these nuclei and their connections appear to be the primary site of damage, input from other brain regions within the circuits, such as the frontal cortex and hippocampus, or more distant regions, including the cerebellum and amygdala, may have a modulatory role on memory function. Further studies to confirm the precise site(s) and extend of brain damage necessary for the memory impairment of KS are required.

Influence of liver pathology on markers of postmortem brain tissue quality.

BACKGROUND: Postmortem brain tissue provides an important resource to investigate various brain disorders, including those resulting from the effects of alcohol abuse. Unlike the traditionally recognized confounders to tissue quality (e.g., coma, hypoxia), our understanding of the effects of liver disease is incomplete. The aim of this study was to determine the effects of liver pathology, and in particular cirrhosis resulting in hepatic encephalopathy (HE), on 2 postmortem brain tissue quality markers, brain pH and RNA integrity. METHODS: We measured tissue quality markers in a cohort of alcohol abuse and control cases collected by the NSW Tissue Resource Centre. Cerebellar tissue was used to evaluate both brain pH and RNA quality (as indicated by the RNA integrity number: RIN). A histological assessment was performed on each case to exclude coexisting pathologies (e.g., cerebrovascular disease, hypoxic encephalopathy, neurodegenerative disease) and to assess the presence or absence of HE. Autopsy reports were reviewed for liver pathology and toxicology. RESULTS: Analysis revealed that cases of alcohol abuse had a lower mean (±SD) brain pH, 6.46 (±0.3) as compared with the control mean 6.64 (±0.2). The mean RIN for the alcohol abuse group was 6.97 (±1.3) and controls 7.66 (±0.5). The severity of liver pathology affected both brain pH (p < 0.0001) and RIN (p < 0.0001). The comparison between cirrhotic cases highlighted increased degradation of RNA in cases with cirrhosis resulting in HE (p = 0.0095). A similar effect was seen on brain pH (p = 0.0019). CONCLUSIONS: The results show that the presence of cirrhosis and, more so, HE reduces the pH and RIN of postmortem brain tissue.

Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation in the brain of human alcoholics.

The genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. Mechanistically, these effects may be integrated through regulation of methylation of CpG dinucleotides overlapping with single-nucleotide polymorphisms (SNPs) associated with a disorder. We addressed this hypothesis by analyzing methylation of prodynorphin (PDYN) CpG-SNPs associated with alcohol dependence, in human alcoholics. Postmortem specimens of the dorsolateral prefrontal cortex (dl-PFC) involved in cognitive control of addictive behavior were obtained from 14 alcohol-dependent and 14 control subjects. Methylation was measured by pyrosequencing after bisulfite treatment of DNA. DNA binding proteins were analyzed by electromobility shift assay. Three PDYN CpG-SNPs associated with alcoholism were found to be differently methylated in the human brain. In the dl-PFC of alcoholics, methylation levels of the C, non-risk variant of 3'-untranslated region (3'-UTR) SNP (rs2235749; C > T) were increased, and positively correlated with dynorphins. A DNA-binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in the brain. The findings suggest a causal link between alcoholism-associated PDYN 3'-UTR CpG-SNP methylation, activation of PDYN transcription and vulnerability of individuals with the C, non-risk allele(s) to develop alcohol dependence.

Clinical and pathological features of alcohol-related brain damage.

One of the sequelae of chronic alcohol abuse is malnutrition. Importantly, a deficiency in thiamine (vitamin B(1)) can result in the acute, potentially reversible neurological disorder Wernicke encephalopathy (WE). When WE is recognized, thiamine treatment can elicit a rapid clinical recovery. If WE is left untreated, however, patients can develop Korsakoff syndrome (KS), a severe neurological disorder characterized by anterograde amnesia. Alcohol-related brain damage (ARBD) describes the effects of chronic alcohol consumption on human brain structure and function in the absence of more discrete and well-characterized neurological concomitants of alcoholism such as WE and KS. Through knowledge of both the well-described changes in brain structure and function that are evident in alcohol-related disorders such as WE and KS and the clinical outcomes associated with these changes, researchers have begun to gain a better understanding of ARBD. This Review examines ARBD from the perspective of WE and KS, exploring the clinical presentations, postmortem brain pathology, in vivo MRI findings and potential molecular mechanisms associated with these conditions. An awareness of the consequences of chronic alcohol consumption on human behavior and brain structure can enable clinicians to improve detection and treatment of ARBD.

Neuroimaging findings in alcohol-related encephalopathies.

OBJECTIVE: Our aim was to review the emergent neuroimaging findings of alcohol-related CNS nontraumatic disorders. Alcohol (ethanol) promotes inflammatory processes, increases DNA damage, and creates oxidative stress. In addition, the accompanying thiamine deficiency may lead to Wernicke encephalopathy. Associated changes in serum osmolarity may lead to acute demyelination. CONCLUSION: Alcohol-related encephalopathies can be life-threatening conditions but can be prevented or treated, if recognized.

Necrotizing vacuolar myopathy presenting with recurrent myoglobinuria.

Myoglobinuria occurs in a variety of systemic and neurological disorders and can pose diagnostic challenges. We report on a 23-year-old man in whom recurrent myoglobinuria was observed due to necrotizing vacuolar myopathy confirmed on muscle biopsy. Histopathologically the intramuscular vacuoles lacked the typical findings reported in vacuolar myopathy due to disorders of glycogen and lipid metabolism. We discuss the management approach to recurrent myoglobinuria. Recurrent myoglobinuria in the absence of toxin or drug exposure and seizure is more often due to primary muscle disease. Recognizing the presence of myoglobinuria and the proximate cause is essential in preventing the development of renal dysfunction and the future recurrence of symptoms.

Selection of reference gene expression in a schizophrenia brain cohort.

OBJECTIVE: In order to conduct postmortem human brain research into the neuropatho-logical basis of schizophrenia, it is critical to establish cohorts that are well-characterized and well-matched. The aim of the present study was therefore to determine if specimen characteristics including: diagnosis, age, postmortem interval (PMI), brain acidity (pH), and/or the agonal state of the subject at death related to RNA quality, and to determine the most appropriate reference gene mRNAs. METHODS: A matched cohort was selected of 74 subjects (schizophrenia/schizoaffective disorder, n = 37; controls, n = 37). Middle frontal gyrus tissue was pulverized, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using quantitative reverse transcription-polymerase chain reaction, nine housekeeper genes were measured and a geomean calculated per case in each diagnostic group. RESULTS: The RINs were very good (mean = 7.3) and all nine housekeeper control genes were significantly correlated with RIN. Seven of nine housekeeper genes were also correlated with pH; two clinical variables, agonal state and duration of illness, did have an effect on some control mRNAs. No major impact of PMI or freezer time on housekeeper mRNAs was detected. The results show that people with schizophrenia had significantly less PPIA and SDHA mRNA and tended to have less GUSB and B2M mRNA, suggesting that these control genes may not be good candidates for normalization. CONCLUSIONS: In the present cohort <10% variability in RINs was detected and the diagnostic groups were well matched overall. The cohort was adequately powered (0.80-0.90) to detect mRNA differences (25%) due to disease. The study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected.

The effect of moderate to heavy alcohol consumption on neuropsychological performance as measured by the repeatable battery for the assessment of neuropsychological status.

BACKGROUND: Excessive alcohol use is associated with damage to the structure and function of the brain and impairment of cognition and behavior. Traditional test batteries used to assess cognitive performance in alcoholics are extensive and costly, limiting their use across various clinical and research settings. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) is a relatively new instrument that attempts to overcome some of these limitations. As yet the individual effect of moderate to heavy alcohol consumption on RBANS performance has not been examined. The primary aim of this study was to explore and quantify differences in performance between controls and drinkers on the RBANS and to examine the influence of age, gender, and alcohol use patterns on test performance. METHODS: Data from a subset of "Using Our Brains" (UoB) donors (n = 28) still actively drinking and meeting criteria for moderate to heavy alcohol use (30 to 80 g of ethanol per day) (Harper, 1988) and 28 matched controls (age, education, and premorbid Intelligence Quotient) were compared. RESULTS: Participants in the alcohol group performed below the healthy control group on the visuospatial and immediate memory index, and also on the RBANS total score p < 0.001 and showed a greater decline in RBANS scores from estimated cross-sectional premorbid levels. There was a positive association between alcohol ingestion in the preceding 12 months and the language index p < 0.03 and the semantic fluency subtest (p < 0.03). Age was negatively associated with story memory (p < 0.02), coding (p < 0.001), list recognition (p < 0.01), story recall (p < 0.03), and figure recall (p < 0.02). CONCLUSION: Our results suggest that the RBANS is able to detect and characterize differences in verbal fluency, visuospatial skills, components of declarative memory, and psychomotor speed between healthy controls and moderate to heavy active alcohol users. Executive functions, commonly affected by alcoholism and not included in the RBANS, require assessment with additional measures.

FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices of human alcoholics.

The transcription factor DeltaFosB is accumulated in the addiction circuitry, including the orbitofrontal and medial prefrontal cortices of rodents chronically exposed to ethanol or other drugs of abuse, and has been suggested to play a direct role in addiction maintenance. To address this hypothesis in the context of substance dependence in humans, we compared the immunoreactivities of FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices (OFC and DLPFC respectively) between controls and alcoholics using semiquantitative immunoblotting. In both structures, we detected three forms of FOSB, one of which was DeltaFOSB, but in neither case did their immunoreactivities differ between the groups. Our results indicate that the DeltaFOSB immunoreactivity in the human brain is very low, and that it is not accumulated in the OFC and DLPFC of human alcoholics, suggesting that it may not be directly involved in addiction maintenance, at least not in ethanol dependence.

Factors that influence decisions by families to donate brain tissue for medical research.

Whilst mainstream transplant literature provides valuable insights into the influences on families to donate organs and tissues for transplant, the relevance of these findings in relation to organ donation for research remain speculative. The present study aims to expand the research donation literature, by exploring factors that influence a family's decision to donate brain tissue to neuroscience research. The verbal responses of the senior available next-of-kin (NOK), to the question of brain donation for research, are analysed. The donation rate was high (54%) over the 5-year-period. NOK relationship to the deceased, and post mortem interval were the main factors associated with a positive donation. Parents were most likely to donate and this may result from a lifetime of decision-making on behalf of the deceased. Also, the longer the interval between death of the potential donor and the question being asked, the greater the likelihood of donation.

Expression of pronociceptin and its receptor is downregulated in the brain of human alcoholics.

Animal studies demonstrated a role of neuropeptide nociceptin (NC) and its receptor (opiate receptor like-1, OPRL1) in ethanol-induced reward; activation of the OPRL1 by natural or synthetic ligands reduced ethanol self-administration and prevented relapse to ethanol drinking. The endogenous NC may function in neuronal circuits involved in reinforcing or conditioning effects of ethanol as a "brake" to limit ethanol intake (Roberto, M., Siggins, G.R. 2006. Nociceptin/orphanin FQ presynaptically decreases GABAergic transmission and blocks the ethanol-induced increase of GABA release in central amygdala. Proc. Natl. Acad. Sci. USA 103. 9715-9720), whereas repeated ethanol intake may downregulate the endogenous NC/OPRL1 system resulting in activation of ethanol consumption. To address this hypothesis, we evaluated whether expression of the pronociceptin (PNOC) and OPRL1 genes is altered in human alcoholics. mRNAs transcribed from these genes were analyzed by quantitative RT-PCR in the prefrontal and orbitofrontal cortices, central amygdala and hippocampal dentate gyrus, structures controlling alcohol consumption. Reduction in PNOC mRNA (1.7-fold) was found in the hippocampus of alcoholics, whereas OPRL1 mRNA levels were decreased (1.4-fold) in the central amygdala. No changes in expression of these genes in other brain areas analyzed were evident. We hypothesise that chronic ethanol intake downregulates PNOC and OPRL1 gene expression in the hippocampus and amygdala, respectively. The findings may be also interpreted as inherited molecular differences between alcoholics and controls. The PNOC/OPRL1 downregulation may underlie impairment of cognitive control over alcohol seeking in alcoholics. Stimulation of the OPRL1 receptors with synthetic agonists may increase threshold for activation of ethanol-related behaviour by environmental cues, and thus may reduce cue- or stress-primed relapse to ethanol consumption.

Tryptophan is a marker of human postmortem brain tissue quality.

Postmortem human brain tissue is widely used in neuroscience research, but use of tissue originating from different brain bank centers is considered inaccurate because of possible heterogeneity in sample quality. There is thus a need for well-characterized markers to assess the quality of postmortem brain tissue. Toward this aim, we determined tryptophan (TRP) concentrations, phosphofructokinase-1 and glutamate decarboxylase activities in 119 brain tissue samples. These neurochemical parameters were tested in samples from autopsied individuals, including control and pathological cases provided by 10 different brain bank centers. Parameters were assessed for correlation with agonal state, postmortem interval, age and gender, brain region, preservation and freezing methods, storage conditions and storage time, RNA integrity, and tissue pH value. TRP concentrations were elevated significantly (p = 0.045) with increased postmortem interval; which might indicate increased protein degradation. Therefore, TRP concentration might be one useful and convenient marker for estimating the quality of human postmortem brain tissue.

The importance of brain banks for molecular neuropathological research: The New South Wales Tissue Resource Centre experience.

New developments in molecular neuropathology have evoked increased demands for postmortem human brain tissue. The New South Wales Tissue Resource Centre (TRC) at The University of Sydney has grown from a small tissue collection into one of the leading international brain banking facilities, which operates with best practice and quality control protocols. The focus of this tissue collection is on schizophrenia and allied disorders, alcohol use disorders and controls. This review highlights changes in TRC operational procedures dictated by modern neuroscience, and provides examples of applications of modern molecular techniques to study the neuropathogenesis of many different brain disorders.

Brain donation: who and why?

Understanding what influences people to donate, or not donate, body organs and tissues is very important for the future of transplant surgery and medical research (Garrick in J Clin Neurosci 13:524-528, 2006). A previous web-based motivation survey coordinated by the New South Wales Tissue Resource Centre found that most people who participated in brain donation were young, female, educated Australians, not affiliated with any particular religion, and with a higher prevalence of medical illness than the general Australian population. It discussed the main motivating factors for brain donation to be "the benefits of the research to medicine and science". This study has been replicated in a paper-based version to capture a broader cross-section of the general population, to find out who they are and what motivates them to donate. All consented and registered brain donors (n = 1,323) were sent a questionnaire via the post and recipients were given 3 months to complete the questionnaire and return it in a reply paid envelope. Results were entered into the original web-based survey and analyzed using SPSS version 10. Six hundred and fifty-eight questionnaires were returned completed, a response rate of 53%. The results show that people from all age groups are interested in brain donation. The over 65's are the largest of the groups (30.7%). The majority of the participants were female (60.6%), married (49.2%) with children (65.8%), employed (52.9%) and have a tertiary education (73.3%). They were either non-religious (48.2%) or Christian (41.6%) and were mostly Australian (65.4%). Most (81%) had pledged to donate other organs and tissues for transplantation. The most commonly cited reasons for the donation were to benefit science (27.6%), to benefit medicine (23.9%), a family illness (17.5%) and to benefit the community (16.6%). This study demonstrates that people across all age groups are interested in brain donation. Recruitment of new brain donors could target the over 65 female Australians, who are not religious or Christian and who have also donated other organs and tissues for transplant purposes. It also indicates the need to make donation for research part of the national transplant donation program.

The neuropathology of alcohol-related brain damage.

Excessive alcohol use can cause structural and functional abnormalities of the brain and this has significant health, social and economic implications for most countries in the world. Even heavy social drinkers who have no specific neurological or hepatic problems show signs of regional brain damage and cognitive dysfunction. Changes are more severe and other brain regions are damaged in patients who have additional vitamin B1 (thiamine) deficiency (Wernicke-Korsakoff syndrome). Quantitative studies and improvements in neuroimaging have contributed significantly to the documentation of these changes but mechanisms underlying the damage are not understood. A human brain bank targeting alcohol cases has been established in Sydney, Australia, and tissues can be used for structural and molecular studies and to test hypotheses developed from animal models and in vivo studies. The recognition of potentially reversible changes and preventative medical approaches are important public health issues.

Dysregulation of cell death machinery in the prefrontal cortex of human alcoholics.

In human alcoholics, the cell density is decreased in the prefrontal cortex (PFC) and other brain areas. This may be due to persistent activation of cell death pathways. To address this hypothesis, we examined the status of cell death machinery in the dorsolateral PFC in alcoholics. Protein and mRNA expression levels of several key pro- and anti-apoptotic genes were compared in post-mortem samples of 14 male human alcoholics and 14 male controls. The findings do not support the hypothesis. On the contrary, they show that several components of intrinsic apoptotic pathway are decreased in alcoholics. No differences were evident in the motor cortex, which is less damaged in alcoholics and was analysed for comparison. Thus, cell death mechanisms may be dysregulated by inhibition of intrinsic apoptotic pathway in the PFC in human alcoholics. This inhibition may reflect molecular adaptations that counteract alcohol neurotoxicity in cells that survive after many years of alcohol exposure and withdrawal.

Increased white matter signal hyperintensities in long-term abstinent alcoholics compared with nonalcoholic controls.

BACKGROUND: The harmful effects of alcohol dependence on brain structure and function have been well documented, with many resolving with sufficient abstinence. White matter signal hyperintensities (WMSH) are thought to most likely be consequences secondary to the vascular (i.e., hypertension and atherosclerosis) effects of AD. We hypothesized that such effects would persist into long-term abstinence, and evaluated them in middle-aged long-term abstinent alcoholics (LTAA) compared with age and gender comparable nonalcoholic controls (NAC). METHODS: Ninety-seven participants (51 LTAA and 46 NAC) underwent cognitive, psychiatric, and structural brain magnetic resonance image evaluations. WMSH were identified and labeled as deep or periventricular by an automated algorithm developed in-house. WMSH volumes were compared between groups, and the associations of WMSH measures with demographic, alcohol use, psychiatric, and cognitive measures were examined within group. RESULTS: Long-term abstinent alcoholics had more WMSH than NAC. There was a significant group by age interaction, with WMSH increasing with age in LTAA, but not in NAC. Within LTAA, WMSH load was independently positively associated with alcohol burden and with age. No associations were evident between WMSH volumes and abstinence duration, family drinking history, years of education, or psychiatric or cognitive variables. CONCLUSION: The magnitude of alcohol abuse was related to increased WMSH volume. The presence of an age effect in the LTAA but not the controls indicates a synergistic effect wherein alcohol advances the onset of aging-related WMSH formation. The increased WMSH load did not appear to have any significant clinical correlates, indicating that the white matter lesions in our sample may not have been severe enough to manifest as cognitive deficits. A limitation of the study is that we did not have data on the presence or severity of lifetime or current indices of vascular risk factors such as hypertension, smoking, or diabetes.