Proceedings of a Mini-Symposium: Substance Abuse and Neuropsychiatric Disorders at The International Workshop on Brain Banking

United States

Sponsored by:  National Institute on Drug Abuse

Introduction

In collaboration with Dr. Jonathan Pollock of the Division of Neurobiology and Behavioral Research (DNBR) and Dr. Joe Frascella of the Division of Treatment Research and Development (DTRD), Dr. Jag Khalsa of NIDA's Center on AIDS and Other Medical Consequences of Drug Abuse (CAMCODA) presented a mini-symposium on Substance Abuse and Neuropsychiatric Disorders at the International Workshop on Brain Banking, March 11-12, 2002. The workshop was organized by Dr. Piotr Kozlowski, a neuropathologist at the National Institute of Neurological Disorders and Stroke (NINDS), (currently Director, New York State Institute for Basic Research in Developmental Disabilities), and co-sponsored by the National Institute on Drug Abuse, and National Institute on Aging of the National Institutes of Health (NIH). More than 60 neuroscientists, neurologists, and neuropathologists presented outstanding research and suggestions for the establishment of brain tissue respositories ("Brain Banks") in the United States and abroad. The mini-symposium participants discussed issues relevant to drug abuse and HIV-related neuropsychiatric and neurological complications and made recommendations for future research (see under RECOMMENDATIONS). The abstracts are being published as a supplement to the Journal of Pathology and Experimental Neurology and a summary of the workshop proceedings is being published elsewhere. The information relevant to drug abuse and HIV infection is summarized below.

In general the participants discussed issues of the need and type of tissue collection, storage, and distribution/shipping. Based on their experiences, they stated that tissues from discrete areas of the brain and every stage of disease are needed for the understanding of pathophysiology of, e.g., Alzheimer's, Parkinson's, schizophrenia or other diseases. A careful dissection of anatomically distinct small regions of the brain is critically important, for example, for the best use of new and sensitive techniques such as massively parallel signature sequencing (MPSS). Further, new techniques also are needed for separating individual cell types since mRNA shows different rates of degradation in different cell populations. But it is also important to note that diseases influenced by a large variety of susceptible genes may require large sample populations. On the subject of tissue collection, it is critical to maintain an excellent symbiotic and collaborative relationship with the local Medical Examiner's (ME) Office and MEs elsewhere in order to collect the post-mortem tissue (whole brain or specific brain areas) in a timely manner and after proper and ethnically sensitive consent from the appropriate relative(s) has been obtained. It may be necessary to educate the ethnically sensitive public in maximizing access to post-mortem tissue for brain research. Publicity may also help in enlisting "control" cases, e.g., brains of non-cognitively impaired famous people following sudden death. It is also essential to use standardized methods of tissue harvesting (timing of collection), preparation, and preservation across as many as ME offices and Brain Banks in the US and abroad. Access to ME cases must be faciliated not just for control cases but because sudden deaths represent, and include, important fields of inquiry, e.g., drug abuse, suicide, neuropsychiatric disorders.

On the subject of storage of brain tissue, it is important to note that a pH of <6 is hypoxic while a pH of >6.4 is considered a better indicator of postmortem (PM) tissue preservation. On the other hand, a wide range of postmortem pH (6.5-7.2) and brain tissue storage has minimal effect on mRNA preservation. Therefore, researchers should be trained in the art of collecting and storing brain tissue, brain tissue handling protocols used by domestic and international brain tissue repositories ("Brain Banks"), and sensitivities and limitations of the current methodology, e.g, microarray technology. Further, in the current genomics age, since there are 30,000-40,000 genes, 100,000 proteins, and 50-70% are expressed in the brain, the human brain tissue is essential for not only determining location and/or function, but also for detecting splice variants. Other critical elements discussed are further elaborated below.

To study the neurological and neuropsychiatric effects of drugs of abuse and or HIV infection, it is important to obtain accurate history and extent of drug exposure because the neurotoxic effects differ with the type of drug. Based on the observation that HIV infection is associated with cognitive impairment/HIV dementia, the role of butyrylcholinesterase (BChE) in Alzheimer's disease and metabolism of cocaine, Royal and his colleagues (see abstract below) successfully examined sections of fixed brain from patients with HIV-related cognitive impairment and from non-infected control subjects, including individuals with Alzheimer's disease, for expression of this enzyme. Their studies further highlight the utility of combining materials for analysis from multiple sources and, therefore, the importance of even more uniform approaches to the procurement, preparation, storage, and distribution of banked materials.

Participants also pointed out that polydrug abuse, that occurs in the real world, should be recognized and investigated as such. Blood samples from each investigated case should be deposited, for example, in the central NIDA-supported repository for genotyping. It is critical to use standardized methods for compiling antemortem clinical data, disease and drug taking histories and toxicology reports; and that clinical data are essential not only for clinicopahological correlation but also for determining subtly different subgroups within patient cohorts. Further, for example, lateral asymmetry between brain hemispheres is important to understand the neuropathological phenotype of drug abuse. Other issues such as gender differences in drug susceptibility and the benefit of newer neuroimaging techniques (MRI, fMRI and PET) were also discussed.

Abstracts

Determining the Molecular Consequences of Cocaine Abuse

Michael J. Bannon, Ph.D. and Donald Kuhn, Ph.D.

Our studies aim to develop a detailed profile of changes in gene expression occurring within the brains of drug abusers. By comparing cocaine overdose subjects with drug-free matched controls, as well as with cocaine-positive subjects dying of other causes, we may be able to develop forensic assays that assist in determining the cause of death. We obtain, under contract with a medical examiner's office, brain tissue sections retained as part of the normal autopsy process. Samples are checked for pH, photographed, flash-frozen, and entered into a computerized tracking system. Upon receipt of toxicology data and a determination of the cause of death, samples are matched for demographic and other variables prior to analysis. Our initial studies have focused on several candidate genes of interest. For example, expression of both the transcription factor NURR1 and the NURR1-responsive dopamine transporter gene are markedly decreased within the dopamine neurons of cocaine abusers. Our more recent studies utilize microarrays to profile gene expression in defined brain regions and in identified cell types isolated by laser capture microdissection. Postmortem assessment of gene expression in drug abusers' brains may extend our knowledge of the molecular basis of addiction and ultimately suggest novel therapeutic strategies.

Challenges and Research Outcomes in Brain Banking at the Drug Abuse/HIV Interface

Jeanne Bell, M.D., Ray Brettle, Vince Egan, Anthony Busuttil and Peter Simmonds

In 1990 a tissue bank was established in Edinburgh, Scotland, for postmortem samples derived from HIV/AIDS subjects. The aim of this project was to discover whether the effects of HIV in the nervous system were significantly different in drug users (the majority of Edinburgh HIV-positive subjects) compared with non-drug users. From the outset, the practice has been to retain non-CNS as well as CNS postmortem tissue samples in both fresh-frozen and fixed form to investigate tissue distribution and viral load. Appropriate control cases, including HIV-negative drug users and patients dying in the earlier stages of the infection, have been ascertained by collaboration with forensic pathologists.

The past decade has seen not only considerable changes in postmortem practice in the United Kingdom, particularly in public attitudes toward this activity, but also in conceptual and methodological advances that have revolutionized pathology-based research. These have certainly impinged on the work undertaken by the Edinburgh HIV tissue bank. In addition much has been learned about the undertaking of detailed high-risk postmortem examinations, the ethics of research in postmortem tissues, and the establishment of national and international brain bank and research networks. The operation of this bank has survived the introduction of effective combination therapy and the changed perceptions of the AIDS epidemic in United Kingdom.

The formula of combining an active multidisciplinary local research program with supply to other research groups worldwide has continued from the outset. The Edinburgh cohort of drug users showed a peculiarly high prevalence of HIV encephalitis, and this has provided a unique opportunity to study the effects of infection with neurovirulent HIV strains combined with opiate and amphetamine misuse. The change in disease pattern following the introduction of effective therapy has generated new questions that in part may be answerable only by a comparison between current and archival cases. There was a window of opportunity between 1988 and 1996 to lay down a resource of tissue samples from untreated patients, study the pathology and neuropathology of a new disease (HIV), and add to what is known of an older problem (drug misuse). The challenge is to circumvent the limitations of working with human post mortem tissue and to support continued progress by providing a high-quality research resource.

Neuroimaging Studies in HIV and Drug Abuse

Linda Chang, M.D.

Advances: Various combinations of stimulant drug abuse, including methamphetamine, cocaine, and MDMA (or ecstasy) are commonly used among those infected with HIV. The mechanisms of interactions between HIV and psychostimulants on brain injury are not well understood. Decreased N-acetyl compounds, a neuronal marker, and increased myoinositol, a glial marker, on proton MR spectroscopy, have been reported in psychostimulant users as well as those additionally infected with HIV. Likewise, decreases in dopamine transporters and receptors have been reported in methamphetamine users. However, the combined effects of HIV and psychostimulant abuse on dopamine function have not been well studied in humans. Challenges: 1) Many of the drug users are poly-drug users; therefore, in order to better define the interaction or combined effects of certain class of drugs with HIV, extensive screening procedures are needed. 2) Another challenge is to assess and ensure an accurate drug use history in relation to when the imaging studies occurred. These problems are common in drug abuse research involving human subjects that often lead to results that are difficult to interpret. Future Directions: 1) In vivo neuroimaging may guide the neuropathological evaluations, with regards to brain regions, in tissues from the brain banks. 2) Ex vivo measurements may allow detailed neurochemical measurements of the combined effects of chronic psychostimulant abuse and HIV. 3) Classical neurochemicals (such as dopamine, serotonin, etc.) as well as structural neurochemicals (including N-acetylaspartate, choline compounds, myoinositol, glutamate/glutamine, etc.) that are visible on in vivo neuroimaging studies should be evaluated in the brain bank specimens in order to facilitate future in vivo monitoring of treatment effects.
(Studies were supported by NIDA: K-20-00280; R01-DA12734; R01-NS38834; and the GCRC MO1 000425).

Brain Collection in the Republic of Macedonia

Aleksej Duma, M.D., Ph.D.

The Republic of Macedonia has a population of 2.2 million people in an area the size of Vermont (approximately 25,000 square miles). There is a single Institute of Forensic Medicine (Medical Examiners Office); three hospitals for patients with mental diseases; and one Institute for Psychiatry, which is located in Skopje, the capital. One psychiatric hospital is located in Bardovci, a village adjacent to Skopje, and the other two are in the southern portion of the country, approximately 3 hours from Skopje by car. There is universal, centralized health care. Most patients with mental diseases are hospitalized when necessary, and a majority of them spend the last years of their lives in one of the psychiatric hospitals, which together can accommodate approximately 2,000 patients. Autopsies are required by law for all hospital deaths and for all deaths from unnatural causes. Although exceptions are made to this requirement, these conditions allow for a population-based collection of brains from individuals with severe psychiatric illnesses. A strong educational system provides a pool of skilled doctors who have a knowledge of English and an interest in research, and who are willing and able to collect the specimens and clinical information.

Neuronal Loss in Intravenous Drug Users

Ian Paul Everall, Ph.D.

The availability of tissue collections from brain banks has been crucial in furthering our understanding of the differences among brain pathology in different risk groups infected with HIV. First, I will present work that we have undertaken assessing neuronal loss in intravenous drug users as well as data demonstrating the correlation of pathological changes in the HIV-infected brain with cognitive impairment, including its relationship with rising brain viral burden. Second, I will use the examples to highlight the importance of standard protocols for tissue handling as well as the importance of prospectively acquired antemortem clinical data. Third, I will propose that future collections facilitate in the provision of viable human brain tissue to promote important studies into pathological mechanisms as well as allow the development of neuroprotective strategies. I will demonstrate this with our work on fibroblast growth factor and currently available antiretroviral agents.

Diagnosis and Clinical Assessment Protocols for Brain Banks

Therese Garrick, R.N.

Clinical Information and Diagnosis: The clinical diagnosis of cases collected for inclusion in the TRC is given the highest priority. Clearly, a poor diagnostic workup of cases will confound any research data derived from TRC tissues. The older neuropathology literature for schizophrenia research is filled with conflicting studies, in part due to errors in the clinical diagnosis of cases (Powers 1999).

To ensure a comprehensive profile of the cases, a standardized protocol has been developed. Relatives of the deceased complete questionnaires, and hospital medical records are reviewed by a trained clinician with psychiatric experience. The treating medical staff are interviewed where possible. Information relating to the primary psychiatric disorder, developmental history, family history, and the medication and treatment history is collated into a structured and formatted treatment summary. The treatment summary is organized in such a way that it is possible to audit the file for interrater reliability purposes. Pathology, radiology reports, and neuropsychological data are collected when available.

The Diagnostic Instrument for Brain Studies (DIBS) is then applied to this information. The DIBS is a multidiagnostic instrument designed for use with medical records and has proven reliable. To include a case in the TRC, it has to meet both Diagnostic and Statistical Manual for Mental Disorders, 4th edition (DSM-IV) and ICD-10 criteria (non-substance use cases). For the substance use disorder (alcohol) cases, they must meet DSM-1V criteria. Random cases are selected for interrater reliability by an independent clinician. Cases are excluded from the TRC if there is inadequate clinical information to reach criteria using the DIBS.

Tissue Donor Programs: Experience in Australia and internationally indicates that people with brain diseases and their families are interested in organ donation for research. Recently, two brain donor programs have been launched in New South Wales. The "Gift of Hope" TDP, which has a strong focus on schizophrenia and allied disorders (1999), and the "Using our Brains" TDP, which targets a broader group of the community, with a strong emphasis on controls (2002). The outstanding advantage of the TDPs is the ability to compare premortem clinical, neuroimaging information with postmortem findings. The clinical protocol for the TDPs involves initial and annual assessments of the donors providing longitudinal data.

A general medical and treatment profile, family history, the Annetts handedness scale, the Diagnostic Instrument for Psychosis, the Repeatable Battery for the Assessment of Neuropsychological Status, and the National Adult Reading Test are carried out on all donors. Donors may also be invited to participate in an MRI scan.

The Department of Pathology at the University of Sydney will ensure that when a donor dies, rapid notification procedures are in place for the retrieval of the donor's tissue.

NCI Tissue Resources and New Challenges

Thea Kalebic, M.D., Ph.D.

Evolving research challenges and broader use of novel high-throughput technologies in basic and clinical studies impose new challenges for developing tissue repositories. Research studies aimed at validating or discovering clinically useful biomarkers require a large number of specimens and collections of specially processed and preserved tissues. Limitations of tissue availability represent a considerable impediment to progress in cancer research.

The National Cancer Institute (NCI) is using various approaches to increase tissue availability and to anticipate future research needs. It supports the NCI Cooperative Human Tissues Network, Cooperative Breast Cancer Tissue Resource, Clinical Trials Cooperative Groups, AIDS and Cancer Specimen Bank, The Cancer Family Registries, and NCI Specimen Resource Locator. Throughout the United States, more than 300 million specimens are stored in various tissue banks.

Nevertheless, the research community encounters many difficulties in obtaining appropriately collected and stored specimens for basic and translational studies. The most common tissue preservation modality-formalin fixation and paraffin embedding-causes the formation of covalent bonds. As a result, the tissue architecture remains well preserved, which is crucially important for histopathological diagnosis. However, the extraction from fixed tissue of macromolecules, such as RNA and proteins, is rather inefficient. To preserve the structural integrity of the tissue while minimizing RNA, DNA, and protein damage that may occur in the process of preservation is a new challenge for developers of tissue repositories.

NCI, under the leadership of the Resource Development Branch, has been assessing novel technologies for tissue preservation as well as modalities to "unfix" fixed tissue. These and other efforts to improve the availability and access to research-driven tissue collections will be discussed. Also, legal and ethical questions related to the use of human specimens will be addressed.

Cellular and Molecular Studies in Schizophrenia

Joel E. Kleinman, M.D., Ph.D. NIMH, NIH.

Studies in the Section on Neuropathology have sought to address two questions. First, is there evidence for a cellular and molecular neuropathology in schizophrenia? Second, how do recently discovered genetic susceptibility factors effect neuropathology? The focus at the cellular level has been on glutamate pyramidal neurons in the mesial temporal lobe and the dorsolateral prefrontal cortex (DLPFC). At the molecular level the focus has been on synapse formation, plasticity and neurodevelopment. Lastly, we have genotyped postmortem human brains to test whether functional polymorphisms for COMT have predicted effects on mRNA expression in midbrain dopamine neurons.

Data will be presented looking at mRNA expression for neuronal and glial glutamate transporter in mesial temporal lobe and DLPFC of brains of schizophrenics and controls. Decreases in the neuronal glutamate transporter in the mesial temporal lobe in schizophrenics appear to be related to decreases in the glial glutamate transporter in the DLPFC. Similar changes in synaptophysin mRNA are also seen in these regions especially in hippocampal subfields, CA3 and 4.

In an attempt to connect these changes to a developmental hypothesis of schizophrenia, we have studied molecules that effect cortical glutamate neuron development and appear to change expression levels in the DLPFC during adolescence and young adulthood, periods linked to the onset schizophrenia. Three candidate genes, GAP-43, BDNF and dopamine type1 receptors change mRNA expression in the DLPFC of normals at these critical times and are further implicated in the neuropathology of schizophrenia.

Lastly, a functional polymorphism in a gene recently shown to be associated with schizophrenia and normal DLPFC cognitive function, COMT, has been shown to effect tyrosine hydroxylase mRNA expression in midbrain dopamine neurons of normals.

NeuroAIDS and Its Progression In India: A Model for an International CNS Tissue Consortium?

Mahendra Kumar, Ph.D.

It was in 1987 that the first case of HIV-infection was reported from India. Over the years, HIV-infection has spread aggressively and according to recent estimates, India has about 4 million HIV-infected individuals, with their numbers nearly doubling almost every 18 months. At this rate of transmission, it is estimated that in 5-10 years the number of HIV infected individuals in India may exceed the present prevalence of the entire world. The primary risk factor for contracting HIV throughout the country appears to be unprotected heterosexual sex, although in Northeast India, injecting drug use is the major risk factor. The vast majority of seropositive men and women belong to the lower SES group but all strata of the society are infected to some extent. Although all strains of the HIV-1 virus are present In India, clad C seems to be the most dominant strain. HIV-2 infection has also been reported from India and it is estimated that up to about 25% of infected individuals may have co-infections of both HIV-1 and HIV-2. The impact of co-infection of HIV-1/2 on the Central Nervous System is scientifically an area of great interest since it has been reported that HIV-2 infection protects against HIV-1 infection.

It is well established that immediately after infection, HIV crosses the blood-brain-barrier and is localized in very high concentrations in the hippocampus and to variable degrees in other areas of the brain. The hippocampus plays an important role in regulating the various hypothalamic-pituitary-endocrine axes and in cognitive functioning. Our earlier investigations support these concepts. The prevalence of HIV infection is very high in Southern India, particularly in the states of Maharashtra, Tamil Nadu, and Karnataka. Recently, in association with the National Institute of Mental Health and Neurosciences (NIMHANS) located in Bangalore, in the state of Karnataka, we have started investigations on the neurological progression of HIV-1+, HIV-1 /2+, and HIV-2+ in men and women. This is a longitudinal study and each participant is proposed to be followed for three years. Every participant will be evaluated every six months for cognitive functioning, mental health outcomes, as well as immune and neuroendocrine status. We have been able to locate a large cohort of seropositive participants (approximately 5,000) for these studies. At present, there is a scarcity of data on neuropathological findings among seropositive individuals in India. NIMHANS is one of the major centers in India known for carrying out autopsies in HIV seropositive individuals but to the best of our knowledge, there is no data available on cognitive functioning during life among the patients coming for post-mortem. Our present funded study can potentially fill this gap in defining the neurological functioning during life of participants in whom autopsy could be performed. This presentation will attempt to provide a model which could be suitable for such studies in developing countries particularly in India, and will take into consideration the cultural, social, religious and belief systems of that country. (Supported by NIH grants # NS 41205, DA 12792 and DA 13550)

Banking on Brains for Cocaine Abusers

Deborah C. Mash, Ph.D.

The transit of cocaine from source nations in the Caribbean and Central and South America through the Bahamian corridor to the United States frequently occurs via the Florida coast. Metropolitan Miami-Dade County, Florida, continues to have one of the highest rates of cocaine-related emergency room episodes and cocaine-related deaths in the United States. Based on a retrospective case control analysis of the toxicology reports, scene descriptions, supplemental background information, and autopsy findings, we have developed a cohort of postmortem neuropathological specimens from chronic cocaine abusers.

Postmortem brain specimens from cocaine-related deaths and cocaine intoxication cases are banked with precision and uniformity. Cases are subdivided into three groups of fatal cocaine intoxication: accidental cocaine overdose, acute high-dose toxicity with seizures, and cocaine intoxication presenting with preterminal excited delirium. Retrospective chart reviews are used to exclude cases from neurochemical study based on evidence of significant underlying cardiovascular pathology, cerebrovascular disorders, and polydrug abuse. Structured interviews with informants are done to assess the patterns of cocaine use in the 30 days prior to death and for lifetime exposure. Cocaine and metabolite levels are measured in blood and brain tissue specimens at the time of death. An overview of the approach used to develop a collection of brain tissues from chronic cocaine abusers and age-matched drug-free control subjects will be presented.

Uncovering Transcriptome Differences With DNA Microarrays in Postmortem Tissue:Technical Considerations

Karoly Mirnics, M.D.

Postmortem brain tissue contains intact mRNA that can be successfully extracted, reverse transcribed, and analyzed using DNA microarrays. It appears that transcript integrity depends more on the agonal state and circumstances of death than on the postmortem interval (PMI): we routinely isolate minimally degraded RNA from brains with >24 hours of PMI.

The appropriate experimental design is essential to perform meaningful comparisons of gene expression patterns. These paradigms consider the individuality for transcriptome of each investigated subject. Furthermore, in brain areas with high phenotypic complexity, DNA microarrays will not be able to assess the expression of the sparsest transcripts, which become "diluted" beyond detection by more abundant RNA species.

Microarray experiments can be compromised at different stages of this complex procedure. Establishing conservative starting material and data quality checkpoints will ensure that the obtained findings represent true biological differences rather than methodological artifacts.

Combined Effects of Drug Abuse and HIV Infection on the Brain: Correlations Between Experimental, Clinical, and Pathological Studies

Avindra Nath, M.D.

Both HIV infection and drugs of abuse have clearly shown independently to have adverse effects on the brain. Furthermore, experimental studies in vitro and using animal models also clearly demonstrate that the combined effects of these drugs of abuse and HIV infection of HIV proteins causes synergistic neurotoxicity, increased viral replication, and immune activation. However, previous clinical and pathological studies have shown conflicting results. The recent advances in technology and the identification of sensitive objective markers of neurological function should allow us to revisit this issue and resolve these questions. Critical to these issues is the proper banking of nervous system tissues from well-characterized cohorts as well as the development of surrogate markers of drug induced central nervous system injury.

The Role of Butyrylcholinesterase in the Pathogenesis of HIV-1 Dementia

Walter Royal III, M.D.

Cognitive impairment in individuals with HIV-1 (HIV) infection appears to be multifactorial, involving the direct effects of viral proteins and toxicity associated with secreted products of immune cells. In patients with Alzheimer's disease, it has been observed that the enzyme butyrylcholinesterase (BChE) is localized to plaques on immunocytochemical staining of tissue. The association of this finding with clinical dementia in these patients has formed part of the rationale for treatment of Alzheimer's disease patients with the drug rivastigmine, a BChE inhibitor. In addition, BChE has been demonstrated to be involved with the metabolism of cocaine and, therefore, may be important in preventing neurotoxicity from this drug.

To investigate the possible role of BChE in the pathogenesis of HIV dementia, we examined sections of fixed brain from patients with HIV-related cognitive impairment and from control subjects for expression of this enzyme. In this presentation we will discuss data from these studies as well as methods that may be useful in examining the possible role of BChE in the development of cognitive impairment in individuals with HIV infection.

A New Technology for Indepth Gene Expression Profiling

Richard Woychik, Ph.D.

Massively parallel signature sequencing (MPSS) is one of the newest tools available to conduct indepth expression profiling. MPSS is an open-ended platform that analyzes the level of expression of virtually all genes in a sample by counting the number of individual mRNA molecules produced from each gene. There is no requirement that genes be identified and characterized prior to conducting an experiment. MPSS has a routine sensitivity at the level of a few molecules of mRNA per cell, and the data sets are in a digital format that simplifies the management and analysis of the data. Therefore, of the various microarray and nonmicroarray technologies currently available, MPSS provides many advantages for generating the type of complete data sets that will help facilitate experiments in systems biology directed at studying the function of specific regions of the brain.

Recommendations for Future Research

The participants suggested that to begin with, NIDA should support the following research activities: (i) Establish a network of forensic pathologists at at least three regional sites for tissue collection, e.g., Florida and Maryland for studying cocaine, opiates, and MDMA-related issues; and California for studying methamphetamine, opiates, and MDMA; (ii) Establish a central holding site with three or four collection sites; (iii) After taking into consideration various state laws, obtain cooperation from physicians who will inform the investigators the availability of the brain tissue as soon as possible; (iv) Develop training manuals; train appropriate staff and develop common protocols for tissue handling; train local staff, nurse or social workers to make the necessary telephone calls and to conduct retrospective evaluations with family members and next-of-kin, and perform consensus review by psychiatrists and forensic pathologists; (v) Develop a procedure such that brains will be sent to one regional storage site for distribution; assign bar codes for all samples; train technicians for brain tissue dissection; and finally (vi) Store separately the brain and hair specimens for toxicology (e.g., cerebellar specimens taken for allele expression, SNPs, etc.). Individualized handling can be accomplished to meet NIDA-funded study protocols (i.e., immunostaing, etc).

Summary:

1. Critical elements for the successful use of postmortem tissue and brain bank resources. 
    
   • Develop a genuine, symbiotic and collaborative working relationship between investigators and the Medical Examiner. 
      
   • Encourage the standardization of tissue harvesting, handling, preparation, and preservation across as many ME offices and Brain Banks as possible. 
      
   • Standardize methods for compiling antemortem clinical data, disease and drug taking histories, and toxicology reports. 
      
   • Develop networks, collaborations, and communications for the maximal use of available tissue. Use sufficiently large sample sizes with appropriately matched subjects (age, gender, history, etc). 
      
   • Use anatomical verification of microarray, Northern blotting, Western blotting and other primary levels of analysis in intact (frozen or fixed) tissue as a secondary or tertiary level of analysis. 
      
   • Psychiatric, neurological, and drug abuse disorders can be studied at molecular and anatomical levels. HIV-induced neurotoxicity and other comorbid conditions often accompanying all of these disorders can also be studied effectively and can yield interpretable results. 
      
   • Viable tissue models can emerge from properly preserved PM tissue and these models can be used to study other than end-stage disease states. For example, HIV infection of cultured cells or slices from PM tissue shows morphological changes that are seen in intact tissue from individuals with HIV. Studies of disease mechanisms, their progression, and protective measures can also be studied. 
      
   • PM tissue studies targeting specific structures, neural systems, proteins and genes for study. 
      
   • Support the use of newer techniques that are being currently used such as: quantitative RT-PCR, in situ hybridization, immunocytochemistry, laser-capture microscopy, and functional studies of enzymes and transporters. Expansion to studies of the genome, the transcriptome, and the proteome are feasible and are entering productive stages through the use of microarray, massively parallel signature sequencing (MPSS), and mass spectrometry technologies. The emerging technologies such as microarray, high-resolution structural studies of the nervous system, MPSS, and mass spectrometry can now detect low copy number transcripts that were previously below the level of detection. 
      
   • PM tissue can be used to develop biomarker profiles that can be linked to specific diseases. This can have important feedback to the ME as part of the autopsy report and can advance the study of diseases of the central nervous system in humans. 
      
   • Results are dispelling the image that PM tissue degrades too rapidly to allow its use in experimentation. In fact, postmortem interval and brain pH, within reasonable limits, do not influence the integrity of mRNA, as one example. While overall yields may decrease under non-ideal conditions, the quality and integrity of harvested mRNA remains high. 
      
   • Use of PM tissue for structural and functional studies of the brain meshes very well with neuroimaging results. 
      
2. Recommendation for NIH to implement: 
    
   • NIH should take a proactive stance in advocating the use of autopsy material for research in diseases of the central nervous system. 
      
   • NIH should "educate" CSR Study Sections to help them appreciate the scientific validity and importance of grant applications using PM tissue. Use of PM tissue is often characterized by study sections as descriptive, or lacking a hypothesis-driven approach. This characterization is often at odds with the publication of data from PM studies in journals with the highest scientific impact. 
      
   • NIH should establish a Special Emphasis Panel to review highly specialized grant applications that focus on the use of PM tissue. 
      
   • NIH should earmark stabilized funding mechanisms for studies of PM tissue. This could include, but is not limited to, RFAs, cutting-edge/high-risk studies, or other multi-center networks. These mechanisms should support banking infrastructures at a minimum to help defray the high expense of obtaining and preserving the highest quality tissue possible. 
      
   • Brain banks and tissue repositories should be networked through a common database so that investigators could query the database for tissue availability, request tissue, share pathological workups, and share data where appropriate. 
      
   • Additional studies should be encouraged to establish optimal conditions for tissue preparation and preservation to include the "unfixing" of already-fixed tissues. Certain repositories have very large numbers of fixed tissues, but their use with some experimental approaches is currently restricted by fixation. 
      
   • NIH should encourage establishment of effective collaborations/sharing of scarce control cases through effective brain bank networking. 
      
   • NIH should encourage international collaborations that allows comparison of very different patient cohorts. Barriers to international collaboration in brain banking may be more perceived than real since mutually acceptable consent procedures and ethical approval for brain banking may already be in place. 
      
   • Finally, postmortem human brain tissue should prove increasingly valuable for any brain related institute in efforts to elucidate normal brain development and/or disease related pathophysiology. This should be of value to the NINDS, NIMH, NIDA, NIAAA, NIA, NICHD, NEI and NIDCD and their intramural research programs.