SECTION 3 - ENABLING TECHNOLOGY

Postmortem Brain Analysis in Sleep Disorder Patients

Background

The postmortem study of brains of patients suffering from sleep disorders has significantly contributed to our understanding of human sleep regulation and its dysfunction. Human brain analysis at autopsy in sleep disorders is important for two major reasons: (1) it generates hypotheses from observations directly in human tissues about the cellular and molecular mechanisms of human disease for testing in animal models, cell culture systems, and genetic models; and (2) it tests the relevance to human disease of observations made in animal models and cell culture systems by examining specific cellular and molecular markers in human tissue samples.

Currently, human neuropathology involves analysis at the structural, neurochemical, cellular, and molecular levels, and its modern tools hold promise of much-needed insights into central and autonomic mechanisms in sleep disorders. A potential revolutionary tool for human brain analysis is microarray analysis of gene expression in autopsied tissues. The potential of this genomic technology in human neuropathology to uncover critical molecular abnormalities is illustrated by its recent application to postmortem brain analysis in schizophrenia. With cDNA microarrays, altered gene expression was found in the frontal cortex in schizophrenic patients compared to autopsy controls. The most changed gene, which was never before linked to schizophrenia, was a regulator of G-protein signaling 4, suggesting schizophrenia is a disease of the synapse and thus providing an opportunity to better understand a devastating disorder whose basic mechanism(s) has been elusive.

Sudden Infant Death Syndrome (SIDS) represents a sleep disorder in which neuropathologic examination with modern neurochemical techniques suggests abnormalities in a specific brainstem region and neurotransmitter system, namely the medullary serotonergic system). These findings from human infant brains will generate hypotheses to be tested in animal models. Narcolepsy, on the other hand, represents a sleep disorder in which seminal observations in genetic animal models resulted in subsequent delineation of the neuropathology in affected human patients, e.g., deficiencies in the hypothalamic hypocretin system.

These two examples underscore the critical need to analyze the human brain at autopsy in patients with sleep disorders. National autopsy networks and brain tissue banks may be needed to collect brain tissues from patients with common, rare, or non-lethal sleep disorders, and to disseminate affected and control brain samples to interested sleep researchers. Some national, NIH-supported brain tissue banks are well established, and have proven vital to the success of human brain research in neurodegenerative disorders such as Alzheimer's disease and genetic disorders such as Rett syndrome). An informal survey of national brain tissue banks, however, reveals virtually no accrual of brains from patients with any primary sleep disorders except SIDS. Specialized training of neuropathologists in the neuroanatomy, neurochemistry, and neuropathology of sleep will be needed, however, to make optimum use of this new research resource.

Progress In The Last 5 Years

- The neuropathologic postmortem evaluation of brains in patients with Narcolepsy confirmed observations from animal models that the major lesion is in the hypocretin neurons of the lateral hypothalamus. A reduced number of hypocretin neurons, associated with gliosis, was reported in the hypothalamus of human patients with narcolepsy, as well as, in a separate study, the absence of hypocretin mRNA in the hypothalamus and lack of hcrt-1 and hcrt-2 levels in the cerebral cortex and pons (target sites).

- The neuropathologic postmortem evaluation of brains in patients with Fatal Familial Insomnia (FFI), a prion disorder, established that the major lesions are in certain subnuclei of the thalamus that are inter-related to the limbic system, and suggested a specific role for these thalamic regions in sleep and autonomic control that can now be tested in animal models.

- Neuropathologic insights from analysis of SIDS and control brains at autopsy revealed that a substantial subset of SIDS victims have abnormalities in serotonergic receptor binding in regions of the medulla involved in chemoreception, respiratory drive, blood pressure responses, and upper airway control. Dysfunction of serotonergic neurotransmission in the medulla in affected infants may put them at risk for sleep-related sudden death when stressed by hypoxia and/or hypercarbia in a critical developmental period.

- Neuropathologic postmortem analysis in patients with dementia and REM Behavior Disorder (RBD) revealed an association between Lewy body dementia and RBD. Lewy bodies were present in affected patients in regions of the brainstem involved in arousal, REM sleep, and autonomic control.

Research Recommendations

- Utilize established national brain tissue banks for increased accrual and dissemination of brain samples and associated histories from patients affected by both primary and secondary sleep disorders (Section V). The spinal cord should be likewise stored in certain cases in which spinal cord involvement is postulated, e.g., Restless Legs Syndrome. The accrual of Central Nervous System (CNS) samples from control cases without sleep disorders will also be needed.

- Establish a National Autopsy Network for Sleep Disorders and perhaps Centers for individual sleep disorders, in order to assure accrual of brains from patients with sleep disorders, including those that are rare and/or non-lethal. Such a network, for example, would alert pathologists that the brain of an individual with Sleep-Disordered Breathing (SDB) or Insomnia dying of an unrelated cause is of interest to sleep scientists and should be obtained and processed as needed for neuropathologic analysis. Blood, cerebrospinal fluid (CSF), and brain and spinal cord tissues should be collected in addition to a detailed sleep history.

- Apply state-of-the-art neurochemical, cellular, and molecular markers to the study of sleep disorders in human brain tissue, especially those without a pathologic correlate at the light microscopic level. Techniques should include gene expression microarray technology, protein analysis with mass spectroscopy, tissue receptor autoradiography, in situ hybridization for mRNA in tissue sections, immunocytochemistry with single and double labeling, stereological cell counting, and electron microscopy combined with immunological markers.

- Apply state-of-the-art genomic approaches to postmortem brain analysis for novel gene discovery and gene expression profiling in sleep disorders. Possible approaches include microarrays, quantitative real-time PCR assays, and serial analysis of gene expression (SAGE). These methods, among many others, provide information about normal and abnormal gene expression in a tissue or cell. Details must be refined regarding applicability of this technology in human brain tissues with variable agonal conditions (e.g., pH changes) and postmortem intervals. The methods for relating expression data to disease-model databases and gene sequence databases (e.g., single nucleotide databases) will also require refinement. Multifactorial and multilevel analyses will need to combine massive gene expression datasets with the clinical diagnosis, medication, family history of the illness, and genetic heritage of each subject.

- Ensure neuropathology limbs to large, multi-institutional studies of human sleep disorders for analysis and storage of specimens (e.g., brain, CSF, blood), and for correlation of neurochemical, cellular, and/or genetic markers with clinical pathophysiology.

- Analyze the spatial and temporal profiles of sleep-related molecules across development including aging in relevant brain regions (e.g., hypothalamus, basal forebrain, brainstem, thalamus, cerebral cortex) in human postmortem brain.