From August 1, 2012 - July 11, 2013, we collected 64 brains through the DC and VA Medical Examiners Offices. We made small refinements to the collection this year by discarding and/or transferring cases out a sum of unusable/depleted cases with a history of primary drug abuse or neurological rule-outs, and increased our number of mood disorder cases. Our collection consists of 1,204 brains, including 157 samples from Stanley Foundation and 150 from agreements. The following counts do NOT include Stanley: SZ 183;Bipolar 93;Major Depression Disorder 182;Controls, Adult 266;Other Mood Disorder 31;Affective Disorders 55;Neonate to Teen 82;Fetal Brains 66;Substance Abuse 16;Other Diagnoses 63;and 65 to be characterized. The major focus of our studies is schizophrenia (SZ), which involves cognitive deficits, negative symptoms, and psychosis, usually starting in late adolescence or early adulthood. The recent discovery of genetic variations associated with SZ not only speaks to a significant component of the etiology, but allows for increased understanding of the neuropsychology, neuroimaging, and neuropathology of the disorder. Postmortem human brain studies guided by genetic advances are a valuable, if not essential component, in elucidating the cellular and molecular pathophysiology of SZ. Improved understanding of a genetic component in SZ may enhance diagnostic abilities and hopefully, lead to new treatments. Postmortem human brain studies are limited by the quality and quantity of the specimens. Brain Procurement: The number of brains collected per year has remained relatively constant for the past ten years and we have become very selective in choosing cases. The entire collection has undergone a case-by-case review leading to a reduction in the number of brains for study. Selected cases all have informed consent from the next-of-kin at time of donation, with extensive medical records available for review. Interviews with the next-of-kin, adds clinical information. The tissue is screened for macro- and microscopic neuropathology, and multiple screens are performed for RNA and protein integrity. Toxicological screens are conducted for substances of abuse, and prescribed psychotropic medications. Clinical Review and Characterization: Informed consent for research on the brain and related tissue samples from the next-of-kin is obtained at or before the time of autopsy includes the brain, blood, and hair specimen (for toxicology) as well as permission to obtain medical records. Next-of-kin are interviewed with a 14-item telephone-screening questionnaire. The next-of-kin are advised that they will receive a follow-up interview in greater depth within several months. This in-depth interview relies upon three screening devices: a SCID (First et al., 1997), a modified psychological autopsy (Kelly and Mann, 1996) and a modified version of the DEAD (Diagnostic Evaluation after Death) (Zalcman et al., 1983) to collect relevant clinical information from the next-of-kin in order to enhance our diagnostic classification. The ME offices provide demographic information (age, gender, race, etc.), a police report, interviews with the next-of-kin, and relevant medical personnel and witnesses, toxicology screens, and a general autopsy report that includes the cause and manner of death. The ME offices perform toxicology screens in the vast majority of cases. If toxicology screens are not performed we have contracted a laboratory to complete the screens. An extensive review of clinical records and all other available information is conducted by trained Section personnel and independently reviewed by two board certified psychiatrists. If the latter agree on diagnosis, the case is formally reviewed at a diagnostic conference attended by the two psychiatrists, our neuropathologist, the clinical interview team, and the database specialist where a DSM-IVR (APA, 2000) final diagnosis is recorded. If the two psychiatrists disagree, a third board certified psychiatrist reviews the case independently prior to the diagnostic conference. No final DSM-IVR diagnosis is assigned at the diagnostic conference unless two psychiatrists concur on diagnoses. Similar rigor is used in designating normal control subjects who must have no history of psychiatric or neurological disorders, no history of significant substance abuse or dependence, a negative toxicology screen at autopsy, and a neuropathological screen free from abnormalities. Nicotine use has been carefully reviewed and is not an exclusionary criterion for normal control subjects. The neuropathological screening involves two components, a macro- and microscopic examination and inspection. A board certified neuropathologist conducts the macroscopic examination at time of tissue collection, weighing and inspecting the whole brain, upper cervical spinal cord, pituitary and pineal glands, dura, and intracranial vasculature. The microscopic examination involves sectioning and staining tissue from the frontal, temporal, and occipital poles, the posterior hippocampus, and the cerebellar vermis, looking for evidence of neurodegeneration or other pathological processes. Sections are routinely stained to reveal neurofibrillary tangles and neuritic plaques that are counted to apply Khachaturian criteria to establish the neuropathological diagnosis of AD (Khachaturian, 1985). Sections are taken from any region that shows signs of macroscopic pathology, such as an apparent infarct or abscess, for additional neuropathological analysis. A formal macro- and microscopic neuropathology report is written from the information obtained from these examinations. Screening for Molecular Biology Studies: The following procedures have been performed on every brain prior to study inclusion: a. Measurement of tissue pH from homogenized cerebellar hemispheric tissue. b. Measurement of mRNA expression of a panel of putatively constitutively expressed genes (such as actin and cyclophilin) using in situ hybridization histochemistry on 14 micron cerebellar hemispheric sections. c. Extraction of total RNA by Qiagen and separation of total RNA using agarose gels to qualitatively determine the integrity and intensity of the 28S and 18S ribosomal RNA bands. d. Quantitative analysis of total RNA integrity and 28S to 18S ratios using capillary electrophoresis on a Bioanalyzer 2100 (Agilent, Inc.). e. Acceptable postmortem intervals (PMI) in human brain for each protein studied with Westerns or ELISAs are inferred from rat studies investigating the PMI when protein levels for that protein significantly decline (Halim et al., 2003). f. To determine effects of medications in the human studies of mRNA and protein expression, we are using brain tissue from rats chronically treated with various doses of antipsychotic drugs (haloperidol and clozapine) (Lipska et al., 2001,2003). g. From these screening measurements, we have developed exclusionary criteria for the use of human brain tissue in our experiments: tissue pH <6.00, PMI >60 hours;abnormal cerebellar actin and cyclophilin expression levels in cerebellar in situ hybridization (greater than two standard deviations from the mean);and a 28S/18S ribosomal RNA ratio <1.2. h. The vast majority of our cases (over 90%) come from ME offices, few, if any, of our brains come from patients who have died with prolonged agonal states. The latter is clearly associated with low pH and increased RNA degradation (Tomita et al., 2004).

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Scientific Cores Intramural Research (ZIC)
Project #
1ZICMH002903-07
Application #
8745789
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$3,068,293
Indirect Cost
Name
U.S. National Institute of Mental Health
Department
Type
DUNS #
City
State
Country
Zip Code
Kunii, Y; Hyde, T M; Ye, T et al. (2013) Revisiting DARPP-32 in postmortem human brain: changes in schizophrenia and bipolar disorder and genetic associations with t-DARPP-32 expression. Mol Psychiatry :
Blasi, Giuseppe; Napolitano, Francesco; Ursini, Gianluca et al. (2013) Association of GSK-3ýý genetic variation with GSK-3ýý expression, prefrontal cortical thickness, prefrontal physiology, and schizophrenia. Am J Psychiatry 170:868-76
Hirunsatit, Rungnapa; George, Elizabeth D; Lipska, Barbara K et al. (2009) Twenty-one-base-pair insertion polymorphism creates an enhancer element and potentiates SLC6A1 GABA transporter promoter activity. Pharmacogenet Genomics 19:53-65
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Montague, D; Weickert, C S; Tomaskovic-Crook, E et al. (2008) Oestrogen receptor alpha localisation in the prefrontal cortex of three mammalian species. J Neuroendocrinol 20:893-903
Agarwal, Varsha; Kommaddi, Reddy P; Valli, Khader et al. (2008) Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite. PLoS One 3:e2337
Halim, Nader D; Lipska, Barbara K; Hyde, Thomas M et al. (2008) Increased lactate levels and reduced pH in postmortem brains of schizophrenics: medication confounds. J Neurosci Methods 169:208-13

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