Abstract
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.
Acknowledgements
Since its establishment, Schizophrenia Research Institute (SRI) has supported schizophrenia research across a broad range of disciplines and has also been a major supporter of the New South Wales Tissue Resource Centre, an infrastructure facility that collects, stores and distributes fixed and frozen postmortem human brain tissue for projects related to schizophrenia. This work was supported by Schizophrenia Research Institute, utilizing infrastructure funding from NSW Health, University of New South Wales School of Psychiatry, and Prince of Wales Medical Research Institute. We thank Dusan Hadzi-Pavlovic for statistical advice.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Methods and Materials
Subjects
The cases were accessed from the New South Wales Tissue Resource Centre (NSW TRC) housed at The University of Sydney (Australia). The focus of this collection is on major psychiatric illnesses, specifically schizophrenia, bipolar disorder, substance use disorders (e.g. alcohol) and those who have no significant history of neuropsychiatric illness or drug abuse as ‘normal’ control cases. Currently, the majority of cases are collected from Coroner's autopsies at the Department of Forensic Medicine (DOFM) in Sydney, Australia. All cases have consent from the next-of-kin (NOK) and authorisations from the Coroner and Designated Officer. The NSW TRC also collects cases through two pre-mortem donor programs, “Using our Brains” (www.braindonors.org) and “Gift of Hope” (www.schizophreniaresearch.org.au). The NSW TRC is a part of the Australian Brain Bank Network, which facilitates brain donations in other Australian states with a variety of pathological, neurological and psychiatric conditions including schizophrenia.
The TRC has approval from the ethics committees of Sydney South West Area Health Service (Protocol number: X07-0074) and The University of Sydney (Ref No. 555). The current TRC selection criteria for DOFM cases prescribes that potential donors (with or without a psychiatric disorder) are aged over 17 years and have no significant history of developmental or other neurological disorder, infectious disease, nor a significant head injury up to 5 years prior to death. Individuals with any history of substance abuse were reviewed on a case by case basis. One patient with a comorbid diagnosis for alcohol dependency and another schizophrenia patient with comorbid drug abuse at the time of death were included in the cohort. Potential donors who had undergone assisted ventilation for more than 24 hours prior to death, or with a prolonged agonal status, were also excluded from the collection.
Postmortem clinical diagnosis
Postmortem clinical diagnosis is a critical and vital first step in the ascertainment of schizophrenia samples. While diagnosing subjects before death may be considered optimal, many brain banks receive non pre-consented donations arising from Coroner's autopsies or from donations by the family after death. For these cases, the brain bank conducts retrospective clinical interviews and record reviews after death. Standards for retrospective postmortem diagnosis have been developed using current diagnostic instruments [Citation1–4]. A postmortem clinical diagnosis is determined for each case primarily through extensive review of medical records by experienced clinicians. All available pre-mortem information is collected in relation to the donor's social, developmental, psychiatric, drug, alcohol and medical treatment history. This information is collated into a formatted, structured clinical summary, as described previously [Citation5,Citation6]. If required, further information is sought from telephone interviews with the next of kin, general practitioners and specialist health personnel.
A DSM-IV diagnosis (American Psychiatric Association 1994) is generated with The Diagnostic Instrument for Brain Studies – Revised (DIBS) that is applied to the clinical summary described above [Citation3]. The DIBS is a semistructured instrument specifically designed for postmortem psychiatric assessment using medical records and informants. It enables diagnosis at a sub-syndrome and symptom-based level, providing comprehensive data for prospective research, whilst increasing the reliability of clinical diagnosis [Citation2–4]. A diagnostic subtype is established through careful examination of the donor's lifetime symptom profile, as rated in individual items in the DIBS. Profiles were cross-matched with DSM-IV diagnostic criteria, to determine the most appropriate subtype for the case. Normal controls are defined as those individuals with no history of significant psychological problems or psychological care, psychiatric admissions or drug detoxification, and with no known history of psychiatric symptoms or substance abuse, as determined by both telephone screening and medical records. Subjects satisfying clinical criteria for a normal control must also have no significant neuropathological changes upon examination of the brain.
Neuroleptics
All neuroleptic medications recorded in available medical records of subjects with schizophrenia were converted to chlorpromazine (CPZ) equivalent doses in milligrams, using the ‘Neuroleptic Conversion Calculator’ software (Version 1.2.1, 2002, Addat Pty Ltd). An average daily dose was determined from an estimate range of a donor's life-time daily neuroleptic medication use (from lowest to highest ever recorded dose) and a CPZ equivalent dose was then calculated. The lifetime dose was estimated by taking the average daily dose in CPZ equivalents and multiplying this by the duration of illness (days). The subject's last recorded medication before death was also documented from the patient's medical records.
Smoking history
Smoking history was collected after review of all available medical records and information from the next of kin. This information is categorised into the following groups: non-smoker, moderate (less than one packet of 25 cigarettes per day), heavy (≥ 1 packet per day), and those who were ex-smokers (with a known period of abstinence of at least 1 year). Smoking histories were available on 59 of 74 cases.
Manner of death
The cause and manner of death, and any contributory causes or significant medical conditions related to death were obtained from Coroner's documents. By definition, cases were not considered as part of the normal control group if their manner of death was suicide. No subjects with schizophrenia were excluded from the study based on the manner of death. The manner of death was classified as: (1) natural (N); (2) accidental (A); (3) suicide (S); (4) homicide (H) and (5) undetermined (U).
Agonal state
The agonal status was assessed based on a 3 point scale: excellent (1), good (2) and poor (3). The rating was assigned to each case based on information from the police report to the Coroner, and the autopsy report as per criteria previously described [Citation7,Citation8]. Donors with a short agonal phase and good physical condition were rated ‘excellent’, those with a moderate agonal phase and average physical condition at death were rated as ‘good’, and donors with a longer agonal phase, and/or poor physical condition prior to death were rated ‘poor’. Since no cases met the criteria for prolonged agonal state (from extended hospital stay before death), this category (rated 4 in previous publications) [Citation7,Citation8] was not used.
Age at death and postmortem interval
Age at death was verified by obtaining both the date of birth and the date of death from next of kin consent forms and medical records. Postmortem interval was calculated from the time a person was last seen alive (or the time of death, if known) to the time when the fresh tissue dissection of the brain commenced.
Tissue dissection
At autopsy, brain weight and volume were determined [Citation9] and the brain was photographed prior to and after tissue preparation. The cerebrum, cerebellum and brainstem were divided in the sagittal plane. The cerebellar hemisphere and brainstem were removed from each cerebral hemisphere by transversely sectioning the brainstem through the rostral midbrain at the level of the superior colliculi. Each cerebral hemisphere and its contralateral cerebellar hemisphere and brainstem were assigned a ‘fresh’ or ‘fixed’ status on a random basis. The fresh hemisphere was cut into ∼1 cm coronal slices and the prefrontal, anterior and posterior cingulate, visual, superior temporal and motor frontal cortices, caudate putamen, basal forebrain, amygdala, thalamus and hippocampus anatomical areas were dissected for separate freezing. The dissected tissue blocks and remaining slices were immediately frozen to a temperature of –80°C. Frozen blocks and slices were then stored at – 80°C (±5°C) in air tight snap-lock plastic bags. The remaining cerebral hemisphere was fixed in 15% buffered formalin for 3 weeks and prepared for histology and neuropathological examination by a qualified neuropathologist as previously described [Citation6]. The macroscopic and microscopic neuropathological reports were used to establish the suitability of each case for research.
pH measurement
To assess tissue integrity 1g of fresh cerebellar tissue was homogenized in 10 mLs of distilled water [Citation10–12]. pH was measured in triplicate using a temperature compensating Mettler Toledo MP220 pH meter (Mettler USA). For this study, an additional pH measurement was taken using 0.1g of pulverized dorsal lateral prefrontal cortex (DLPFC) tissue from the same tissue block used for RNA and protein extractions.
Brain anatomical dissections
Frozen tissue was dissected on a dry ice platform using a moderate speed (up to 40,000 rpm) dental drill (Cat# UP500-UG33, Brasseler, USA) to minimize heat generation during the dissection. For the DLPFC dissections (average weight of tissue ∼0.5g grey matter tissue from the crown of the middle frontal gyrus was obtained from the coronal slab corresponding to the middle one-third (rostral–caudally) found anterior to the genu of the corpus callosum.