Tissues from 19 depressed subjects and 21 age-matched psychiatrically healthy control subjects were obtained at autopsy from the Coroner’s Office of Cuyahoga County, Cleveland, Ohio, USA. An ethical protocol approved by the Institutional Review Board of the University Hospitals of Cleveland was used, and informed written consent was obtained from the next-of-kin for all subjects. Blood and urine samples from all subjects were examined by the coroner’s office for psychotropic medications and substances of abuse.
Retrospective, informant-based psychiatric assessments were performed for all depressed and control subjects (see and ). A trained interviewer administered the Schedule for Affective Disorders and Schizophrenia: lifetime version (SADS-L) to knowledgeable next-of-kin of 15 of the depressed subjects, as previously described (Stockmeier et al 2002
). The Structured Clinical Interview for DSM-IV Psychiatric Disorders (SCID) was administered to next-of-kin of the four remaining depressed subjects (First et al 1996
). Axis I psychopathology was assessed and consensus diagnosis was reached in conference using information from the interview and medical records. Responses from the 15 subjects evaluated with the SADS-L were also recorded in the SCID, and these subjects met DSM-IV criteria for MDD using information collected with either structured diagnostic interview. Eighteen subjects met DSM-IV criteria for an MDD episode within the last 2 weeks of life, and one subject with depression was in remission. Five depressed subjects were comorbid for panic disorder with agoraphobia; agoraphobia; benzodiazepine abuse; sedative anxiolytic hypnotic related disorder, not otherwise specified; pathologic gambling; or delusional disorder. Two depressed subjects met diagnostic criteria for alcohol abuse at 2 and 24 years before their deaths.
Characteristics of the Control Subjects
Characteristics of the Depressed Subjects
The depressed subjects consisted of 7 women and 12 men. The deaths of 13 of the 19 depressed subjects were ruled to be suicide by the coroner. Of the subjects with depression, reveals that seven had a prescription for an antidepressant drug filled in the last month of life, and the antidepressant drug sertraline was detected postmortem in two of these subjects. includes information on whether the depressed subjects were ever treated with an antidepressant drug. That an antidepressant drug was present in the blood of so few depressed subjects or suicide victims has been noted by others as well (Isometsa et al 1994
; Marzuk et al 1995
; Oquendo et al 1999
The control subjects, consisting of 9 women and 11 men, did not meet criteria for an Axis I disorder at the time of their deaths and were closely age-matched with the depressed subjects. Two control subjects met diagnostic criteria for alcohol abuse at 10 and 30 years before their deaths.
The hippocampal formation was dissected from the right temporal lobe at autopsy. Two coronal cuts were made: at the interface between the anterior and posterior segments of the uncus and at 2 cm posterior to the first cut. The body of the hippocampal formation was dissected, frozen in dry-ice-cooled isopentane, and stored at −80°C. Tissue samples from age-matched pairs of control and depressive subjects were coded throughout all histologic procedures, image processing and morphometric analysis so that laboratory personnel were not aware of the psychiatric diagnoses assigned to the samples. Coded and anonymous blocks of tissue from the two groups of subjects were alternatively selected and sectioned. Care was taken that coded blocks of tissue from both cohorts were sectioned in an alternating manner to avoid a possible difference in histologic treatment of tissue. From the anterior surface of each coded block four frozen sections were cut on an IEC microtome at a setting of 40 µm thickness by the same experienced technician. The sections were thaw-mounted on chrome-alum subbed microscope slides and air dried before staining. Three of these sections were processed for routine staining Nissl substance with cresyl violet. The remaining section was fixed in Millonig’s buffer (Dowlatshahi et al 2000
) and processed by the Timm’s sulfide silver method to facilitate detection of hippocampal subregions (Danscher 1981
). Regions of the hippocampal formation were identified (Amaral and Insausti 1990
). The pyramidal neuron layer of CA3 makes a sharp bend extending toward the hilus of the dentate gyrus and folds back on its own self. In this study, the portion of CA3 extending between CA2 and the sharp bend toward the hilus is termed CA3, and the portion of CA3 extending toward the hilus and enclosed in the granule cell layer is termed CA3-internal (CA3i) ().
Figure 1 Brightfield photomicrographs of coronal sections of the postmortem human hippocampal formation. (A) Cresyl violet–stained section from a 70-year-old male control subject (postmortem interval = 20 hours) and (B) an adjacent section processed by (more ...)
After staining, the section thickness was determined by differential focusing using an oil-immersion high-powered objective. An experienced observer using these criteria focused from the top to the bottom of all sections at the selected points (Gardella et al 2003
; Uylings et al 1986
). The vertical movement of the microscope stage was measured by a microcator (Heidenhain, Germany). For each section, the thickness was measured at three randomly selected points in the CA areas, avoiding the edges of the section, and mean values were determined (Andersen and Gundersen 1999
; Dorph-Petersen et al 2001
). Because these three measurements per section in the CA subareas were very similar, no more measurements per section were performed. The coefficient of variance (CV = ± SD/mean) for intrasection thickness was 3% (controls) and 5% (MDD). The CV for intersection thickness was 4% (control) and 5% (MDD). These coefficients of variance are much smaller than the difference in section thickness for the two cohorts (~20%). The differential shrinkage in depressed subjects was in the z axis, because the sections were thaw-mounted on glass slides immediately after cutting.
The number of glia and neurons (pyramidal neurons, granule cell neurons of the dentate gyrus) per volume unit was estimated with the optical disector (Pakkenberg and Gundersen 1988
). Cell measurements were made with a 63.5X oil objective (N.A. 1.4). The horizontal x axis and y axis dimensions of the three-dimensional disector counting boxes in CA1–CA3 were 150 × 150 µm, and in the granule cell layer of the dentate gyrus they were 50 × 50 µm. These counting boxes were positioned in a systematic, randomly placed manner in three sections per subject. The counting unit of a cell was the center of the nucleus defined by focusing on the clear nuclear edge and the most clearly defined nuclear chromatin and nucleolus (e.g., Gardella et al 2003
; Gundersen et al 1988
; Howard and Reed 1998
). A nucleolus is present in pyramidal cells but not in glia. Using this counting unit, the height of the counting box was the thickness of the pertinent section at the counting sites (see Results for thickness values in the two cohorts). In each brain in CA1–CA3, 12–15 counting boxes per region per subject were examined, and in the granule cell layer of the dentate gyrus, 7–15 boxes were examined per subject. In all the three-dimensional boxes for CA1, 135 pyramidal neurons and 180 glia were counted on average per subject; for CA3, 135 pyramidal neurons and 210 glia were counted on average per subject. For the granule cell layer of the dentate gyrus, an average of 80 neurons and 38 glia were counted per subject in all three-dimensional boxes. In addition, to correct for the differential shrinkage along the z axis between MDD and control subjects, the probe volumes for cell densities were multiplied by 40 µm, divided by the actual section thickness, so that the height of the counting box became equal to the “section thickness” setting of the cryostat. The cell densities thus were differentially corrected relative to the uncorrected values, that is, relatively more in the MDD cases. The advantage of this correction for calculating cell density is that it assesses density of neurons and glia in the original sections before histologic processing and thus before differential shrinkage of MDD as opposed to control sections. This permits the comparison of cell density between cohorts without the confounding influence of group differences in section thickness.
Somatic size of neurons and glial nuclear size was indicated from projected surface area measurements in the absence of the vertical section design (Gundersen et al 1988
; Uylings and van Pelt 2002
) applying a 63.5X oil objective (N.A. 1.4).
Least squares adjusted means and SE estimates are presented. The main statistical analysis used was a repeated-measure analysis of variance (ANOVA; SAS PROC Mixed), with diagnosis as a between-subjects effect; CA regions as a within-subjects effect; and age, postmortem interval, tissue pH, and brain weight as covariates (entered separately). Gender was included as a factor in some analyses. Size and density data for neurons and glia in the granule cell layer of the dentate gyrus were analyzed separately from data gathered in the CA regions because the scale of these measures was markedly different from the scale of data from the CA regions. The potential effect of being an active smoker before death, having an antidepressant medication prescription within the last month of life, or of dying by suicide was assessed individually by evaluating the depressives with one of these potential confounds verses the depressives without these confounds. Bonferroni corrections were used to test for statistically significant effects between the two subject groups; a p value of .05 was divided by eight, representing the anatomic variables being assessed (CA neuron density, CA neuron soma size, CA glial density, CA glial nuclear size, DG neuron density, DG neuron soma size, DG glial density, and DG glial nuclear size).
Pearson correlations were calculated to examine potential interactions between age, postmortem interval, tissue pH, age at onset, and the duration of the depressive illness on the eight neuronal and glial density and size measures. A Bonferroni-corrected p value of .00125 was necessary for there to be a statistically significant effect of these variables on neuronal and glial measures.