Department of Clinical Neurology
Department of Psychiatry
Department of Clinical Neurology, University of Oxford, UK
Correspondence: Professor M. M. Esiri, Neuropathology Department, Radcliffe Infirmary, Oxford OX2 6HE, UK.Tel: 01865 224403; fax: 01865 224508; e-mail: margaret.esiri{at}clneuro.ox.ac.uk
Declaration of interest None. Funding detailed in Acknowledgements.
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ABSTRACT |
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Aims To measure the size of hippocampal pyramidal neurons in the brains of people with and without schizophrenia.
Method Pyramidal neuron size in hippocampal subfields was estimated stereologically from sections taken at 5 mm intervals throughout the whole length of right and left hippocampi from the brains of 13 people with schizophrenia and 16 controls. Results were assessed using repeated-measures analysis of covariance looking for a main effect of diagnosis and gender, and interactions of these with side.
Results We were unable to detect significant differences related to diagnosis, gender or side for any hippocampal subfield for this series of cases.
Conclusions For this series of brains, hippocampal cell size is unchanged in schizophrenia.
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INTRODUCTION |
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Reduction in size of a brain structure may reflect a reduced size of the constituent glial cells and neurons and their processes as well as (or as an alternative to) a reduced number of neurons. Thus, it is important to document estimates of cell size as well as cell number in brain structures that are of interest in schizophrenia. Here we present our findings with respect to pyramidal cell volume, estimated stereologically, in the hippocampus on both sides of brains taken from 13 people with schizophrenia and 16 controls.
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METHOD |
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The temporal lobes were dissected away from the rest of the brain, and sliced into 5 mm coronal slices throughout their entire length, such that the entirety of the hippocampus was available for histological examination. Each slice was embedded in paraffin wax, and a 25 µm section was cut from its anterior face, mounted on a coated slide, stained with cresyl violet and luxol fast blue, and coverslipped. The outlines of four cytoarchitecturally defined hippocampal subfields were delineated in the manner described by West & Gundersen (1990):
The volume density (Vv) of these subfields was measured on both sides of the brain, using stereological point-counting techniques (Howard & Reed, 1998). Volume density in this study refers to the proportion of each hippocampal subfield that is occupied by pyramidal neuronal cell bodies.
The prepared slides were examined using a 660 objective and an Olympus BX50
microscope mounted with a JVC TK-C1380 colour video camera and stage motor,
which in turn were controlled and viewed on a computer running the Olympus
Cast-Grid 2.0 stereology sampling software. On each slide, each subfield was
examined at specific points positioned in a raster search pattern array which
covered the entirety of the subfield (Fig.
1). The search pattern was 0.5x0.5 mm2 for the
hilus and CA2/3 subfields, 1x1mm2 for the CA1 subfield, and
0.75x0.75 mm2 for the subiculum. Each subfield appeared on an
average of 6.7 slides per case (range 315; in a few cases the
hippocampus was cut obliquely, which meant it appeared in few sections,
although this did not alter the intensity of the sampling). Neuron density
(Nv) was counted in a mean 57 dissectors and
volume density (Vv) was assessed with a mean 88
frames per case; a mean of 89 neurons were counted for
Nv, and a mean of 135 points were counted for
Vv estimation per case. At each such point a
plane within the section was brought into focus, and an array of 36 random
test points thrown over the microscope image. The number of test points that
fell over pyramidal cell bodies was counted, and the mean number of point
counts ([notdef]) per image
calculated. Pyramidal cells were identified on the basis of their position,
orientation shape, presence of an apical dendrite and prominent, single
nucleolus. The volume density was calculated for each subfield by
Vv =
/36.
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In a previous study, the neuron density (number of cells per unit volume, Nv) within each hippocampal subfield had been estimated (Walker et al, 2002; further details available from the author upon request). Using these data, the mean pyramidal cell body volume (VN) was calculated using the formula VN = Vv/Nv.
Statistical methods
The cell volume for each subfield was assessed by repeated-measures
analysis of covariance (ANCOVA), with diagnosis and gender as between-subject
factors and side as a within-subject factor, using SAS version 6.12 for
Macintosh. As there was a significant difference between the brains from the
control and schizophrenia groups in the duration spent in formalin prior to
histological preparation, this variable was entered as a covariate in the
ANCOVA model. The main effect of diagnosis, gender, and interactions of these
with side, gender, and gender and side together were tested for. Given the
number of effects tested for each measure, was set at 0.01. Thus, for
an effect to be significant, it had to generate a value for P of 0.01
or below.
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RESULTS |
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Effects of diagnosis, gender and side
A bar chart of mean cell volume is given in
Fig. 2. The mean cell volumes
(standard deviations in parentheses) for the subfields were as follows:
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There was no significant effect for diagnosis, gender or side for any
subfield. Thus, for the hilus, all F(1,24) 1.22,
P
0.2021; for the CA2/3 subfield, all
F(1,24)
3.25, P
0.0842; for the CA1
subfield, all F(1,23)
1.35, P
0.2574;
for the subiculum, all F(1,23)
2.19, P
0.1522.
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DISCUSSION |
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We addressed the potential of regional specificity of changes in schizophrenia by dividing the hippocampus into four subfields. We did not further divide our hippocampal subfields into anterior and posterior halves. It is thus possible that changes in one half (anterior or posterior) of a subfield might have been masked or diluted by variance in the other half. In a meta-analysis of hippocampal volumes in schizophrenia assessed by MRI it was found that inclusion of the amygdala, abutting on the anterior hippocampus, in the region of interest significantly increased the size of the reduction in volume seen in schizophrenia. The recommendation was made that in future research relative alterations in anterior and posterior hippocampus in schizophrenia should be assessed separately (Nelson et al, 1998). It is also possible that our study might have failed to detect a true reduction in cell size in some hippocampal subfields because of the small sample size (type II error).
Decreases in neuronal size have been reported for other regions of the brain in schizophrenia the dorsolateral prefrontal cortex, anterior cingulate cortex, cerebellar Purkinje cells, substantia nigra and locus caeruleus but not in the motor cortex or calcarine cortex (reviewed by Harrison, 1999). Further studies will be needed before the primacy of these changes in the disease can be judged.
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Clinical Implications and Limitations |
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LIMITATIONS
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ACKNOWLEDGMENTS |
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Received for publication January 24, 2003. Revision received June 2, 2003. Accepted for publication June 4, 2003.
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