Wolfson Research Centre, Institute for Ageing and Health, Newcastle General Hospital, Newcastle upon Tyne, UK
Correspondence: Alan J. Thomas, Wolfson Research Centre, Institute for Ageing and Health, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE, UK. Tel: 0191256 3323; fax: 0191 219 5051; e-mail: a.j.thomas{at}ncl.ac.uk
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ABSTRACT |
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Aims We investigated the expression of cell adhesion molecules (CAMs) in the prefrontal cortex in depression.
Method Immunohistochemistry to localise CAMs in post-mortem tissue from 20 subjects with major depression and 20 controls, and image analysis to quantify their expression.
Results We found significant increases in CAMs in the grey matter of the DLPFC in the depression group but no comparable differences in the ACC or occipital cortex. In the white matter there was a non-significant increase in intercellular adhesion molecule-I in the DLPFC in the depression group but no increase in the other areas or for vascular cell adhesion molecule-I in any area. Paired tests showed specificity for the DLPFC in the depression group only.
Conclusions The increase in CAM expression in the DLPFC suggests an inflammatory reaction and is consistent with ischaemia.
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INTRODUCTION |
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METHOD |
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All subjects in the depression group had undergone extensive assessments, including a full history, mental state examination, physical examination, screening blood tests, cognitive tests and, in some cases, computed tomography or magnetic resonance imaging scans. All had received standard antidepressant treatment regimes, with selective serotonin reuptake inhibitors or tricyclic antidepressants singly or often in combination with other agents, and 11 had received electroconvulsive therapy. No control subject had taken any antidepressant or antipsychotic medication. The case notes on all subjects were examined to see if they had a history of hypertension sufficient to need treatment with antihypertensive medication. All subjects had had a full post-mortem assessment (except one whose body was unavailable for autopsy) and the post-mortem delay was recorded.
Tissue
After death the right hemisphere was fixed in 10% formalin and the brains
were dissected in a standard manner. To obtain tissue blocks for analysis,
tissue was selected from three areas: the DLPFC (Brodmann areas (BA) 9 and
46), the ACC (BA 24) and the occipital cortex (BA 19 and 39) as a comparison
area. The DLPFC blocks were chosen by carefully selecting the coronal slice
from each subject to include BA 9 and 46 according to a standard map
(Perry, 1993). Owing to the
variation in humans, this may have included BA 10 in some subjects. The ACC
block was taken from BA 24 just rostral to the genu of the corpus callosum or,
occasionally, just including the rostral tip of the genu. These blocks were
chosen to examine the areas identified as reduced in function in depression in
the DLPFC (Bench et al,
1992) and ACC (Drevets et
al, 1997). The occipital cortex block was chosen to determine
whether any changes in the prefrontal areas were specific for these areas or
occurred throughout the brain. These blocks were embedded in paraffin wax, the
duration of fixation of these large blocks was recorded and they were
sectioned using a sledge microtome into 10-µm sections (one per subject) on
large slides (3 x 2''). These slides were coded so that all
analysis could be carried out blind to diagnosis.
Immunocytochemistry
Sections were processed for immunocytochemical localisation in a standard
manner. Briefly, sections were dewaxed in xylene, rehydrated and microwaved in
0.01% citrate buffer (pH 6.0) to optimise antigen retrieval. They were
immersed in hydrogen peroxide, blocked with an appropriate serum and incubated
for 1 h at room temperature with the primary antibody. The primary antibodies
used were a polyclonal antibody to intercellular adhesion molecule-1 (ICAM-1)
(R&D Systems; 1:500 dilution), a polyclonal antibody to vascular cell
adhesion molecule-1 (VCAM-1) (R&D Systems; 1:800 dilution) and a
monoclonal antibody to collagen IV (Sigma; 1:500 dilution).
Immunocytochemistry was carried out using slides with code numbers to ensure
blindedness and with a random order of depression and control cases. Slides
were processed together for each primary antibody in order to ensure that the
immunocytochemistry conditions were the same for both groups. The expression
of ICAM-1 and VCAM-1 is increased by ischaemia
(Kim, 1996) in cerebral
endothelial cells in vitro and studies of human ischaemic stroke have
also shown increased ICAM-1 expression in the microvessels in association with
the stroke lesions (Lindsberg et
al, 1996). Both ICAM-1 and VCAM-1 were therefore chosen as
putative markers of cerebral ischaemia. Collagen IV (a basement membrane
protein) is a marker of the density of the microvascular tree and was measured
to determine whether any differences in ICAM-1 or VCAM-1 reflected alterations
in the whole vascular tree rather than in ICAM-1 or VCAM-1 expression in the
vessels (Kalaria & Hedera,
1995). Appropriate secondary antibodies were then applied followed
by avidin-biotinylated horseradish peroxidase complex (Vector Laboratories
Ltd) and diaminobenzidine (DAB) as a chromagen. All sections were lightly
counterstained with haematoxylin and were examined using a light microscope to
check the quality of staining before proceeding to quantitative analysis.
Quantitative analysis
Analysis was conducted on one section per anatomical area per subject,
based on calculations of the variability between fields and between different
immunocytochemical assays. The mean coefficient of error for five fields (from
five subjects) was 6.8% and there was very little difference whether fields
were taken from one section per subject (mean coefficient of error=6.6% for
ten fields from five subjects) or from two sections per subject run in
different immunocytochemistry assays (mean coefficient of error=7.3% for ten
fields, five per section, from five subjects).
Images were captured using a x 10 objective lens on a Zeiss Axloplan 2 light microscope coupled to a three-chip CCD true-colour video camera (JVC KY F55B), producing a field size of 185 000 µm2 (0.185 mm2). For each antibody, five images selected randomly were captured from the grey matter and ten from the white matter on each section from the DLPFC and the occipital cortex, and ten from the grey matter and fifteen from the white matter in the ACC. Images were obtained randomly by the operator selecting a field by moving the stage while not looking at the section. If the field was outside the area of interest (grey or white matter) then this was repeated until the field included the required tissue. This ensured that each field was selected independently of the microscopic appearance of its microvessels. They were analysed blind to diagnosis on a monitor using a standard software program (Image Pro Plus, version 4.0; Media Cybernetics). This involved measuring the area of DAB staining, expressed as a percentage of the total image area (areal fraction), and then calculating a mean score for the grey and white matter for each cortical area. The areal fraction was not obtained using thresholding because this method was found to lead to inappropriate inclusion of structures that were not stained with the CAM antibodies. Instead, the dropper method was used, in which the operator selected DAB-stained microvessels by eye in each field according to their exact redgreenblue colour characteristics; this avoided the thresholding problem of including other structures. Interrater and intrarater reliability ratings were calculated (from 48 images on 16 patients) for the two raters using the intraclass correlation coefficient and the coefficient of variation, respectively.
Statistical analysis
Statistical comparisons were carried out using SPSS software (Version 9.0).
Tests for normality were conducted and un-paired, two-way Student's
t-tests or MannWhitney tests were used, as appropriate, to
compare ICAM-1 and VCAM-1 expression in the depression and control groups. To
examine whether any changes in ICAM-1 and VCAM-1 expression showed specificity
for the prefrontal areas, paired t-tests and Wilcoxon signed rank
tests were used, as appropriate, for within-group comparisons. Secondary
analyses were conducted to examine possible confounders. These included
comparison of collagen IV expression in the two groups and possible effects of
treatment and hypertension on CAM expression. Pearson correlation
coefficients, t-tests or MannWhitney tests, and analysis of
variance were used, as appropriate, for these analyses.
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RESULTS |
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Comparison of depression and control groups
The ICAM-1 expression was significantly higher in the depression group in
both the grey matter (t=2.77, d.f.=38, P=0.009) and the
white matter (t=2.28, d.f.=38, P=0.029) in the DLPFC, but
not in the ACC (grey matter: t=0.86, d.f.=38, P=0.394; white
matter: t=1.15, d.f.=38, P=0.258). The VCAM-1 expression
also was significantly higher in the grey matter of the DLPFC (W=321,
P=0.017) but not in the white matter (W=362,
P=0.199). In the occipital cortex, ICAM-1 was significantly increased
in depression in the grey matter (t=2.10, d.f.=38, P=0.042)
but not in the white matter (t=1.55, d.f=38, P=0.128). There
were no significant differences in VCAM-1 immuno-reactivity in either the ACC
(grey matter: W=397, P=0.725; white matter: W=468,
P=0.123) or the occipital cortex (grey matter: W=368,
P=0.262; white matter: W=4.17, P=0.871). A
Bonferroni correction was set at 0.017, based on testing three areas in each
subject, and the increase in the grey matter of the DLPFC remained significant
for both ICAM-1 and VCAM-1. Such corrections were not made for using both
ICAM-1 and VCAM-1 or for assessing grey and white matter, because we had
hypothesised a priori that these measures would be highly correlated
and correcting for these would have been too stringent.
Comparison of areas within groups
In the grey matter, paired t-tests showed that ICAM-1 expression
in the DLPFC in the depression subjects was highly significantly elevated
compared with both the ACC (t=3.05, P=0.01) and the
occipital cortex (t=2.72, P=0.01), whereas there was no
difference between the ACC and the occipital cortex (t=1.17,
P=0.26). Similarly in the white matter, there was a highly
significant increase in ICAM-1 in the DLPFC compared with the ACC
(t=3.85, P=0.001) and occipital cortex (t=4.08,
P=0.001) but no difference between the ACC and the occipital cortex
(t=1.77, P=0.09). In contrast, in grey matter in the control
group there were no significant differences between the DLPFC and the ACC
(t=1.26, P=0.22), the DLPFC and the occipital cortex
(t=1.71, P=0.10) or the ACC and the occipital cortex
(t=0.02, P=0.98). In the white matter of the control group
there was a significant increase in ICAM-1 in the DLPFC compared with the ACC
(t=2.59, P=0.02) but no significant differences between the
DLPFC and the occipital cortex (t=1.76, P=0.10) or between
the ACC and the occipital cortex (t=1.39, P=0.18). Similarly
for VCAM-1, there was a significant increase in the grey matter of the
depression group in the DLPFC compared with both the ACC (Z=-3.44,
P=0.001) and the occipital cortex (Z=-1.98, P=0.05)
but not in the ACC compared with the occipital cortex (Z=-1.34,
P=0.18). In the white matter of the depression subjects VCAM-1 was
highly significantly elevated in the DLPFC compared with both the ACC
(Z=-3.58, P<0.001) and the occipital cortex
(Z=-3.06, P=0.002) but there was no difference in VCAM-1
expression between the ACC and occipital cortex (Z=-1.53,
P=0.13). In contrast, in the control subjects there were no
differences in VCAM-1 expression in the grey matter between the DLPFC and the
ACC (Z=-1.32, P=0.19), the DLPFC and the occipital cortex
(Z=-0.85, P=0.40) or the ACC and the occipital cortex
(Z=-0.06, P=0.96). In the control subjects' white matter
there was a significant elevation of VCAM-1 in the DLPFC compared with the ACC
(Z=-2.20, P=0.03) and a trend towards significance between
the DLPFC and the occipital cortex (Z=-1.83, P=0.07) but no
difference between the ACC and the occipital cortex (Z=-0.43,
P=0.67).
Assessment of potential confounders
There were no significant group differences in the expression of collagen
IV in any area in either grey matter (t<0.85, d.f.=38,
P>0.399) or white matter (t<1.43, d.f.=38,
P>0.16). In the total group studied there was no correlation
between age, post-mortem delay or duration of fixation and any of the four
significant results (age: r<0.095, n=40,
P>0.56; post-mortem delay: r<0.14, n=40,
P>0.37; fixation: r<0.233, n=40,
p>0.148). Hypertension did not account for the group differences
(adjusted R2 for contribution of hypertension <0.053)
and a post hoc comparison of those with and without hypertension
(regardless of whether or not they had depression) found no group differences
for either ICAM-1 (t<1.07, d.f.=30, P>0.291) or VCAM-1
(t<0.78, d.f.=30, P>0.439) in any area for either grey
or white matter.
In the depression group we found no correlation between age of onset of depression or any of the four significant outcome measures (r<0.224, n=20, P>0.343). The group had a late age of onset of depression (see Table 1), with only six subjects having onset before 60 years and only three before 45 years. Using MannWhitney tests there was no difference between those who had received (n=11) and those who had not received (n=9) electroconvulsive therapy (W>108, P>0.552), nor was there any difference between those who had (n=13) and those who had not (n=7) received anti-depressant treatment within a week of death (W>60, P>0.275). Two suicides were included in the depression group and none of the results was changed by re-analysis without their inclusion.
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DISCUSSION |
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Increase in CAM expression is consistent with ischaemia
We chose to test the vascular depression hypothesis
(Alexopoulos et al,
1997) by measuring the expression of ICAM-1 and VCAM-1 in the
prefrontal cortex because these two molecules are increased by ischaemia
in vitro (Kim, 1996)
and studies of human ischaemic stroke have also shown increased ICAM-1
expression in the microvessels in association with the stroke lesions
(Lindsberg et al,
1996). There is increasing evidence that ischaemic brain damage
develops over a much longer period than was previously believed
(Stoll et al, 1998) and our findings are therefore comparable with a chronic reduction in cerebral
perfusion to the DLPFC as well as with acute events. A recent study in the
DLPFC suggested that elderly people with depression have increased astrocytes
(Miguel-Hidalgo et al,
2000), which would be consistent with our finding of increased CAM
expression in the DLPFC because post-ischaemic inflammatory changes would
increase astrocyte activity. Studies in younger patients with depression have
reported a reduction in glia in both the ACC
(Ongur et al, 1998)
and the DLPFC (Rajkowska et al,
1999), which could lead to a lack of glial support for neurons and
increased neuronal vulnerability. In elderly subjects chronic and/or acute
ischaemia could lead to an inflammatory response causing neuronal damage and
subsequent increased glial activity. However, the extent to which these
findings can be generalised to late-life depression in general is unclear; our
depression subjects were all hospital patients who had had multiple episodes
of depression.
Other possible explanations for the findings
Although we carried out the study to test for evidence of ischaemia, we
cannot exclude the possibility that depression mediates increases in CAM
expression by other mechanisms, although the weight of evidence associating
depression with vascular diseases, especially in the elderly
(Alexopoulos et al,
1997), favours prefrontal ischaemia as the most likely explanation
of the current results. Another possible pathway leading to increased CAM
expression could be the increased circulating levels of cytokines (e.g.
interleukin 1 and tumour necrosis factor alpha), which have also been
described in depression (Connor &
Leonard, 1998). These cytokines stimulate the expression of CAMs
and thus form a possible link.
The two groups were similar in age, gender, post-mortem delay and duration of tissue fixation, and our analysis of the data found no evidence that the results could be explained by any of these factors. In a previous study (Thomas et al, 2001) we found no differences in clinical measures of vascular risk (e.g. hypertension) between the two groups and so the results do not appear to be due to such confounders. Age of onset of depression did not affect the results, but because most of the depression group had a late age of onset we could not fully explore the extent to which increased CAM expression might differ in an early-onset group. We also found no evidence that treatments unique to the depression group (antidepressants and electroconvulsive therapy) could account for the group differences. Because this was a case-note study we have been unable to examine fully all possible confounding factors and it is possible that the up-regulation of the CAMs was due to some other factor or factors that we have been unable to exclude as a confounder. Increased CAM expression occurs in association with amyloid plaques in Alzheimer's disease and after stroke (Kim, 1996), but our neuropathological assessment has excluded such confounders as explanations. We measured the expression of collagen IV to examine the possibility that any differences in CAM expression were due to quantitative differences in the cerebral endothelium (Kalaria & Hedera, 1995) but because no differences in collagen IV were found we have ruled out this possible explanation as well.
Importance of DLPFC in depression in the elderly
Our early findings (Thomas et
al, 2000) showed increased ICAM-1 expression in the DLPFC in
depression. We have now extended this by replicating the finding with VCAM-1
and found such increased immunoreactivity to show some specificity for the
DLPFC that is not found in the ACC. One possible explanation for this pattern
is that the depressive symptomatology in our subjects was produced by
disproportionate dysfunction in the frontal-subcortical circuit linking the
DLPFC to the head of the caudate nucleus rather than in the circuit linking
the ACC to the nucleus accumbens (Alexander
et al, 1986). Alternatively, because the DLPFC lies in an
area of the prefrontal cortex between the territories of the anterior and
middle cerebral arteries, it appears to be more vulnerable to ischaemia,
especially owing to haemodynamic alterations
(Chui & Willis, 1999). Our
results are consistent with this because we found a higher CAM
immunoreactivity in the DLPFC in both groups, but significantly more so for
subjects with depression. These two alternatives could be related because
ischaemic damage to the DLPFC would produce a characteristic pattern of
depressive symptomatology involving retardation, poor concentration and
dysexecutive failure. Such a pattern of executive dysfunction has been
associated with poor response to antidepressant treatment and with relapse and
recurrence in elderly subjects with depression
(Alexopoulos et al,
2000), which suggests that our findings have clinical
importance.
Although further investigation is required, our findings potentially have major implications for the aetiology and management of depression in the elderly. If further research shows that post-ischaemic inflammation is involved in late-life depression, the use of anti-inflammatory treatments (e.g. non-steroidal anti-inflammatory agents or Cox-2 inhibitors) may become indicated to reduce inflammation and prevent further tissue injury.
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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 June 8, 2001. Revision received April 5, 2002. Accepted for publication April 9, 2002.
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