ICAM-1-LIKE IMMUNOREACTIVITY IN MDD AND NON-PSYCHIATRIC COMPARISON SUBJECTS
In all subjects blood vessels were easily identified by their strong ICAM-1 immunoreactivity ( and ). Immunoreactive structures without blood vessel morphology were sparse in subjects younger than 60, but were common in all cortical layers in subjects over 60 (). This extravascular ICAM-1 immunoreactivity, described previously (Miguel-Hidalgo et al., 2007
), takes the appearance of round immunoreactive regions between 100 and 300 μm in diameter, at times fused into larger conglomerates (). The average total area fraction of ICAM-1 immunoreactivity, including vascular and extravascular immunoreactivity, was lower in subjects with MDD than in non-psychiatric comparison subjects (F(1,31)=6.35, p<0.02) (); ANCOVA using age, pH, postmortem interval, and storage time as covariates). The area fraction of vascular and extravascular ICAM-1 immunoreactivity was also significantly lower in subjects with MDD as compared to non-psychiatric comparison subjects (vascular F(1,31)=8.86 p<0.01; extravascular F(1,31)=4.85, p<0.04) ().
Figure 1 Low power micrographs of ICAM-1 immunoreactivity (I to VI between dashed lines indicate the extent of the six cortical layers in all the pictures) in area 47 of 4 human subjects of different age. A) 30 years old non-psychiatric comparison subject (CONTROL); (more ...)
Figure 2 High power magnification micrographs of ICAM-1 immunoreactivity in layer III of cortical area 47. The top panels correspond to younger non-psychiatric comparison subject (CONTROL)(A) and subjects with MDD (B), ages 30 and 34 at the time of death, respectively. (more ...)
Figure 3 Bar graphs representing the average total area fraction of all ICAM-1-IR structures combined (A), the area fraction of immunoreactive structures with vascular morphology (B), and the area fraction of extravascular patches of ICAM-1 immunoreactivity (C) (more ...)
ICAM-1 IMMUNOREACTIVITY IN AGE SUBGROUPS
To asses whether differences between the comparison group and subjects with MDD are age-dependent, each diagnostic group was divided into one subgroup of subjects younger than 60 and another subgroup of subjects older than 60, following a previously reported criterion (Miguel-Hidalgo et al., 2007
). The cut-off at 60 was chosen because in another study on ICAM-1 in the PFC subjects were considered elderly above 60 years old (25) an over that age there are significant increases in GFAP. Furthermore several neuroimaging studies also consider 60 years old as a cutoff for studying elderly patients (Lai et al., 2000
; Steffens et al., 2003
; Taylor et al., 2004
). Two-way ANCOVA using age group (older vs. younger) and disease group (subjects with MDD vs. comparison subjects) as fixed factors revealed a significant difference in the total area fraction (F(1,30)=5.75, p=0.023), and in the area fraction of the vascular (F(1,30)=7.67, p<0.01) and extravascular compartments (F(1,30)=3.51, p=0.071) of ICAM-1 immunoreactivity (with brain pH, storage time, and postmortem delay used as covariates) between subjects with MDD and subjects in the comparison group. There was a strong significant interaction between age group and disease group for total ICAM-1 (F(1,30)=11.835, p<0.005) and for extravascular ICAM-1 (F(1,30)=10.35, p<0.005) indicating a much higher level of ICAM-1 in the older subjects of the comparison group. ANCOVAs for the four resulting groups revealed a significant difference in the total area fraction of ICAM-1 (F(3,30)=16.17, p<0.0005) and the area fraction of the vascular (F(3,30)=4.00, p<0.02) and extravascular compartments of ICAM-1 (F(3,30)=14.77, p<0.001)(with brain pH, storage time, and postmortem interval used as covariates)
Pairwise univariate contrasts showed that the area fraction of vascular ICAM-1 immunoreactivity was significantly higher in older comparison subjects than in older subjects with MDD by 40% (F(1,30)=10.59, p< 0.005) (). The area fraction of vascular ICAM-1 in the comparison group was not significantly higher in older subjects as compared to younger subjects. There was also no significant difference in older subjects with MDD as compared to younger subjects with MDD. There was a tendency for a lower area fraction in young subjects with MDD as compared to young subjects of the comparison non-psychiatric group, but in the posthoc univariate analysis the difference was not statistically significant (F(1,30)=1.124, p=0.298).
Figure 4 Bar graphs representing the area fraction of vascular (A) and extravascular ICAM-1 (B) immunoreactivity according to age (young are subjects younger than 60, old are subjects 60 and older). There is very little or no change in vascular area fraction in (more ...)
In the non-psychiatric comparison groups the area fraction of extravascular ICAM-1 in older subjects was dramatically higher than in younger subjects (F(1,30)=37.59, p< 0.0005) by 1496%. This area fraction in older subjects with MDD was also significantly lower as compared to older comparison subjects (F(1,30)=13.86, p<0.001) by 247% (). The area fraction in older subjects with MDD, although with a trend for higher values, was not significantly different from that in younger subjects with MDD (F(1,30)=3.099, p<0.089). The area fraction of extravascular ICAM-1 immunoreactivity in younger subjects with MDD was not significantly different from younger comparison subjects (F(1,30)=0.698, p< 0.410).
ASSOCIATION WITH SUICIDE
In the MDD group 11 subjects had died by suicide, so the possibility existed that ICAM-1 immunoreactivity might differ in subjects with MDD dying by suicide as compared to those dying by other causes. For the extravascular compartment of ICAM-1 immunoreactivity, 2-way ANCOVA (with age group and suicide as factors, and pH, PMI and storage time as covariates) revealed a significant difference between suicides and non-suicides (F(1, 11) = 21.85 , p<0.005), and a significant interaction between suicide and age (F(1, 11) = 19.36 , p<0.005), with those subjects dying by suicide after 60 having significantly lower values of ICAM-1 immunoreactivity than subjects with MDD not dying by suicide. For the vascular ICAM-1 compartment there was no difference between suicide and non-suicide subjects with MDD (F(1, 11) = 1.66 , p=0.224), and no significant interaction between age group and suicide (F(1, 11) = 2.02 , p=0.183).
EFFECTS OF ONSET AND DURATION OF DEPRESSION
To determine whether the estimated onset of depression correlated with area fraction of vascular and extravascular ICAM-1, partial correlations were determined between age of onset and area fraction of ICAM-1 immunoreactivity, controlling for age at time of death, postmortem interval, brain pH and storage time. We observed a strongly significant negative correlation between area fraction of extravascular ICAM-1 and age at onset (r = -0.712, p = 0.004, df = 12). Controlling for the same variables, a significant positive correlation was found between extravascular ICAM-1 and the estimated duration of depression. However, the area fraction of vascular ICAM-1 was not significantly correlated with the age of onset (r= -0.186, p = 0.525, df = 12) or the duration of depression (r = 0.179, p = 0.541, df = 12).
EFFECT OF ANTIDEPRESSANT TREATMENT
Thirteen out of 18 subjects with MDD received antidepressant (AD) treatment prior to death. Nine of the 18 subjects had not taken AD medication during the last month of life. To determine whether there was a difference in ICAM-1-I area fraction between AD-medicated and non-AD-medicated subjects with MDD, we first compared the 13 subjects with AD treatment (MDD+AD) to the 5 subjects with MDD without AD treatment; we then compared the 9 subjects with AD treatment during the last month of life (MDD+AD[final month]) to those without treatment, using ANCOVA for both comparisons (covariates: age, brain pH, postmortem interval and storage time). There was no significant effect of AD treatment on the area fraction of either vascular or extravascular ICAM-1 immunoreactivity (for no AD treatment vs. AD treatment F(1,12)=0.117, p=0.738 vascular and F(1,12)=1.785, p=0.206 extravascular; for no treatment during last month vs. treatment within last month, F(1,12)=0.362, p=0.559 vascular and F(1,12)=1.202, p=0.294 extravascular).
COLOCALIZATION OF ICAM-1 IMMUNOREACTIVITY WITH GFAP-POSITIVE ASTROCYTES
We previously found that extravascular ICAM-1 structures were spatially associated almost exclusively with GFAP immunoreactive processes in non-psychiatric comparison subjects (10). We therefore examined whether extravascular ICAM-1 immunoreactivity in subjects with MDD was associated with GFAP-immunofluorescent processes in sections near those used for non-fluorescent labeling of ICAM-1 (three sections per brain). Then, we determined the area percentage of ICAM-1-I extravascular structures in spatial register with GFAP immunoreactive structures () In all subjects we found no less than 96% of ICAM-1 extravascular immunoreactivity in register with GFAP immunoreactive structures.