Inflammation is increased with anxiety- and depression-like signs in a rat model of spinal cord injury

Abstract

Spinal cord injury (SCI) leads to increased anxiety and depression in as many as 60% of patients. Yet, despite extensive clinical research focused on understanding the variables influencing psychological well-being following SCI, risk factors that decrease it remain unclear. We hypothesized that excitation of the immune system, inherent to SCI, may contribute to the decrease in psychological well-being. To test this hypothesis, we used a battery of established behavioral tests to assess depression and anxiety in spinally contused rats. The behavioral tests, and subsequent statistical analyses, revealed three cohorts of subjects that displayed behavioral characteristics of (1) depression, (2) depression and anxiety, or (3) no signs of decreased psychological well-being. Subsequent molecular analyses demonstrated that the psychological cohorts differed not only in behavioral symptoms, but also in peripheral (serum) and central (hippocampi and spinal cord) levels of pro-inflammatory cytokines. Subjects exhibiting a purely depression-like profile showed higher levels of pro-inflammatory cytokines peripherally, whereas subjects exhibiting a depression- and anxiety-like profile showed higher levels of pro-inflammatory cytokines centrally (hippocampi and spinal cord). These changes in inflammation were not associated with injury severity; suggesting that the association between inflammation and the expression of behaviors characteristic of decreased psychological well-being was not confounded by differential impairments in motor ability. These data support the hypothesis that inflammatory changes are associated with decreased psychological well-being following SCI.

Keywords: Anxiety; Depression; Inflammation; Spinal cord injury.

Figure 1

Experimental schedule.

Figure 2

Recovery as a function of injury severity. Commensurate with the severity of their injury, the groups showed significantly different converted BBB scores across the recovery period (A). Recovery of locomotor function decreased as injury severity increased. Similarly, the severe injury group showed significantly less weight gain than all other groups (B), and consumed less food than the moderate and intact groups (C). The mean (± SEM) performance of the intact, mild, moderate and severe injury groups is depicted.

Figure 3

The panels of graphs depict the mean (± SEM) behavioral performance of the intact, mild, moderate and severe injury groups on the tests of depression and anxiety: Sucrose preference (A), Open field activity (B), Social exploration (C), Forced swim (D), Center activity (E) and Shock probe burying (F). No significant differences were found among injury severity groups on any of the measures.

Figure 4

Hierarchical cluster dendrogram. The dendrogram illustrates the results of the hierarchical cluster analysis, which separated the sample into three main cohorts (Group 1 (n=15), Group 2 (n=12), Group 3 (n=20)), based on the subjects’ average performance on the behavioral tests retained by the principal component analyses. The numbers on the y-axis represent the individual subjects.

Figure 5

Comparison of baseline measures of anxiety and depression (Mean ± SEM), across the psychological well-being groups. There were no significant baseline differences across groups for sucrose preference (A). However, the DEP and ANX/DEP showed lower activity in an open field relative to HEALTHY subjects at baseline (B), and the DEP group exhibited less social exploration than the HEALTHY group (C). Baseline differences were not found for the measure of passive anxiety, center time in the open field (D). * p <.05.

Figure 6

Post hierarchical cluster differences: The panels of graphs depict the mean (± SEM) behavioral performance of the ANX/DEP, DEP, and HEALTHY groups on the tests of depression and anxiety: Sucrose preference (A), Open field activity (B), Social exploration (C), and Shock probe burying (D). ANX/DEP subjects showed decreased sucrose preference relative to the HEALTHY group on Days 10 and 21, as well as increased shock probe burying relative to the DEP and HEALTHY groups on Days 5 and 20. The DEP group showed decreased open field activity relative to the ANX/DEP and HEALTHY group on Day 10 and relative to the HEALTHY group on Day 21. The DEP group also showed decreased social exploration on Day 10 relative to the HEALTHY group. * p <.05.

Figure 7

Psychological well-being membership by injury severity. This figure shows group membership by injury severity, and indicates the percentage of subjects identified as either anxious/depressed or depressed for each injury severity level.

Figure 8

Center activity as measure of passive anxiety. The mean (± SEM) performance of the ANX/DEP, DEP, and HEALTHY for center activity in an open field is depicted for Days 10 and 21. On Day 21, the ANX/DEP and DEP groups showed decreased center activity relative to the HEALTHY group. * p <.05.

Figure 9

Recovery measures by psychological well-being. No differences were found among ANX/DEP, DEP and HEALTHY groups for converted BBB scores (A), weight gain (B), or food consumption (C).

Figure 10

No correlations were found between BBB scores and performances on behavioral tests of depression [open field (A) and (B)] and anxiety [center activity in an open field (C) and (D)].

Figure 11

Tactile withdrawal (A) and vocalization thresholds (B) measures by psychological well-being, on Days 11 and 22. The mean (± SEM) performance of the ANX/DEP, DEP, and HEALTHY groups are shown on the tactile tests. The ANX/DEP group showed a lower paw withdrawal threshold than the HEALTHY group on Day 22. Similarly, both the ANX/DEP and the DEP group showed a lower vocalization threshold than the HEALTHY group. * p <.05.

Figure 12

Thymus weight by psychological well-being groups (intact subjects excluded). The mean (± SEM) thymus weight of the DEP group was significantly lower than that of the HEALTHY group. * p <.05.

Figure 13

Alpha-2 macroglobulin levels in serum on Days 1 and 24 by psychological well-being. The mean (± SEM) alpha-2 macroglobulin levels, normalized to intact subjects, are shown by psychological well-being. A trend for both the ANX/DEP and DEP groups to show increased levels of serum alpha-2 macroglobulin on Day 1 relative to the HEALTHY group was found, p = .16 and .09 respectively.

Figure 14

The serum expression levels of pro- and anti-inflammatory cytokines in the serum on Days 1 (A) and 24 (B). The mean (± SEM) concentration of analytes that differed significantly across psychological well-being groups are shown. The graph depicts the original data, with the HEALTHY group separated into healthy contused subjects and healthy intact subjects to denote possible HEALTHY group differences due to intact subjects. The statistical differences reported here are based on post-hoc Sidak comparisons between the ANX/DEP, DEP and HEALTHY groups, with intacts removed. The ANX/DEP and DEP groups showed increases in both pro-inflammatory cytokines and chemokines on both days 1 and 24. * p <.05.

Figure 15

The mean (± SEM) concentration of analytes in lysate (normalized to total protein concentration) found to differ by psychological well-being are shown. The graph depicts the original data, with the HEALTHY group separated into healthy contused subjects and healthy intact subjects to identify possible HEALTHY group differences driven by intact subjects (rather than psychological well-being). The statistical differences reported here are based on post-hoc Sidak comparisons between the ANX/DEP, DEP and HEALTHY groups, with intacts removed. As the graphs indicate, even with intacts removed, the ANX/DEP group shows increases in both pro-inflammatory cytokines and chemokines.*p<.05.

Figure 16

The mean (± SEM) concentration of analytes in lysate (normalized to total protein concentration) found to differ by psychological well-being are shown. The graph depicts the original data, with the HEALTHY group separated into healthy contused subjects and healthy intact subjects to denote possible HEALTHY group differences due to intact subjects alone. The statistical differences reported here are based on post-hoc Sidak comparisons between the ANX/DEP, DEP and HEALTHY groups, with intacts removed. As the graphs indicate, even with intacts removed, the ANX/DEP group shows increases in both pro-inflammatory cytokines and chemokines. * p <.05.

Figure 17

The mean (± SEM) concentrations of pro-inflammatory cytokines and chemokines in lysate (normalized to total protein concentration) are shown for the intact, mild, moderate and severe injury groups. Analytes are separated into graphs A and B to clearly show differences for cytokines/ chemokines with higher or lower concentrations. * p <.05.