Transient blockage of the CD11d/CD18 integrin reduces contusion volume and macrophage infiltration after traumatic brain injury in rats

Abstract

The early inflammatory response to traumatic brain injury (TBI) may result in secondary damage. The purpose of this study was to evaluate the effects of a transient treatment employing a blocking monoclonal antibody (mAb) to the CD11d/CD18 integrin on histopathological outcome and macrophage infiltration following TBI. A parasagittal fluid percussion (FP) brain injury (1.8-2.1 atm) was induced in male Sprague-Dawley rats. Rats were randomized into two trauma groups, treated (N=7) and nontreated (N=8) animals. In the treated group, a mAb to the CD11d subunit of the CD11d/CD18 integrin was administered 30 min, 24 and 48 h after brain injury. Control animals received an isotype-matched irrelevant mAb using the same dose and treatment regimen. At 3 days after TBI, animals were perfusion-fixed for histopathological and immunocytochemical analysis. The anti-CD11d mAb treatment reduced contusion areas as well as overall contusion volume compared to vehicle treated animals. For example, overall contusion volume was reduced from 2.7+/-0.5 mm(3) (mean+/-SEM) to 1.4+/-0.4 with treatment (p<0.05). Immunocytochemical studies identifying CD68 immunoreactive macrophages showed that treatment caused significant attenuation of leukocyte infiltration into the contused cortical areas. These data emphasize the beneficial effects of blocking inflammatory cell recruitment into the injured brain on histopathological outcome following traumatic brain injury.

Figure 1

Hematoxylin and eosin (H&E) stained paraffin sections of cortical contusions at 3 days post-TBI in vehicle and anti-CD11d treated animals. (A) Vehicle treated animal shows a large hemorrhagic contusion at the gray-white matter interface. (B) anti-CD11d treated animal has a much more focal area of injury after TBI. The upper boundaries of the contusion area are denoted with arrowheads (10X).

Figure 2

CD68 stained immunopositive cells in the medial cortex (A) TBI vehicle animal with a large number of infiltrating/activated cells (10x) (B) Higher magnification of the medial cortex of a vehicle treated TBI animal. Arrowheads point to representative CD68 immunopositive cells (40X). (C) TBI anti-CD11d mAb treated animal. There are a smaller number of CD68 immunopositive cells within the medial cortex of antibody treated animals (10X). (D) Higher magnification of anti-CD11d treated animal showing reduced number of microglia/macrophges in this vulnerable cortical area (40x).

Figure 3

Bar graph of mean contusion areas and volume +SEM. Repeated measures ANOVA was significant (p<0.05) for group, bregma level and group x level. (A) Anti-CD11d treatment significantly reduced contusion area at bregma level −3.3, −4.3, −5.8 and −6.8 compared to vehicle treated animals. (*p<0.05). (B) Animals treated with the anti-CD11d antibody had smaller contusion volumes compared to vehicle treated animals. (*p<0.05)

Figure 4

Bar graph of mean +SEM of total number of CD68 immunopositive cells in the medial and lateral cerebral cortex. Anti-CD11d treatment significantly (*p<0.05) reduced the number of activated microglia/macrophages within each cortical area ipsilateral to the injury site.

Figure 5

Bar graph of mean +SEM of total number of CD68 immunopositive cells in the ipsilateral hippocampus. Anti-CD11d treated animals had significantly (p<0.05) lower numbers of microglia/macrophages compared to vehicle treated animals.