Acute, regional inflammatory response after traumatic brain injury: Implications for cellular therapy

Abstract

Background: Although cellular therapy has shown promise in the management of traumatic brain injury (TBI), microenvironment interactions between the intracerebral milieu and therapeutic stem cells are poorly understood. We sought to characterize the acute, regional inflammatory response after TBI.

Methods: Rats underwent a controlled cortical impact (CCI) injury or sham injury, were killed at 6, 12, 24, 48, and 72 hours, and intracerebral fluid (IF) was isolated from the direct injury, penumbral, ipsilateral frontal, and contralateral regions. Cortical and hippocampal areas were also isolated. Regional cytokine levels were measured. Polymorphonuclear cell (PMN) oxidative burst and marker expression were assessed after incubation with the IF. Immunohistochemistry was used to identify intracerebral CD68(+) cells (microglia/macrophages).

Results: The proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, and tumor necrosis factor-alpha were significantly elevated after CCI in the injury and penumbral regions. Increases in the same cytokines were localized to the cortex and the hippocampus. Increased PMN expression of CD11b and L-selectin was identified after incubation with injury or penumbral area IF, without change in PMN oxidative burst. CD68(+) cells were noted in the direct injury and penumbral areas.

Conclusion: The local cerebral milieu in the first 48 hours after TBI is highly proinflammatory. This response is most pronounced in areas at or proximal to the direct injury. The local, acute proinflammatory response after TBI may serve as a therapeutic target of early cell therapy or, conversely, may create an unfavorable local milieu, limiting the efficacy of early cellular therapy.

Figure 1. Intracerebral areas isolated

Four intracerebral areas were isolated, minced, centrifuged, and the supernatant was collected for analysis of cytokines and stimulation of PMN’s.

Figure 2. Elevated intracerebral cytokines identified in specific areas and at specific time points relative to the TBI

The pro-inflammatory cytokines IL-1α (A), IL-1β (B), IL-6 (C), and TNF-α (D) were significantly elevated six hours after CCI in the injury and penumbral regions when compared to sham animals (*p<0.01 for all). IL-1α, IL-1β, and IL-6 remained elevated through 12, 12, and 24 hrs, respectively (*p<0.01 or †p<0.05). In the frontal area, IL-6 was significantly increased at 24 hours (33–50 fold, p<0.01, Dunnett’s test), but not at 6 or 12 hours after TBI.

Figure 3. Elevated intracerebral cytokines identified at specific anatomic locations (1. cortex and 2. hippocampus) at 6 hours after TBI

The pro-inflammatory cytokines IL-1α (A), IL-1β (B), IL-6 (C), and TNF-α (D) were significantly elevated six hours after CCI in the ipsilateral cortex and hippocampus when compared to sham animals (*p<0.05 for all). TNF-α was also elevated in the contralateral cortex and hippocampus. Note – brain section taken from Paxinos and Watson, 2005.

Figure 4. Increased PMN expression of CD11b and L-selectin in the presence of intracerebral interstitial fluid after TBI

The expression of CD11b (A) and Lselectin (B) on unstimulated PMN’s was significantly increased in the area of direct injury and penumbral area at 6 and12 hours, respectively, in the presence of the intracerebral interstitial fluid.

Figure 5. Macrophages/microglia identified in the area of injury

Brain tissue sections were isolated after TBI, stained with DAPI (blue) to identify nuclei, and incubated in FITC-conjugated anti-CD68 antibodies (green) to identify macrophages/microglia. Numerous CD68 positive cells were identified in the area of injury, a moderate number in the penumbral area, and the number of cells decreased rapidly in areas further away from the injury. A complete coronal section, after nissl staining, is shown for perspective.

Figure 6. Macrophages/microglia were not identified in the contralateral hemisphere

Brain tissue sections were isolated after TBI, stained with DAPI (blue) to identify nuclei, and incubated in FITC-conjugated anti-CD68 antibodies (green) to identify macrophages/microglia. The contralateral hemisphere contained few CD68 positive cells.