Neuroinflammation and tissue damage
Consistent with previous findings from our laboratory , repeated mLFP in the rat induced a long-term neuroinflammatory response involving increases in activated neutrophils, microglia/macrophages, and astrocytes within the injured brain. This response occurred concurrently with increased lipid peroxidation, axonal injury, neuronal loss, and behavioral impairments. The oxidative burst of activated neutrophils and macrophages generates free radicals that can lead to secondary CNS injury in the form of peroxidation of lipid membranes [19, 35, 36]. The enzyme MPO is part of the pathway that generates oxidative stress via formation of hydrogen peroxide and other oxidative molecules, and is present in large quantities in granules within neutrophils and, to a lesser extent, macrophages [37, 38]. Activated astrocytes are also active participants in neuroinflammation . Astrocytes respond to a host of pro-inflammatory mediators released by activated leukocytes, and in turn release additional cytokines that can regulate microglia, neurons, and other astrocytes .
Anti-CD11d mAb treatment significantly reduced activated neutrophils, macrophages, and astrocytes in the SR condition relative to repeated mLFP rats treated with a 1B7 control mAb. Analysis of neuronal loss, as monitored by immunohistochemistry and western blot analysis of brain homogenates, revealed a significant increase in NeuN expression in the CD11d-SR rats compared to the 1B7-SR rats. Additionally, analysis of axonal injury, as monitored by immunohistochemical and western blot analysis of the corpus callosum, revealed a significant decrease in APP accumulation in the CD11d-SR rats compared to the 1B7-SR rats. Reductions in neuronal loss and axonal injury by anti-CD11d mAb are both likely mechanisms for the behavioral recovery associated with the treatment. As the anti-CD11d mAb treatment also reduced MPO levels and lipid peroxidation in the injured brain, oxidative stress and lipid peroxidation appear to be key mechanisms of inflammatory-mediated secondary damage and associated behavioral impairments in repeated mLFP. We have previously reported that the anti-CD11d treatment changes the nature of the cytokine/chemokine response from a pro-inflammatory response to a wound healing response when delivered acutely after spinal cord injury . Thus the reduced recruitment of neutrophils and macrophages to the lesion site may have led to altered levels of cytokine and chemokine production at the lesion and consequently to reduced levels of leukocyte activation and immune-mediated tissue damage.
At 8 weeks post-injury, the anti-CD11d mAb-treated rats continued to exhibit significant reductions in astrogliosis, axonal injury, and behavioral impairments relative to 1B7-treated rats. However, the anti-CD11d mAb-treated rats were impaired relative to sham controls on some behavioral measures, and exhibited significant neuronal loss, axonal injury, and lipid peroxidation. As the anti-CD11d treatment reduced but did not completely prevent neutrophil and macrophage infiltration of the injured brain, a small inflammatory response may have led to these effects. As the current study is the first to use the anti-CD11d mAb in the complex pathophysiological setting of repeated concussion, the treatment regimen applied may not have been optimal, generating a less robust outcome than may be possible. Other mechanisms of secondary injury are also likely to contribute to the pathology of repeated concussion and might account for some of the detrimental effects observed here . Nonetheless, anti-CD11d mAb treatment at 2 h and 24 h after each of three mLFP significantly reduced neuroinflammation, lipid peroxidation, neuronal loss, axonal injury, and behavioral impairments, indicating that neuroinflammation may be a key contributor to secondary damage in repeated concussions and associated neurodegenerative conditions such as chronic traumatic encephalopathy.
The anti-CD11d mAb treatment improved cognitive function following repeated concussion, as CD11d rats outperformed 1B7 rats in the water maze. During the SR condition, the CD11d rats displayed no impairments in the water maze compared to sham-injured rats and performed better than the 1B7 rats on all measures. Consistent with previous findings from our laboratory, long-term cognitive impairments were present in rats with repeated concussion at 8 weeks post-injury relative to sham controls , regardless of treatment. However, the CD11d rats continued to outperform the 1B7 rats, requiring significantly less time to locate the hidden platform during both acquisition and reversal compared to the 1B7 group.
The 1B7-treated rats also displayed a significant increase in anxiety-like behavior in both the SR and LR conditions as they spent significantly less time in the open arm of the elevated-plus maze compared to the sham controls [32, 42], while the CD11d-treated groups did not . These findings are consistent with previous findings from our laboratory indicating that repeated mLFP increase anxiety-like behavior, and that the anti-CD11d mAb treatment reduces the development of anxiety in rats after TBI .
The 1B7-LR group also exhibited significantly more slips and falls on the beam task compared to sham-LR rats, while the CD11d-LR group did not. These findings are consistent with previous findings from our laboratory indicating that anti-CD11d mAb treatment preserves motor function following TBI . The finding that the 1B7 group displayed deficits on the beam task in the LR condition suggests that locomotor ability should be considered when interpreting water maze and elevated-plus maze results. However, although 1B7 rats displayed increased slips and falls in the LR condition, they were not impaired on the measures of beam traverse times, swim speed in the water maze, and the number of closed arm entries in the elevated-plus maze, suggesting that the impairments suffered by 1B7 rats represent a fine motor or gait deficit rather than a gross motor abnormality . Furthermore, impairments in the water maze and elevated-plus maze also occurred in the 1B7-SR group in the absence of any evidence of motor abnormalities. Thus, it appears unlikely that any motor impairments suffered by the 1B7 group directly accounted for the water maze and the elevated-plus findings.
These results are similar to the findings of other studies in which antibody treatments targeted the infiltration of peripheral leukocytes after single severe TBI in rats [18, 23, 44, 45]. Specifically, we have previously reported that anti-CD11d mAb treatment reduced leukocyte levels, astrocyte activation, lipid peroxidation, and neuronal loss in a severely injured brain, and these changes were accompanied by improved cognitive, emotional, and sensorimotor outcomes relative to injured rats treated with a control mAb [18, 23]. Although key methodological differences limit direct comparisons between these experiments and the current study, it is important to note that anti-CD11d mAb therapy had similar benefits in both mild and severe TBI settings. Furthermore, despite the overall reduction of neutrophil and macrophage populations in the injured brains, animals treated with anti-CD11d mAb do not appear to suffer infections in the CNS or elsewhere . This likely reflects the acute nature of the treatment schedule (i.e. completed by 48 h after each injury), thus limiting the time window during which the anti-CD11d mAb-treated rats might be more vulnerable to infection due to compromised leukocyte trafficking .