J. C. Alonso-Escalante1, D. F. Hamade2, C. P. Sodhi1, W. Fulton1, D. J. Hackam1, I. W. Nasr1 1Johns Hopkins University School Of Medicine,Pediatric Surgery,Baltimore, MD, USA 2American University Of Beirut School Of Medicine,Beirut, BEIRUT, Lebanon
Introduction:
The morbidity of traumatic brain injury (TBI) develops after the convergence of acute and chronic neuroinflammatory pathways that cause axonal injury. The role of the innate and adaptive immune systems in these processes remains incompletely understood. We now hypothesize that innate toll-like receptor 4 (TLR4), and adaptive immune cells are decisive in the development of TBI in mice, and sought to address this by development of an experimental model of TBI in 4-8 week-old male mice.
Methods:
A Controlled Cortical Impact (CCI) murine TBI model using C57BL/6 wild-type (WT), adaptive immune deficient Rag1 KO, and innate immune receptor TLR4 knockout (KO) mice, was performed by shooting a 3-mm diameter piston over intact dura mater with a speed of 2m/s, and a depth of 1.5mm. The volume of brain injury was assessed using H&E stained sections and quantitative microscopy, and MRI of total cerebral cortex. Using confocal immunofluorescence (IF) stained for CD3, TUNEL and NeuN, sections of injured cortex were labeled to determine the extent of CD3+ cell infiltration and neuronal apoptosis. All analyses were performed at 1 day and 7 days after CCI.
Results:
The Volume of brain injury, as measured by MRI, showed a mean lesion volume of 81±2mm3 in WT C57BL/6 mice (n=2), 140±7mm3 in Rag1 KO mice (n=3), and 156±2mm3 in TLR4 KO mice (n=3) 1 day after cortical impact. After 7 days, the mean lesion volumes were 50±3mm3 in WT C57BL/6 mice (n=2), 132±3mm3 in Rag1 KO mice (n=2), and 36±1mm3 in TLR4 KO mice (n=2). H&E results were positively correlated with MRI results and demonstrated a volume of 90±5mm3 and 56±8mm3 in C57BL/6 WT, 148±4mm3 and 160±5mm3 in Rag1 KO, 180±6mm3 and 28±4mm3 in TLR4 KO mice, all at 1 day and 1 week post-TBI respectively. TLR4 KO mice showed a statistically significant increase in the number of apoptotic neurons (208±26cells/hpf) compared to WT C57BL/6 (87±6cells/hpf) 1 day after CCI. After 1 week of trauma, TLR4 KO mice showed a significant decrease in apoptotic neurons (126±1cells/hpf) when compared to WT C57BL/6 (210±7cells/hpf) (p<0.05). CD3+ cell infiltration was significantly greater at 1 day after CCI in TLR4 KO mice (14±1cells/hpf) vs WT C57/BL6 mice (8±2 cells/hpf) (p<0.05). Strikingly, after 1 week of CCI, CD3+ cell infiltration was significantly decreased in TLR4 KO mice (6.3±1cells/hpf) vs WT C57BL/6 mice (12±2cells/hpf) (p<0.05) suggesting a significant role for the innate immune system in the pathogenesis of this disease.
Conclusion:
We now conclude that in accordance with our hypothesis, the development of TBI in mice requires the input of the innate and adaptive immune system, and that these findings are time dependent. These results raise the possibility that strategies that modulate the innate and adaptive immune systems may have a role in reducing the severity of brain injury in post-TBI patients.