Authors :
Presenting Author: Emmanuel Green, (BS) – University of California Riverside
Deepak Subramanian, PhD – Postdoc-Employee, Molecular Cell & Systems Bio Dept, University of California Riverside; Erick Contreras, BS – LAB AST 3, Molecular Cell & Systems Bio Dept, University of California Riverside; Vijayalakshmi Santhakumar, PhD – Vice Chair & Professor, Molecular Cell & Systems Bio Dept, University of California Riverside
Rationale:
Traumatic Brain Injuries (TBI) often lead to a strong innate immune response characterized by sterile inflammation. Early inflammatory cascade after TBI is thought to drive excitotoxic cell death, altered neuronal excitability, and network connectivity in the hippocampus resulting in post-traumatic epilepsy (PTE). We previously identified a role for Toll-like receptor 4 (TLR4) in increasing dentate gyrus network excitability and seizures following concussive brain injury. Similarly, aberrant Matrix metalloproteinase-9 (MMP-9) activity, an enzyme critical for synaptic remodeling and plasticity has been implicated in the development of post-traumatic neurological changes. Here we examined the effects of early inhibition of TLR4 signaling and MMP-9 activity on dentate hilar cell loss, granule cell dispersion and mossy fiber sprouting, three hallmarks of epilepsy, after concussive brain injury in juvenile rats.
Methods:
Male and Female Juvenile Wistar rats (p23) underwent moderate (1.8 - 2 atm) lateral Fluid Percussion Injury (FPI) or sham injury and were treated with vehicle (DMSO) or TLR4 antagonist CLI-095 (0.5 mg/kg, i.p.) or MMP-9 antagonist SB3CT (50 mg/kg, i.p.) at 30 min, 8 hrs, and 24 hrs after injury. Six weeks post injury, rats were transcardially perfused with 4% paraformaldehyde and coronal hippocampal brain slices (50μm) were obtained for immunostaining. Nissl staining was performed to quantify dentate hilar cell population and granule cell dispersion. Immunostaining for zinc transporter (ZnT3) was performed to detect mossy fiber sprouting. Hilar cell counts and granule cell layer width were performed on Nissl stained slices using Stereoinvestigator. Znt3 Stained slices were then imaged on a monochrome epifluorescence Zeiss microscope at a ten times objective and analyzed on Image J.
Results:
Our results revealed a 56.25% decrease in hilar cell population in injured animals compared to their sham-injured counterparts (Sham: 916.2 ± 77.65, FPI: 392.3 ± 31.94, n = 3/group). Additionally, we observed a significant dispersion of the granule cell layer in injured animals compared to sham controls (width in microns Sham:108.1 ± 4.35, FPI: 174.1 ± 9.26, n = 3-5/group). However, hilar cell loss and granule cell dispersion were prevented by early inhibition of TLR4 signaling or MMP-9 activity suggesting that these cascades are involved in loss of neurons following FPI. Despite significant cell loss and granule cell dispersion, we did not observe mossy fiber sprouting in the inner molecular layer of injured animals. (p = 0.162, by unpaired t-test).
Conclusions:
Our results suggest that TLR4 signaling and MMP-9 activity after brain injury contribute to the promotion of long term consequences of TBI including hilar cell loss and granule cell dispersion. However, whether these molecular cascades are related remains to be examined. The surprising absence of mossy fiber sprouting in our model supports the idea that morphological changes following brain insult could be age dependent. Together, our results highlight the importance of early immune response in dictating long-term outcomes of TBI.
Funding:
AES BRIDGE, R01NS069861 (VS), DoD W81XWH-21-1-0684 (DS)