Abstracts

Rationale: Over a million people sustain traumatic brain injury (TBI) in the USA each year. Patients sustaining brain injury have an increased risk for developing epilepsy. Closed head injury causes an acute and sterile inflammatory response including the activation of innate immune receptors and microglia. Here, we propose that increases in toll-like receptor (TLR) signaling following brain injury can modulate neuronal activity and augment post-traumatic dentate network excitability. Methods: Young adult Wistar rats (25-27 days) were subject to moderate (2 atm) lateral fluid percussion injury (FPI) or sham injury using standard methods (Gupta et al., 2012). Western blot and immunohistochemical analyses were used to examine post-traumatic changes in the expression of TLR2 and TLR4 in the ipsilateral hippocampus 4 hr, 24 hr, and 7 days after FPI. Field potential recordings were performed on acute hippocampal slices prepared 3-7 days after injury. Network activity was evoked using a bipolar stimulation electrode placed in the perforant path and recordings were obtained in the dentate granule cell layer. In each slice, the granule cell population spike amplitude in control ACSF was compared to responses recorded following a 2 hour incubation in either control ACSF or 1 μM LPS-RS, a selective TLR4 antagonist. Results: Western blots revealed that, when compared to sham-controls, the expression of TLR2 and TLR4 in the hippocampus was increased 4 and 24 hours after FPI but not 7 days post-injury. In preliminary immunostaining studies, both the intensity of labeling and number of cells expressing TLR2 and TLR4 were increased in hippocampal sections obtained 4 hours after FPI . While TLR2 was expressed in GFAP-labeled astrocytes, TLR4 was expressed in NeuN-positive neuronal profiles in dentate gyrus, CA1 and CA3. Large TLR4 labeled neurons were observed in the dentate hilus and stratum radiatum of CA1 and CA3, suggesting that certain classes of GABAergic interneurons may express TLR4. In slices from FPI rats, LPS-RS decreased the amplitude of the granule cell population spike evoked by a 4mA stimulus, to 30% of the amplitude in ACSF (amplitude in mV, FPI in ACSF: 1.29 ± 0.27, FPI in LSP-RS: 0.38 ± 0.11mA, t-test, p<0.005, n=15). The reduction of evoked population spike amplitude by LPS-RS was specific to slices from FPI rats and was not present in slices from sham-controls incubated in LPS-RS (amplitude in mV, sham in ACSF: 0.08 ± 0.06, sham in LPS-RS: 0.06 ± 0.06, t-test, p>0.05, n=8) or in slices incubated in ACSF. Conclusions: These data demonstrate an early and transient increase in the expression of the innate immune receptors TLR2 and TLR4 in the post-traumatic hippocampus. The distribution of TLR4-labeled profiles suggests regional and cell-type specific increase TLR2 and TLR4 after brain injury. Physiological studies indicate that TLR4 signaling contributes to the early increase in dentate excitability after TBI. These findings pave the way for elucidating the role of innate immune receptor signaling in the enhanced risk for epilepsy following brain injury.