Undercut model of Traumatic Brain Injury shows changes in inflammatory markers but not cell death 3 days after injury

Following traumatic brain injury (TBI), there is a risk of developing post-traumatic epilepsy (PTE), but no preventative treatment is known for PTE. In various models of PTE, an increased incidence of epileptogenic activity is correlated with neuronal cell death, gliosis, and inflammation. The focus of the current literature is on the role of cell death and glial cell reactivity in epileptogenesis, while the robust inflammatory process after TBI is less characterized. αB-crystallin (Cryab) is a protein upregulated in multiple sclerosis lesions that has anti-inflammatory and neuroprotective effects. Previous studies have shown its utility as a treatment modality in a rodent stroke model. Cryab decreases levels of key pro-inflammatory cytokines and decreases neuronal cell death. This project explores the inflammatory response 3 days after Undercut, a partial neocortical isolation model of TBI. Additionally, the second focus of this study is whether exogenous Cryab can be a treatment for epileptogenesis.
After making the Undercut lesion, Cryab was administered via intraperitoneal injection daily for a total of three days. Immunohistochemical techniques were used to anaylyze free-floating brain sections and measure how markers of cell death (cleaved caspase-3, NeuN), gliosis, and neuronal density were affected by Cryab treatment. Additionally, levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and the anti-inflammatory cytokine IL-10 were measured at different time points and brain locations (whole brain and Undercut region) 1 and 3 days after Undercut.
Preliminary data suggest that the Undercut lesion increases concentrations of IL-1β [p=0.1] in the Undercut region of the brain tissue 3 days after injury. Additionally, following Undercut concentration of IL-6 decreases [p=0.09] and TNF-α increases [p=0.08] in splenocytes harvested 3 days after injury and cultured with lipopolysaccharide (LPS). Cryab treatment significantly reduced concentrations of IL-1β [p=0.02] in brain tissue in the Undercut. Splenocyte cultures harvested from Cryab-treated Undercut animals have significantly increased concentrations of IL-6 [p=0.03] and significantly decreased concentrations of TNF-α [p=0.01]. Additionally, both astrocytes [p=0.005] and microglia [p=0.0001] were significantly activated in Undercut rats compared to control rats, and Cryab treatment significantly decreased microglia density [p=0.03]. Cryab did not significantly decrease markers of caspase-mediated apoptosis or neuronal counts at this 3-day time point.
This study shows that there is an inflammatory response in the neocortex of the Undercut model of TBI, including microglial activation and increases in pro-inflammatory cytokines. Understanding the inflammatory response in the Undercut model of PTE may contribute to our understanding of the pathological mechanisms associated with development of hyperexcitable brain circuitry. These findings can pave the way for novel avenues for preventative treatment.

Keywords:  Department of Biology
Prince, David A
post-traumatic epilepsy
traumatic brain injury
undercut
aB-Crystallin
inflammation
cytokines
electrophysiology
Department of Biology
Stanford University
Thesis