Abstracts

Rationale: In this study, we examined the neuroprotective potential of the antiepileptic drug (AED) levetiracteam (LEV) and the calpain inhibitor leupeptin (LEUP) to prevent posttraumatic epileptogenesis (PTE) and neuronal degeneration, using an in vitro model of traumatic brain injury (TBI). We also examined neocortical PTE following controlled cortical impact (CCI) injury in whole rats, as a prelude to in vivo testing of these compounds. Methods: In the in vitro injury model, acute coronal slices (400 μm) of rat neocortex (P21-30) were injured via a lateral surgical cut that separates the superficial layers (I, II, and part of III) from the deeper regions. This model produces a purely mechanical injury that leads to persistent epileptiform activity and cell necrosis within 1-2 h after trauma. Slices were maintained in a standard recording chamber at 31°C. Randomly selected slices were treated after injury with either LEV (250-500 μM) or LEUP (200 μg/100 mL), which were bath-applied for a duration of 1 h and then washed-out. Slices were examined for epileptiform activity via intracellular and extracellular recordings. Neuronal degeneration was assessed by Fluoro-Jade B (FJB) staining. Results: Post-injury treatment of slices with either LEV or LEUP prevented the development of PTE, significantly reducing (>50%) the proportion of injured slices that exhibited evoked epileptiform burst firing. LEV also increased the stimulus intensity needed to trigger epileptiform bursts by 2-4 fold, suggesting it inhibits PTE, in part, by raising burst threshold. We also found that LEV is protective when administered at times up to 60 min post-injury, and thus may have a substantially longer (2-3 fold) therapeutic window than other AEDs tested in this model, e.g., valproate. Histological analyses with FJB showed that both LEV and LEUP significantly reduced the levels of cell necrosis, and thus provide anatomical protection as well. Although encouraging, the findings from these in vitro studies need to be validated using in vivo models of TBI. We have thus begun to examine PTE in rats experimentally injured in the CCI model of non-penetrating cortical neurotrauma. Rats received a severe cortical injury (2 mm depth) and after a 7-14 d period, neocortical slices were prepared and examined for physiological and anatomical pathologies. At 7 d post-injury, evoked epileptiform activity was observed, similar to the responses observed in slices injured in vitro. At 14 d after injury, both spontaneous and evoked epileptiform activity were observed, indicating a progressive development of seizure-like activity in the neocortex after trauma. FJB staining revealed significant neocortical cell degeneration, comparable to the levels observed in slices injured in vitro. Thus, the CCI model is an appropriate in vivo system for studying PTE. Conclusions: These results demonstrate the potential of LEV and calpain inhibitors as neuroprotective therapies to prevent posttraumatic epileptogenesis and cell necrosis, which we will assess further using the CCI model of in vivo cortical trauma.