Experimental investigations of single mild brain injury (SMI) show relatively little resultant cognitive impairment. However, repeated mild brain injuries (RMI), as those sustained by athletes (e.g., football, hockey, and soccer players) may have cumulative effects on cognitive performance and neuropathology. Numerous clinical studies show persistent, latent, and long-term consequences of RMI, unlike the episodic nature of SMI. The nature of repeated traumatic brain injury (TBI) introduces confounding factors in invasive and even semiinvasive animal models of brain injury (e.g., scar formation). Thus, the present study characterizes SMI and RMI in a noninvasive mouse weight drop model and the cumulative effects of RMI on cognitive performance. Investigation of drop masses and drop distances revealed masses of 50, 100, and 150 g dropped from 40 cm resulted in 0% mortality, no skull fracture, and no difference in acute neurological assessment following sham injury, SMI, or RMI. Cumulative effects of RMI were examined following four mild brain injuries 24 h apart induced by 50-, 100-, or 150-g masses dropped from 40 cm through histological measures, mean arterial pressure, and measures of complex/spatial learning. RMI produced no overt cell death within the cortex or hippocampus, no evidence of blood-brain barrier compromise, and no significant change in mean arterial pressure. Following testing in the Morris water maze (MWM) on days 7-11 after initial injury, mice in the RMI 100-g and RMI 150-g groups had significantly longer MWM goal latencies compared to sham, SMI 150-g, and RMI 50-groups. Additionally, the evident cognitive deficit manifested in the absence of observed cell death. This is the first study to show complex/spatial learning deficits following RMI, similar to the visual/spatial perception and planning deficits observed in clinical studies.

Primary septo-hippocampal cell cultures were incubated in varying concentrations of tumor necrosis factor (TNF-alpha; 0.3-500 ng/ml) to examine proteolysis of the cytoskeletal protein alpha-spectrin (240 kDa) to a signature 145 kDa fragment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3. The effects of TNF-alpha incubation on morphology and cell viability were assayed by fluorescein diacetate-propidium iodide (FDA-PI) staining, assays of lactate dehydrogenase (LDH) release, nuclear chromatin alterations (Hoechst 33258), and internucleosomal DNA fragmentation. Incubation with varying concentrations of TNF-alpha produced rapid increases in LDH release and nuclear PI uptake that were sustained over 48 hr. Incubation with 30 ng/ml TNF-alpha yielded maximal, 3-fold, increase in LDH release and was associated with caspase-specific 120-kDa fragment but not calpain-specific 145-kDa fragment as early as 3.5 hr after injury. Incubation with the pan-caspase inhibitor, carbobenzosy- Asp-CH(2)-OC (O)-2-6-dichlorobenzene (Z-D-DCB, 50-140 microM) significantly reduced LDH release produced by TNF-alpha. Apoptotic-associated oligonucleosomal-sized DNA fragmentation on agarose gels was detected from 6 to 72 hr after exposure to TNF-alpha. Histochemical changes included chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Results of this study suggest TNF-alpha may induce caspase-3 activation but not calpain activation in septo-hippocampal cultures and that this activation of caspase-3 at least partially contributes to TNF-alpha-induced apoptosis.

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen-glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of alpha-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3-mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen-glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.

Tissue-type transglutaminase (tTG, EC 2.3.2.13) has been implicated in various disease paradigms including neurodegenerative disease. In these studies, tTG induction after traumatic brain injury was studied using a rat cortical impact model. Using western blots, two forms of tTG protein expression were identified ± a 79-kDa primary form (tTG-L) and a less abundant 70-kDa form (tTG-S). Both forms of tTG protein were elevated after injury. In ipsilateral cortex, peak induction of tTG-L protein [561% 80% of control (n 5)] was observed five days after injury, with expression remaining elevated after two weeks. Peak induction of tTG-S protein [302% 81% of control (n 5)] was observed three days after injury. Lesser tTG protein induction was observed in hippocampus. Northern blot analysis demonstrated two tTG transcripts in the ipsilateral cortex with peak induction of tTG-L mRNA three days after injury. However, tTG-S mRNA was not identified in control samples and only faintly detected in injured tissue. To facilitate analysis of low abundance transcripts in smaller tissue samples, a semiquantitative realtime PCR strategy was used. Semi-quantitative PCR analysis of tTG-L mRNA induction in ipsilateral cortex (peak after three days; 414% 21% of control, n 3) confirmed tTG-L mRNA induction determined by northern blot (410% of control).