DNA fragmentation in leukocytes following repeated low dose sarin exposure in guinea pigs.

The objective of this study was to determine levels of DNA fragmentation in blood leukocytes and parietal cortex from guinea pigs following repeated low-level exposure to the chemical warfare nerve agent (CWNA) sarin. Guinea pigs were injected (s.c.) once a day for 10 days with saline, or 0.1, 0.2, or 0.4 LD50 (50% mean lethal dose) sarin dissolved in sterile physiological saline. Blood and parietal cortex was collected after injection at 0, 3, and 17 days recovery and evaluated for DNA fragmentation using single-cell gel electrophoresis (Comet assay). Cells were imaged using comet analysis software and three parameters of DNA fragmentation measured: tail length, percent DNA in the tail, and tail moment arm. Repeated low-dose exposure to sarin produced a dose-dependent response in leukocytes at 0 and 3 days post-exposure. There was a significant increase in all measures of DNA fragmentation at 0.2 and 0.4 LD50, but not at 0.1 LD50. There was no significant increase in DNA fragmentation in any of the groups at 17 days post-exposure. Sarin did not produce a systematic dose-dependent response in parietal cortex at any of the time points. However, significant increases in DNA fragmentation at 0.1 and 0.4 LD50 were observed at 0 and 3 days post-exposure. All measures of DNA fragmentation in both leukocytes and neurons returned to control levels by 17 days post-exposure, indicating a small and non-persistent increase in DNA fragmentation following repeated low-level exposure to sarin.

Systemic administration of a calpain inhibitor reduces behavioral deficits and blood-brain barrier permeability changes after experimental subarachnoid hemorrhage in the rat.

Increases in intracellular calcium and subsequent activation of calcium-activated proteases (e.g., calpains) may play a critical role in central nervous system injury. Several studies have implicated calpain activation following subarachnoid hemorrhage (SAH). This study evaluated the effect of a calpain inhibitor administration following SAH in the rat on behavioral deficits (postinjury days 1-5, employing a battery of well-characterized assessment tasks), and blood-brain barrier permeability changes (48 h post-SAH, quantifying the microvascular alterations according to the extravasation of protein-bound Evans Blue using a spectrophotofluorimetric technique). Rats were injected with 400 microl of autologous blood into the cisterna magna to induce SAH. Within 5 min after the surgical procedure, Calpain Inhibitor II or vehicle was continuously administered intravenously for 2 days. Results indicated that Calpain Inhibitor II treatment after SAH significantly improved (a) beam balance time (day 1, p < 0.05), but not beam balance score, (b) latency to traverse the beam on days 1-4 (day 1-3, p < 0.001; day 4, p < 0.01), and (c) loss in body weight on days 4-5 (p < 0.05). Evans Blue dye extravasation was significantly less in SAH Calpain Inhibitor II-treated rats compared to SAH vehicle-treated rats in seven out of the eight brain regions studied (p < 0.001, 0.01, and 0.05). These results suggest that pharmacological inhibition of a relatively selective, membrane-permeant calpain inhibitor can significantly reduce some pathophysiological SAH consequences, and indicate that the inhibition of calpain may be a beneficial therapeutic approach to reduce post-SAH global brain dysfunction.

Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures.

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.

Nefiracetam improves Morris water maze performance following traumatic brain injury in rats.

Nefiracetam, a pyrrolidone derivative, is a nootropic agent that has facilitated cognitive function in a wide variety of animal models of cognitive dysfunction. The purpose of this study was to investigate the efficacy of the chronic postinjury administration of nefiracetam (DM-9384) in improving cognitive performance following central fluid percussion brain injury in rats. Twenty-four hours following surgical preparation, a sham injury or a moderate fluid percussive injury (2.1 atm) was delivered. Nefiracetam was administered chronically (0 or 9 mg/kg, po, for sham animals and 0, 3, or 9 mg/kg for injured animals) on postinjury days 1-15. Cognitive performance was assessed using the Morris water maze (MWM) on postinjury days 11-15. Chronic administration of 3 and 9 mg/kg nefiracetam attenuated MWM deficits produced by central fluid percussive brain injury. Importantly, the MWM performance of the injured animals treated with 9 mg/kg did not significantly differ from uninjured, sham animals. The 9-mg/kg dose of nefiracetam did not have a positive or negative effect on MWM performance of uninjured animals. The results of the present experiment suggest that a nootropic such as nefiracetam may be an appropriate treatment for trauma-induced cognitive dysfunction.

TNF-alpha stimulates caspase-3 activation and apoptotic cell death in primary septo-hippocampal cultures.

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.

Positive and negative modulation of the GABA(A) receptor and outcome after traumatic brain injury in rats.

Glutamate-mediated excitotoxicity has been shown to contribute to cellular dysfunction following traumatic brain injury (TBI). Increasing inhibitory function through stimulation of gamma-aminobutyric acid (GABA(A)) receptors may attenuate excitotoxic effects and improve outcome. The present experiment examined the effects of diazepam, a positive modulator at the GABA(A) receptor, on survival and cognitive performance in traumatically brain-injured animals. In experiment 1, 15 min prior to central fluid percussion brain injury, rats (n=8 per group) were injected (i.p.) with saline or diazepam (5 mg/kg or 10 mg/kg). Additional rats (n=8) were surgically prepared but not injured (sham-injury). Rats pre-treated with the 5 mg/kg dose of diazepam had significantly lower mortality (0%) than injured, saline-treated rats (53%). Also, diazepam-treated (5 mg/kg) rats had significantly shorter latencies to reach the goal platform in the Morris water maze test performed 11-15 days post-injury. In experiment 2, at 15 min post-injury, rats were given either saline (n=5) or 5 mg/kg diazepam (n=6). Rats treated with diazepam did not differ in mortality from injured rats treated with vehicle. However, rats treated with diazepam at 15 min post-injury had significantly shorter latencies to reach the goal platform in the Morris water maze than injured, vehicle-treated rats. In experiment 3, the post-injury administration of bicuculline (1.5 mg/kg, n=8), a GABA(A) antagonist, increased Morris water maze goal latencies compared to injured animals treated with saline (n=8). These results suggest that enhancing inhibitory function during the acute post-injury period produces beneficial effects on both survival and outcome following experimental TBI.

Central amygdaloid lesions attenuate cardiovascular responses to acute stress in rats with borderline hypertension.

The present study tested the hypothesis that lesions of the central nucleus of the amygdala (CeA) would reduce the cardiovascular responses to acute stress in a rodent model that is genetically predisposed toward hypertension. Male borderline hypertensive rats (BHR) were given bilateral electrolytic lesions directed to destroy the CeA or were subjected to a sham procedure. Direct measurements of blood pressure and heart rate were recorded during rest, during 10 min of acute stress, and for 10 min following stress. Analysis of the data revealed that BHR with CeA lesions had a significant attenuation of the stress-induced pressor response compared to sham-operated subjects. Behavioral measures taken in an open field chamber before and after lesions revealed no differences in numbers of squares crossed or rearings. These results suggest that the CeA is an important neural structure in mediating cardiovascular responses to acute stress in a model susceptible to environmentally induced hypertension.