Test repositioning for functional assessment of neurological outcome after experimental stroke in mice.

Stroke is a cerebrovascular pathology for which the only approved treatment is fibrinolysis. Several studies have focused on the development of new drugs but none has led to effective therapies to date, due, among others, to the difficulty to evaluate clinical deficits in experimental animal models. The present study aims to explore the applicability of known behavioral tests not commonly used in ischemia for the neurological assessment of mice after experimental stroke in different brain areas. A total of 225 CD1 male mice were randomly assigned to permanent middle cerebral artery occlusion by ligature (pMCAO) or permanent anterior cerebral artery occlusion by photothrombosis (pACAO) models. Modified neuroseverity score, footprint test, forced swim test and elevated plus maze were performed. Under these experimental conditions, modified neuroseverity score showed neurological impairment early after experimental stroke in both models. By contrast, the footprint test and the elevated plus maze detected short-term neurological deterioration in the pMCAO model but not in the pACAO model. Furthermore, the forced swim test identified depression-like behavior in mice after ischemia only when the left hemisphere was affected. In conclusion, we propose the repositioning of known neurobehavioral tests, but not commonly used in the stroke field, for the fast detection of neurological impairments early after ischemia, and even specific to discriminate the territory affected by arterial occlusion as well as the hemisphere where brain damage occurs. All these findings may prove useful to improve the experimental design of neuroprotective drugs in order to bridge the gap between experimental studies and clinical trials.

Amelioration of ischemic brain damage by peritoneal dialysis.

Ischemic stroke is a devastating condition, for which there is still no effective therapy. Acute ischemic stroke is associated with high concentrations of glutamate in the blood and interstitial brain fluid. The inability of the tissue to retain glutamate within the cells of the brain ultimately provokes neuronal death. Increased concentrations of interstitial glutamate exert further excitotoxic effects on healthy tissue surrounding the infarct zone. We developed a strategy based on peritoneal dialysis to reduce blood glutamate levels, thereby accelerating brain-to-blood glutamate clearance. In a rat model of stroke, this simple procedure reduced the transient increase in glutamate, consequently decreasing the size of the infarct area. Functional magnetic resonance imaging demonstrated that the rescued brain tissue remained functional. Moreover, in patients with kidney failure, peritoneal dialysis significantly decreased glutamate concentrations. Our results suggest that peritoneal dialysis may represent a simple and effective intervention for human stroke patients.

The coexistence of multiple receptors in a single nerve terminal provides evidence for pre-synaptic integration.

Excitatory synaptic transmission is inhibited by G protein coupled receptors, including the adenosine A(1), GABA(B), and metabotropic glutamate receptor 7. These receptors are present in nerve terminals where they reduce the release of glutamate through activating signaling pathways negatively coupled to Ca(2+) channels and adenylyl cyclase. However, it is not clear whether these receptors operate in distinct subpopulations of nerve terminals or if they are co-expressed in the same nerve terminals, despite the functional consequences that such distributions may have on synaptic transmission. Applying Ca(2+) imaging and immunocytochemistry, we show that these three G protein coupled receptors coexist in a subpopulation of cerebrocortical nerve terminals. The three receptors share an intracellular signaling pathway through which their inhibitory responses are integrated and coactivation of these receptors produced an integrated response. Indeed, this response was highly variable, from a synergistic response at subthreshold agonist concentrations to an occluded response at high agonist concentrations. The presence of multiple receptors in a nerve terminal could be responsible for the physiological effects of neurotransmitter spillover from neighboring synapses or alternatively, the co-release of transmitters by the same nerve terminal.

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals.

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.