Background and Purpose-Genetically engineered mice are used to study the role of single genes in cerebral ischemia, but inherent, strain-dependent differences in neuronal vulnerability may affect experimental end points. To examine this possibility, tissue injury resulting from focal ischemia and its relationship to cerebral hemodynamics were determined in 3 common mutant mouse strains. Methods-Permanent middle cerebral artery ligation was performed in male C57BL/6J, Balb/C, and 129X1/SvJ mice.Mean arterial blood pressure, blood gases, basal and postischemic cortical blood flow ([ 14 C]iodoantipyrine autoradiography and laser-Doppler flowmetry), posterior communicating artery patency, and infarct size were determined. Results-Basal cortical blood flow did not differ among strains. Ten minutes after middle cerebral artery ligation, relative red cell flow in the ischemic cortex was 6% to 7% of preischemic flow in every strain. Despite similar hemodynamics, cortical infarcts in Balb/C mice were 3-fold larger than those in 129X1/SvJ and C57BL/6J mice; infarct size in the latter 2 strains was not significantly different. The posterior communicating artery was either poorly developed or absent in Ͼ90% of the Balb/C and C57BL/6J but in Ͻ50% of the 129X1/SvJ mice. Conclusions-The extent of ischemic injury differed markedly between the 3 strains. The presence and patency of posterior communicating arteries, although variable among strains, did not affect preischemic or postischemic cortical blood flow or bear any relationship to ischemic injury. Therefore, intrinsic factors, other than hemodynamic variability, may contribute to the differences in ischemic vulnerability among strains. These findings underscore the importance of selecting genetically matched wild-type controls. (Stroke. 2000;31:2707-2714.)

Neuroprotection against cerebral ischemia can be realized if the brain is preconditioned by previous exposure to a brief period of sublethal ischemia. The present study was undertaken to test the hypothesis that nitric oxide (NO) produced from the neuronal isoform of NO synthase (NOS) serves as a necessary signal for establishing an ischemia-tolerant state in brain. A newborn rat model of hypoxic preconditioning was used, wherein exposure to sublethal hypoxia (8% oxygen) for 3 hours renders postnatal day (PND) 6 animals completely resistant to a cerebral hypoxic-ischemic insult imposed 24 hours later. Postnatal day 6 animals were treated 0.5 hour before preconditioning hypoxia with the nonselective NOS inhibitor L-nitroarginine (2 mg/kg intraperitoneally). This treatment, which resulted in a 67 to 81% inhibition of calcium-dependent constitutive NOS activity 0.5 to 3.5 hours after its administration, completely blocked preconditioning-induced protection. However, administration of the neuronal NOS inhibitor 7-nitroindazole (40 mg/kg intraperitoneally) before preconditioning hypoxia, which decreased constitutive brain NOS activity by 58 to 81%, was without effect on preconditioning-induced cerebroprotection, as was pretreatment with the inducible NOS inhibitor aminoguanidine (400 mg/kg intraperitoneally). The protective effects of preconditioning were also not blocked by treating animals with competitive [3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate; 5 mg/kg intraperitoneally] or noncompetitive (MK-801; 1 mg/kg intraperitoneally) N-methyl-D-aspartate receptor antagonists prior to preconditioning hypoxia. These findings indicate that NO production and activity are critical to the induction of ischemic tolerance in this model. However, the results argue against the involvement of the neuronal NOS isoform, activated secondary to a hypoxia-induced stimulation of N-methyl-D-aspartate receptors, and against the involvement of the inducible NOS isoform, but rather suggest that NO produced by the endothelial NOS isoform is required to mediate this profound protective effect.

Summary: This study used the brain dialysis technique to test the hypothesis that the adenosine concentration of cerebral interstitial fluid increases during situations in which cerebral oxygen supply is inadequate for oxygen demand, Sealed 300-ILm hollow dialysis fibers were im planted in the caudate nucleus of pentobarbital-anesthe tized rats and perfused at 2 ILl/min with artificial cerebro spinal fluid, In vitro tests indicated the recovery of aden osine, inosine, and hypoxanthine from the external medium to be �20% at 2 ILl/min and close to 100% at 0, I ILl/min, Three in vivo interventions were tested: hypoxia/ hypotension (PaOZ = 41.9 mm Hg; MABP = 42.8 mm Metabolic regulation of CBF is widely accepted (Kuschinsky and Wahl, 1978; Berne et aI., 1981h; Kontos, 1981;Wahl, 1985), although the mediators involved are still controversial (Wahl, 1985). The purine nucleoside adenosine, a by-product of ATP metabolism and known cerebral blood vessel di lator (Berne et aI. , 1974;Wahl and Kuschinsky, 1976; Gregory et aI. , 1980; Berne et aI. , 1981a) 522Hg; n = 9), local potassium infusion (n = 4), and cere bral anoxia/ischemia (n = 10). These interventions pro duced 10-, 4-, and 30-fold increases in perfusate adeno sine concentration, respectively, as well as increases in perfusate concentrations of inosine and hypoxanthine. A separate group of rats (n = 9) perfused at 0. 1 ILl/min yielded estimates of cerebral interstitial fluid adenosine, inosine, and hypoxanthine concentrations of 1. 26, 3.30, and 7. 19 ILM, respectively. These results are consistent with the adenosine hypothesis for the regulation of CBF.

Cerebrovascular inflammation contributes to secondary brain injury following ischemia. Recent in vitro studies of cell migration and molecular guidance mechanisms have indicated that the Slit family of secreted proteins can exert repellant effects on leukocyte recruitment in response to chemoattractants. Utilizing intravital microscopy, we addressed the role of Slit in modulating leukocyte dynamics in the mouse cortical venular microcirculation in vivo following TNFα application or global cerebral ischemia. We also studied whether Slit affected neuronal survival in the mouse global ischemia model as well as in mixed neuronal-glial cultures subjected to oxygenglucose deprivation. We found that systemically administered Slit significantly attenuated cerebral microvessel leukocyte-endothelial adherence occurring 4 h after TNFα and 24 h after global cerebral ischemia. Administration of RoboN, the soluble receptor for Slit, exacerbated the acute chemotactic response to TNFα. These findings are indicative of a tonic repellant effect of endogenous Slit in brain under acute proinflammatory conditions. Three days of continuous systemic administration of Slit following global ischemia significantly attenuated the delayed neuronal death of hippocampal CA1 pyramidal cells. Moreover, Slit abrogated neuronal death in mixed neuronal-glial cultures exposed to oxygen-glucose deprivation. The ability of Slit to reduce the recruitment of immune cells to ischemic brain and to provide cytoprotective effects suggests that this protein may serve as a novel anti-inflammatory and neuroprotective target for stroke therapy.