Here we show that ischemia induces sustained contraction of pericytes on microvessels in the intact mouse brain. Pericytes remain contracted despite successful reopening of the middle cerebral artery after 2 h of ischemia. Pericyte contraction causes capillary constriction and obstructs erythrocyte flow. Suppression of oxidative-nitrative stress relieves pericyte contraction, reduces erythrocyte entrapment and restores microvascular patency; hence, tissue survival improves. In contrast, peroxynitrite application causes pericyte contraction. We also show that the microvessel wall is the major source of oxygen and nitrogen radicals causing ischemia and reperfusion-induced microvascular dysfunction. These findings point to a major but previously not recognized pathophysiological mechanism; ischemia and reperfusion-induced injury to pericytes may impair microcirculatory reflow and negatively affect survival by limiting substrate and drug delivery to tissue already under metabolic stress, despite recanalization of an occluded artery. Agents that can restore pericyte dysfunction and microvascular patency may increase the success of thrombolytic and neuroprotective treatments.

Background and Purpose-Sonic hedgehog (Shh) protein is required for the maintenance of neural progenitor cells (NPCs) in the embryonic and adult hippocampus. Brain ischemia causes increased proliferation of hippocampal NPCs. We therefore examined whether Shh regulates the increase in proliferation of NPCs after ischemia/hypoxia. Methods-Male SV129 mice were exposed to a 20-minute middle cerebral artery occlusion; hippocampi were then analyzed for Shh mRNA and protein expression by real-time polymerase chain reaction, immunoblot, and immunohistochemistry. Primary cell cultures of neurons, astrocytes, and NPCs were exposed to 16 hours of hypoxia (1% O 2 ) and analyzed by real-time polymerase chain reaction and immunoblot for Shh expression. Proliferation of NPCs, in vivo and in vitro, was measured by bromodeoxyuridine incorporation. Results-Among the cell types examined in vitro, only NPC and neurons increased Shh mRNA under hypoxic conditions. Furthermore, hypoxia increased proliferation of NPCs and this proliferation was enhanced by the addition of recombinant Shh or blocked by the pathway-specific inhibitor, cyclopamine. Middle cerebral artery occlusion was associated with a transient 2-fold increase in the mRNA encoding both Shh and its transcription factor, Gli1, 0.5 days after ischemia. Within the hippocampus, Shh protein was increased approximately 3-fold 3 and 7 days after ischemia and was observed predominantly within cells in the CA3 and hilar regions. Shh was expressed only in mature neurons. In vivo, cyclopamine suppressed ischemia-induced proliferation of subgranular NPCs. Conclusion-The

Sphingosine-1-phosphate (S1P) is a lipid mediator that exerts multiple cellular functions through activation of a subfamily of G-protein-coupled receptors. Although there is evidence that S1P plays a role in the developing and adult CNS, little is known about the ability of brain parenchyma to synthesize this lipid. We have therefore analyzed the brain distribution of the enzymatic activity of the S1P synthesizing enzyme, sphingosine kinase (SPHK) [EC:2.7.1.91], as well as mRNA distribution for one of the two isoforms of this enzyme, sphingosine kinase 2. SPHK activity, measured by the conversion of [ 3 H]sphingosine to [ 3 H]S1P, is highest in cerebellum, followed by cortex and brainstem. Lowest activities were found in striatum and hippocampus. Sensitivity to 0.1% Triton-X suggests that this activity is accounted for by SPHK2. RT-PCR and in situ hybridization studies show that mRNA for this isoform has a distribution similar to that of SPHK activity. In vivo and in vitro ischemia increase SPHK activity and SPHK2 mRNA levels. These results indicate that SPHK2 is the predominant S1P-synthesizing isoform in normal brain parenchyma. Sphingosine-1-phosphate (S1P) exerts a wide variety of biological activities in many vertebrate cell types. It was initially proposed to act as a second messenger, based on the ability of extracellular growth factors to activate sphingosine kinase (SPHK) [EC:2.7.1.91] and increase intracellular S1P levels. However, the putative site that mediates these intracellular actions of S1P has not been identified. The discovery and cloning of five S1P receptors (S1P 1 /EDG 1 , S1P 2 /EDG 5 , S1P 3 /EDG 3 , S1P 4 /EDG 6 , and S1P 5 /EDG 8 ) has stimulated the notion that S1P acts as an extracellular signaling molecule, regulating a host of cellular functions such as proliferation, immunomodulation, apoptosis, migration, cytoskeletal organization, and differentiation/morphogenesis (Hla 2003 The present address of Kamil Topalkara is the Department of Neurology, Faculty of Medicine, Cumhuriyet University, Sivas, 58140 Turkey. 3 The present address of Margherita Popolo is the Service de Neurologie, Hôpital Pasteur, BP69, Av de la voie Romaine, 06002 Nice Cedex 1, France.Abbreviations used: BSA, bovine serum albumin; MCA, middle cerebral artery; OGD, oxygen glucose deprivation; PBS, phosphatebuffered saline; S1P, sphingosine-1-phosphate; SDS, sodium dodecyl sulphate; SPHK, sphingosine kinase.Journal of Neurochemistry, 2007Neurochemistry, , 103, 509-517 doi:10.1111Neurochemistry, /j.1471Neurochemistry, -4159.2007 Kleuser et al. 1998;Shu et al. 2002). Activated platelets represent a major source of S1P (English et al. 2000(English et al. , 2001Yatomi et al. 2000). S1P levels in serum are considerably higher than in plasma (0.4 vs. 0.1 lmol/L, respectively). However, the interaction of S1P with low-density lipoprotein (Kimura et al. 2001) might reduce the free concentration of S1P, possibly as a protection mechanism from full activation of vascular S1P receptors.The fact that the brain is ...