title: Systemic L-citrulline prevents cerebral vasospasm in haptoglobin 2-2 transgenic mice after subarachnoid hemorrhage.
abstract: BACKGROUND
  Nitric oxide (NO) depletion and periadventitial inflammation contribute to the pathogenesis of cerebral vasospasm. L-Citrulline increases L-arginine levels, thereby raising NO synthesis. Transgenic C57Bl6 mice with a haptoglobin (Hp) 2-2 genotype develop more severe vasospasm than wild-type (Hp 1-1) mice after subarachnoid hemorrhage (SAH).
  
  
  OBJECTIVE
  To evaluate the toxicity of systemic L-citrulline and its effect on basilar artery (BA) vasospasm, neurobehavioral scores, and inducible NO synthase (iNOS)/endothelial NO synthase (eNOS) expression after SAH in Hp 2-2 mice.
  
  
  METHODS
  The Hp 2-2 genotypes were confirmed by reverse-transcriptase polymerase chain reaction. Toxicity was assessed with escalating L-citrulline doses. To test efficacy, Hp 1-1 and Hp 2-2 mice (n = 64) were divided into 4 groups (n = 32 per genotype): sham surgery (n = 8), SAH with no treatment (n = 8), SAH + vehicle (n = 8), and SAH + L-citrulline (200 mg/kg IP every 8 hours; n = 8). Post-SAH neurobehavioral scores were recorded at 24 hours; animals were perfused; and BAs were processed for analysis. Expression of iNOS and eNOS was determined by reverse-transcriptase polymerase chain reaction.
  
  
  RESULTS
  The administration of L-citrulline resulted in higher BA lumen patencies in both genotypes (Hp 1-1: SAH + vehicle, 77.8 ± 3.2% vs SAH + L-citrulline, 91.8 ± 5.9% [mean ± SEM]; P < .05; Hp 2-2: SAH + vehicle, 67.1 ± 2.0% vs SAH + L-citrulline, 86.9 ± 2.2%; P < .001). Neurobehavioral scores were higher in Hp 2-2 mice treated with L-citrulline (SAH + vehicle, 1.2 ± 0.2 vs SAH + L-citrulline, 2.4 ± 0.2; P < .01). Expression of iNOS and eNOS increased in Hp 2-2 mice after L-citrulline treatment, but limited sample sizes prevented further statistical analysis. L-Citrulline was not toxic even at the highest dose.
  
  
  CONCLUSIONS
  L-Citrulline is safe; increases BA patency, neurobehavioral scores, and NOS expression in Hp 2-2 mice after SAH; and is a potential agent for treatment of vasospasm after SAH.
authors.affiliation: The Johns Hopkins University School of Medicine, Department of Neurosurgery, Baltimore, Maryland 21287, USA.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Pradilla
authors.forename: Tomas
authors.initials: T
authors.lastname: Garzon-Muvdi
authors.forename: Jacob J
authors.initials: JJ
authors.lastname: Ruzevick
authors.forename: Matthew
authors.initials: M
authors.lastname: Bender
authors.forename: Lindsay
authors.initials: L
authors.lastname: Edwards
authors.forename: Eric N
authors.initials: EN
authors.lastname: Momin
authors.forename: Reid C
authors.initials: RC
authors.lastname: Thompson
authors.forename: Rafael J
authors.initials: RJ
authors.lastname: Tamargo
mesh_terms: D000818:Animals; D001120:Arginine / Q000097:blood; D001488:Basilar Artery / Q000187:drug effects / Q000502:physiology; D002560:Cerebrovascular Circulation / Q000187:drug effects / Q000502:physiology; D002956:Citrulline / Q000494:pharmacology* / Q000633:toxicity; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005838:Genotype; D006242:Haptoglobins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009569:Nitric Oxide / Q000097:blood; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000235:genetics; D013345:Subarachnoid Hemorrhage / Q000150:complications / Q000188:drug therapy*; D020301:Vasospasm, Intracranial / Q000209:etiology / Q000235:genetics / Q000517:prevention & control*
chemical_list: D006242:Haptoglobins; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse