3-nitrotyrosine and Subarachnoid-Hemorrhage

Hydrogen gas has been demonstrated to neutralize free radicals and reduce oxidative stress recently. Our objective was to determine the therapeutic effect of H2 inhalation and its antioxidative activity on early brain injury after subarachnoid hemorrhage.. Controlled in vivo laboratory study.. Animal research laboratory.. One hundred thirty-seven adult male Sprague-Dawley rats weighing 280-350 g.. Subarachnoid hemorrhage was induced by endovascular perforation method in rats. Subarachnoid hemorrhage rats were treated with 2.9% hydrogen gas inhaled for 2 hrs after perforation. At 24 and 72 hrs, mortality, body weight, neurologic deficits, and brain water content were assessed. Blood-brain barrier permeability and apoptosis were also measured at 24 hrs. To investigate the antioxidative activity of hydrogen gas, the expression of malondialdehyde, nitrotyrosine, and 8-hydroxyguanosine, which are oxidative markers of lipid, protein, and DNA damage, respectively, were measured at 24 hrs.. Hydrogen gas significantly alleviated brain edema and blood-brain barrier disruption, reduced apoptosis, and improved neurologic function at 24 hrs but not 72 hrs after subarachnoid hemorrhage. These effects were associated with the amelioration of oxidative injury of lipid, protein, and DNA.. Hydrogen gas could exert its neuroprotective effect against early brain injury after subarachnoid hemorrhage by its antioxidative activity.

Topics: Administration, Inhalation; Animals; Antioxidants; Apoptosis; Blood-Brain Barrier; Brain; Brain Chemistry; Brain Injuries; Guanosine; Hydrogen; Lipid Peroxidation; Male; Malondialdehyde; Oxidative Stress; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Tyrosine; Water

We studied whether endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled in large cerebral arteries after subarachnoid hemorrhage (SAH) and also whether this causes cerebral vasospasm in a mouse model of anterior circulation SAH. Control animals underwent injection of saline instead of blood (n=16 SAH and n=16 controls). There was significant vasospasm of the middle cerebral artery 2 days after SAH (lumen radius/wall thickness ratio 4.3 ± 1.3 for SAH, 23.2 ± 2.1 for saline, P<0.001). Subarachnoid hemorrhage was associated with terminal deoxynucleotidyl transferase dUTP nick-end labeling, cleaved caspase-3, and Fluoro-Jade-positive neurons in the cortex and with CA1 and dentate regions in the hippocampus. There were multiple fibrinogen-positive microthromboemboli in the cortex and hippocampus after SAH. Transgenic mice expressing lacZ under control of the eNOS promoter had increased X-gal staining in large arteries after SAH, and this was confirmed by the increased eNOS protein on western blotting. Evidence that eNOS was uncoupled was found in that nitric oxide availability was decreased, and superoxide and peroxynitrite concentrations were increased in the brains of mice with SAH. This study suggests that artery constriction by SAH upregulates eNOS but that it is uncoupled and produces peroxynitrite that may generate microemboli that travel distally and contribute to brain injury.

Topics: Animals; Arterioles; Blotting, Western; Caspase 3; Cerebral Arteries; Fibrinogen; Immunohistochemistry; In Situ Nick-End Labeling; Intracranial Thrombosis; Lac Operon; Mice; Mice, Transgenic; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Peroxynitrous Acid; Subarachnoid Hemorrhage; Superoxides; Tyrosine; Vasospasm, Intracranial

In the present study, we investigated the in vivo effects of melatonin on SAH-induced cerebral vasospasm and oxidative stress, resulting from SAH in an experimental rat model. Twenty-eight rats (225-250 g) were divided into four groups equally: group 1; control, group 2; SAH, group 3; SAH plus placebo, and group 4; SAH plus melatonin. We used double haemorrhage method for SAH groups. Beginning 6 h after SAH, 20 mg/kg melatonin or equal volume of 0.9% saline was administered intraperitoneally twice daily for 5 days to groups 3 and 4, respectively. Melatonin or 0.9% saline injections were continued up to fifth day after SAH and rats were sacrificed at the end of this period. Brain sections at the level of the pons were examined by light microscopy. The lumen diameter and the vessel wall thickness of basilar artery were measured using a micrometer. The serum levels of cerebral vasodilator nitric oxide (NO), the brain levels of an intrinsic antioxidant superoxide dismutase (SOD) and a NO regulator arginase activities were measured. The brain levels of inducible nitric oxide (iNOS) and nitrotyrosine, a nitrosative stress parameter immunohistochemiacally determined. In conclusion, melatonin administration ameliorated cerebral vasospasm by increasing serum NO level and decreasing the brain the levels of arginase and oxidative stress. It is therefore possible that increased brain arginase activity after SAH may also have a significant role in the pathogenesis of vasospasm by limiting the availability of arginine for NO production.

Topics: Animals; Antioxidants; Arginase; Basilar Artery; Brain; Constriction, Pathologic; Free Radicals; Immunohistochemistry; Male; Melatonin; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Rats; Rats, Wistar; Subarachnoid Hemorrhage; Superoxide Dismutase; Tyrosine; Vasospasm, Intracranial

The present study determined whether gene transfer of human copper/zinc superoxide dismutase-1 (Cu/Zn SOD-1) prevented the autoregulatory impairment of CBF induced by subarachnoid hemorrhage (SAH). After application of recombinant adenovirus (100 microL of 1 x 10(10) pfu/mL, intracisternally) encoding human Cu/Zn SOD-1 3 days before experiments, Cu/Zn SOD-1 activity significantly increased in association with increase in Cu/Zn SOD-1 mRNA and protein expression in the cerebral vasculature of both sham-operated and SAH rats as determined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry, and SAH-induced increase in superoxide anion was markedly reduced in accordance with increased nitric oxide production. In line with these findings, rats that received human Cu/Zn SOD-1 therapy showed the prominent restoration of blunted vasodilation of the pial artery in response to calcitonin gene-related peptide and levcromakalim, and the recovery of impaired autoregulatory vasodilation in response to acute hypotension, thereby leading to significant restoration of CBF autoregulation. These results provide a rational basis for application of Cu/Zn SOD-1 gene therapy for protection of the impairment of autoregulatory CBF during the acute stage of SAH.

Topics: Adenoviridae; Animals; Arteries; Blood Vessels; Cerebrovascular Circulation; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Humans; Male; NADPH Oxidases; Nitrates; Nitrites; Pia Mater; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Superoxide Dismutase; Superoxide Dismutase-1; Superoxides; Transgenes; Tyrosine; Vasomotor System

Subarachnoid hemorrhage (SAH) increases production of vascular extracellular superoxide anion (*O2-). We examined whether overexpression of murine extracellular superoxide dismutase (EC-SOD) alters SAH-induced cerebral vasospasm, oxidative stress, and neurological outcome.. Mice exhibiting a 2-fold increase in vascular EC-SOD and wild-type (WT) littermates were subjected to sham surgery or SAH by perforation of the right anterior cerebral artery. Neurological deficits were scored 72 hours later. Middle cerebral artery (MCA) diameter was measured or immunohistochemically stained for nitrotyrosine.. MCA diameter (mean+/-SD) was greater in EC-SOD versus WT mice after SAH but not sham surgery (EC-SOD SAH=56+/-10 microm; WT SAH=38+/-13 microm [P<0.01]; EC-SOD sham=99+/-16 microm; WT sham=100+/-15 microm). SAH decreased median (range) neurological score (scoring scale, 9 to 39; no deficit=39) versus shams, but there was no difference between EC-SOD and WT groups (EC-SOD SAH=26 [23 to 30]; WT SAH=23 [19 to 29] [P=0.27]; EC-SOD sham=39 [39]; WT sham=39 [39]). Sensory-motor deficits correlated with MCA diameter (P<0.001) but worsened primarily between 60 and 50 micro m, plateauing below this threshold. The percentage of mice with MCA nitrotyrosine staining increased after SAH in WT (sham=29%; SAH=100% [P<0.05]) but not EC-SOD (sham=33%; SAH=44% [P=0.80]) mice.. Endogenous overexpression of EC-SOD attenuated vasospasm and oxidative stress but failed to reduce neurological deficits after SAH. Extracellular *O2- likely plays a direct role in the etiology of vasospasm.

Topics: Animals; Aorta; Brain; Disease Models, Animal; Enzyme Activation; Extracellular Space; Humans; Immunohistochemistry; Male; Mice; Mice, Transgenic; Middle Cerebral Artery; Oxidative Stress; RNA, Messenger; Subarachnoid Hemorrhage; Superoxide Dismutase; Tyrosine; Vascular Patency; Vasospasm, Intracranial

The involvement of de novo nitric oxide synthase (NOS) induction in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH) was examined using a rat model of SAH. SAH was induced by endovascular perforation with Nylon thread. The rats were killed at different time intervals, from one day to seven days after endovascular perforation. Inducible NOS messenger RNA (mRNA) expression was determined by reverse-transcription polymerase chain reaction (RT-PCR) and the distribution of iNOS positive cells was immunohistochemically examined. In the vascular tissue with a subarachnoid membrane, iNOS mRNA was expressed from one day to seven days after SAH. Inducible NOS positive cells were mainly recognized in the vascular tissue, but not in the brain parenchyma. The distribution of nitrotyrosine, an indicator of peroxynitrite production was also examined immunohistochemically and nitrotyrosine-positive cells were observed almost at the same sites of iNOS induction. To determine the role of iNOS in the development of cerebral vasospasm, we measured the diameter of the middle cerebral artery in animals either treated or not treated with aminoguanidine (AG), a selective inhibitor of iNOS. AG ameliorated the vasoconstrictive change after SAH. These results are thus considered to provide molecular and immunohistochemical evidence showing that iNOS expression following SAH and NO produced by iNOS can develop cerebral vasospasm after SAH.

Topics: Animals; Antibodies; Cerebral Arteries; Cerebrovascular Circulation; DNA Probes; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Guanidines; Immunohistochemistry; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Subarachnoid Hemorrhage; Tyrosine; Vasoconstriction