The oxygen free radicals originating from mitochondrial complex I contribute to oxidative brain injury following hypoxia-ischemia in neonatal mice.

Oxidative stress and Ca(2+) toxicity are mechanisms of hypoxic-ischemic (HI) brain injury. This work investigates if partial inhibition of mitochondrial respiratory chain protects HI brain by limiting a generation of oxidative radicals during reperfusion. HI insult was produced in p10 mice treated with complex I (C-I) inhibitor, pyridaben, or vehicle. Administration of P significantly decreased the extent of HI injury. Mitochondria isolated from the ischemic hemisphere in pyridaben-treated animals showed reduced H(2)O(2) emission, less oxidative damage to the mitochondrial matrix, and increased tolerance to the Ca(2+)-triggered opening of the permeability transition pore. A protective effect of pyridaben administration was also observed when the reperfusion-driven oxidative stress was augmented by the exposure to 100% O(2) which exacerbated brain injury only in vehicle-treated mice. In vitro, intact brain mitochondria dramatically increased H(2)O(2) emission in response to hyperoxia, resulting in substantial loss of Ca(2+) buffering capacity. However, in the presence of the C-I inhibitor, rotenone, or the antioxidant, catalase, these effects of hyperoxia were abolished. Our data suggest that the reperfusion-driven recovery of C-I-dependent mitochondrial respiration contributes not only to the cellular survival, but also causes oxidative damage to the mitochondria, potentiating a loss of Ca(2+) buffering capacity. This highlights a novel neuroprotective strategy against HI brain injury where the major therapeutic principle is a pharmacological attenuation, rather than an enhancement of mitochondrial oxidative metabolism during early reperfusion.

C3a receptor antagonist attenuates brain injury after intracerebral hemorrhage.

Neuroprotective therapy targeting the complement cascade may reduce injury associated with intracerebral hemorrhage (ICH). We investigated the role of C3a-receptor antagonist (C3aRA) after ICH in mice. Autologous whole blood was infused into the right striatum of mice that were treated with C3aRA or vehicle, using both a pre- and postinjury dosing regimen. Hematoma volume, brain water content, and inflammatory cell profile were assessed at 72 h post-ICH. Neurologic dysfunction was assessed by evaluating both spatial memory and sensorimotor capacity. Animals pretreated with C3aRA showed significantly improved neurologic function, brain water content, and granulocyte infiltration relative to vehicle-treated animals when assessed at 72 h. There was no significant difference in hemorrhagic/nonhemorrhagic ratio of microglial activation among all groups. Hematoma volumes were also not significantly different between C3aRA-treated and vehicle-treated animals. Administration of C3aRA beginning 6 h postinjury afforded significant amelioration of neurologic dysfunction as well as a reduction in brain water content. Treatment with C3aRA improved neurologic outcome while reducing inflammatory cell infiltration and brain edema formation after experimental ICH in mice. Results of this study suggest that the C3a receptor may be a promising target for therapeutic intervention in hemorrhagic stroke.

C3a receptor modulation of granulocyte infiltration after murine focal cerebral ischemia is reperfusion dependent.

The complement anaphylatoxin C3a contributes to injury after cerebral ischemia in mice. This study assesses the effect of C3a receptor antagonist (C3aRA) on leukocyte infiltration into the ischemic zone. Transient or permanent middle cerebral artery occlusion (MCAO) was induced in wild-type C57Bl/6 mice. Intraperitoneal C3aRA or vehicle was administered 45 mins before or 1 h after occlusion. Twenty-four hours after occlusion, we harvested brain tissue and purified inflammatory cells using flow cytometry. Soluble intercellular adhesion molecule (ICAM)-1 protein levels were assessed using enzyme-linked immunosorbent assays, and ICAM-1 and C3a receptor (C3aR) expression was confirmed via immunohistochemistry. In the transient MCAO model, animals receiving C3aRA showed smaller strokes, less upregulation of C3aR-positive granulocytes, and less ICAM-1 protein on endothelial cells than vehicle-treated animals; no significant differences in other inflammatory cell populations were observed. C3a receptor antagonist-treated and vehicle-treated animals showed no differences in stroke volume or inflammatory cell populations after permanent MCAO. These data suggest that blocking the binding of C3a to C3aR modulates tissue injury in reperfused stroke by inhibiting the recruitment of neutrophils to the ischemic zone. It further establishes antagonism of the C3a anaphylatoxin as a promising strategy for ameliorating injury after ischemia/reperfusion.

A mouse model of intracerebral hemorrhage using autologous blood infusion.

The development of controllable and reproducible animal models of intracerebral hemorrhage (ICH) is essential for the systematic study of the pathophysiology and treatment of hemorrhagic stroke. In recent years, we have used a modified version of a murine ICH model to inject blood into mouse basal ganglia. According to our protocol, autologous blood is stereotactically infused in two stages into the right striatum to mimic the natural events of hemorrhagic stroke. Following ICH induction, animals demonstrate reproducible hematomas, brain edema formation and marked neurological deficits. Our technique has proven to be a reliable and reproducible means of creating ICH in mice in a number of acute and chronic studies. We believe that our model will serve as an ideal paradigm for investigating the complex pathophysiology of hemorrhagic stroke. The protocol for establishing this model takes about 2 h.

O-desulfated heparin improves outcome after rat cerebral ischemia/reperfusion injury.

OBJECTIVE: Inflammatory cascades play a significant role in progressive neurological injury after transient cerebral ischemia. It has been demonstrated that heparin, a potent anticoagulant, also possesses anti-inflammatory properties that diminish postreperfusion damage after stroke. However, the potential for heparin to induce hemorrhagic transformation of an infarct has deterred its use in cases of focal cerebral ischemia. In this study, we examined whether or not administration of a novel O-desulfated heparin (ODSH), with significantly decreased anticoagulant activity but active anti-inflammatory effects, would ameliorate inflammatory neurological injury without increasing intracerebral hemorrhage in a rat model of transient middle cerebral artery occlusion. METHODS: Rats were injected immediately before ischemia with phosphate-buffered saline or ODSH (5 mg/kg, intravenously) and then every 12 hours (15 mg/kg, subcutaneously) for 72 hours. The animals were assessed for neurological function using a foot fault test and modified Bederson scale on Days 1, 2, and 5; plasma samples were analyzed for activated clotting time at multiple time points after the initial ODSH dose. After sacrifice on Day 5, infarct volume was determined and brain tissue was examined for evidence of hemorrhage both grossly and using a previously validated spectrophotometric hemoglobin assay. RESULTS: ODSH-treated animals demonstrated significantly improved foot fault performance (P = 0.03) on Day 5 and reduced stroke volumes (P = 0.03) relative to controls. Although the brains of ODSH-treated rats exhibited significantly higher hemoglobin levels in a standardized assay (P = 0.01), there were no incidences of gross hemorrhage observed in either group, and activated clotting time measurements for the treated animals were not significantly elevated over baseline at any time point. CONCLUSION: Our findings indicate that ODSH can be administered at a dose that provides postischemic anti-inflammatory neuroprotection without an increased risk of intracerebral hemorrhage.

Preclinical evaluation of the neuroprotective effect of soluble complement receptor type 1 in a nonhuman primate model of reperfused stroke.

OBJECT: Postischemic cerebral inflammatory injury has been extensively investigated in an effort to develop effective neuroprotective agents. The complement cascade has emerged as an important contributor to postischemic neuronal injury. Soluble complement receptor Type 1 (sCR1), a potent inhibitor of complement activation, has been shown to reduce infarct volume and improve functional outcome after murine stroke. Given numerous high-profile failures to translate promising antiinflammatory strategies from the laboratory to the clinic and given the known species-specificity of the complement cascade, the authors sought to evaluate the neuroprotective effect of sCR1 in a nonhuman primate model of stroke. METHODS: A total of 48 adult male baboons (Papio anubis) were randomly assigned to receive 15 mg/kg of sCR1 or vehicle. The animals were subjected to 75 minutes of middle cerebral artery occlusion/reperfusion. Perioperative blood samples were analyzed for total complement activity by using a CH50 assay. Infarct volume and neurological scores were assessed at the time the animals were killed, and immunohistochemistry was used to determine cerebral drug penetration and C1q deposition. An interim futility analysis led to termination of the trial after study of 12 animals. Total serum complement activity was significantly depressed in the sCR1-treated animals compared with the controls. Immunostaining also demonstrated sCR1 deposition in the ischemic hemispheres of treated animals. Despite these findings, there were no significant differences in infarct volume or neurological score between the sCR1--and vehicle-treated cohorts. CONCLUSIONS: A preischemic bolus infusion of sCR1, the most effective means of administration in mice, was not neuroprotective in a primate model. This study illustrates the utility of a translational primate model of stroke in the assessment of promising antiischemic agents prior to implementation of large-scale clinical trials.

Complement component C3 mediates inflammatory injury following focal cerebral ischemia.

The complement cascade has been implicated in ischemia/reperfusion injury, and recent studies have shown that complement inhibition is a promising treatment option for acute stroke. The development of clinically useful therapies has been hindered, however, by insufficient understanding of which complement subcomponents contribute to post-ischemic injury. To address this issue, we subjected mice deficient in selected complement proteins (C1q, C3, C5) to transient focal cerebral ischemia. Of the strains investigated, only C3-/- mice were protected, as demonstrated by 34% reductions in both infarct volume (P<0.01) and neurological deficit score (P<0.05). C3-deficient mice also manifested decreased granulocyte infiltration (P<0.02) and reduced oxidative stress (P<0.05). Finally, administration of a C3a-receptor antagonist resulted in commensurate neurological improvement and stroke volume reduction (P<0.05). Together, these results establish C3 activation as the key constituent in complement-related inflammatory tissue injury following stroke and suggest a C3a anaphylatoxin-mediated mechanism.

C1q-deficiency is neuroprotective against hypoxic-ischemic brain injury in neonatal mice.

BACKGROUND AND PURPOSE: This study was undertaken to determine whether the initial component of the classical complement (C) activation pathway contributes to hypoxic-ischemic brain injury in neonatal mice. METHODS: Hypoxia-ischemia (HI) was produced in C1q(-/-) and wild-type (WT) neonatal mice. At 24 hours after HI, neonatal mouse reflex performance and cerebral infarct volume were assessed. Long-term outcomes were measured by water-maze performance and degree of cerebral atrophy at 7 to 8 weeks after HI. Activation of circulating neutrophils, and C1q, C3, and neutrophil deposition in brains were examined. RESULTS: C1q(-/-) mice were significantly protected against HI (mean+/-SE infarct volume in C1q(-/-) mice=17.3+/-5.5% versus 53.6+/-6.8% in WT mice; P<0.0001) and exhibited significantly less neurofunctional deficit compared with WT mice. Immunostaining revealed significantly greater deposition of C3 (and C1q) as well as granulocytes in the infarcted brains in WT mice compared with C1q(-/-) animals. Activation of circulating leukocytes was significantly decreased in C1q(-/-) mice compared with WT mice, which correlated strongly (r=0.7) with cerebral infarct volumes. CONCLUSIONS: Cerebral deposition of C1q and C3 after hypoxic-ischemic insult is associated with significantly greater neurologic damage in WT mice compared with C1q(-/-) mice, providing strong evidence that the classical C pathway contributes to the hypoxic-ischemic brain injury. Significantly decreased activation of circulating neutrophils associated with diminished local accumulation and attenuation of brain injury in C1q(-/-) mice suggests a potential cellular mechanism by which C1q mediates neurodegeneration in HI.