Elsevier

Brain Research

Volume 1298, 28 October 2009, Pages 171-177
Brain Research

Research Report
Synergistic neuroprotective effects of C3a and C5a receptor blockade following intracerebral hemorrhage

https://doi.org/10.1016/j.brainres.2009.04.047Get rights and content

Abstract

Background: Intracerebral hemorrhage (ICH) is associated with neurological injury that may be ameliorated by a neuroprotective strategy targeting the complement cascade. We investigated the role of C5a-receptor antagonist (C5aRA) solely and in combination with C3a-receptor antagonist (C3aRA) following ICH in mice.

Methods: Adult male C57BL/6J mice were randomized to receive vehicle, C5aRA alone or C3aRA and C5aRA 6 and 12 h after ICH, and every 12 h thereafter. A double injection technique was used to infuse 30 μL of autologous whole blood into the right striatum. A final group of mice received a sham procedure consisting only of needle insertion followed by vehicle injections. Brain water content and flow cytometry analysis for leukocyte and microglia infiltration and activation in both hemispheres were measured on day 3 post ICH. Neurological dysfunction was assessed using a Morris water-maze (MWM), a 28-point scale, and a corner test at 6, 12, 24, 48 and 72 h  after ICH induction.

Results: Neurological deficits were present and comparable in all three cohorts 6 h after ICH. Animals treated with C5aRA and animals treated with combined C3aRA/C5aRA demonstrated significant improvements in neurological function assessed by both the corner turn test and a 28-point neurological scale at 24, 48 and 72 h  relative to vehicle-treated animals. Similarly, C5aRA and C3aRA/C5aRA-treated mice demonstrated better spatial memory retention in the Morris water-maze test compared with vehicle-treated animals (C3aRA/C5aRA: 23.4 ± 2.0 s p  0.0001 versus vehicle: 10.0 ± 1.7 s).

Relative to vehicle-treated mice, the brain water content in C3aRA/C5aRA-treated mice was significantly decreased in the ipsilateral cortex and ipsilateral striatum (ipsilateral cortex: C3aRA/C5aRA: 0.755403 ± 0.008 versus 0.773327 ± 0.003 p = 0.01 striatum: 0.752273 ± 0.007 versus 0.771163 ± 0.0036 p = 0.02). C5aRA-treated mice and C3aRA/C5aRA-treated mice had a decreased ratio of granulocytes (CD45+/CD11b+/Ly-6G+) in the hemorrhagic versus non-hemorrhagic hemispheres relative to vehicle-treated animals (C5aRA: 1.78 ± 0.36 p = 0.02 C3aRA/C5aRA: 1.59 ± 0.22 p = 0.005 versus vehicle: 3.01).

Conclusions: While administration of C5aRA alone provided neuroprotection, combined C3aRA/C5aRA therapy led to synergistic improvements in neurofunctional outcome while reducing inflammatory cell infiltration and brain edema. The results of this study indicate that simultaneous blockade of the C3a and C5a receptors represents a promising neuroprotective strategy in hemorrhagic stroke.

Introduction

Hemorrhagic stroke accounts for approximately 15% of all strokes but is associated with a disproportionate degree of morbidity. Studies have indicated that inflammatory processes and complement activation in particular may be involved in exacerbating brain injury after the hemorrhagic event (Hua et al., 2000). Early attempts at complement inhibition using cobra venom factor, C1-esterase inhibitor, and soluble complement receptor-1 revealed reduced edema formation in hemorrhagic models (Xi et al., 2001; Xi et al., 2002) and decreased infarct volumes in animal stroke models (Figueroa et al., 2005, De Simoni et al., 2003, Vasthare et al., 1998). However, the lack of specificity of these agents left it unclear as to which complement components are most relevant in the pathogenesis of cerebral injury.

C3a and C5a, collectively known as the anaphylotoxins, induce a wide variety of detrimental biochemical responses, including neutrophil recruitment, formation of free radicals, and increased permeability of the blood–brain barrier (Elsner et al., 1994). Recent studies have revealed that functional inhibitors, as well as genetic knockouts of C3, were protective against ICH-induced edema (Yang et al., 2006) and cerebral ischemic/reperfusion injury (Mocco et al., 2006). However, attempts to inhibit C5 have met with conflicting results. Genetic knockouts of C5 have shown increased vulnerability to ICH (Nakamura et al., 2004), ischemic stroke (Mocco et al., 2006), and excito-toxic injury (Pasinetti et al., 1996). In contrast, functional inhibition of C5 and the C5a receptor have shown neuroprotection against ischemia–reperfusion injury in MCAO models (Costa et al., 2006; Kim et al., 2008) as well as visceral (Fleming et al., 2003) and renal models (de Vries et al., 2003). Recently, our group showed that blocking C3a using a receptor antagonist was protective in a murine model of ICH (Rynkowski et al., 2009). Given that C5a presented a similarly promising target with overlapping and possibly compensatory properties to C3a, we examined the effects of C5a receptor blockade alone and in combination with C3a receptor blockade to determine if dual therapy had synergistic benefits.

Section snippets

Neurological function and behavioral tests

At 6 h after ICH, when the first drug injection was given, there were significant behavioral deficits in each cohort over sham (n = 11) as assessed using the 28-point scoring system and corner turn test (Fig. 1). All tests demonstrated a gradual recovery of function in C5aRA-treated (n = 13) and C3aRA/C5aRA-treated mice (n = 16) over 72 h  after ICH. Compared with vehicle-treated animals (n = 17), C5aRA-treated mice, and C3aRA/C5aRA-treated mice showed significant improvement at 24, 48 h  in the turn

Discussion

The complement cascade has a key role regulating the inflammatory process through its influence on neutrophil recruitment, disruption of the blood–brain barrier with subsequent edema formation (Murphy et al., 1992; Peerschke et al., 1993). The development of clinically useful therapies targeting this cascade has been hindered, however, by insufficient understanding of which complement subcomponents contribute to post-hemorrhagic injury. Given the wide range of detrimental cellular responses

Animal preparation and intracerebral infusion

All procedures were approved by the Columbia University Institutional Animal Care and Use Committee. Adult male C57BL/6J mice weighing 23–30 g were randomized to receive intraperitoneal injection of either C5aRA (1 mg/kg) (hexapeptide-derived macrocycle AcF[OPdChaWR]), combined C3aRA (SB290157, Calbiochem, Darmstadt, Germany) and C5aRA (1 mg/kg of each drug) or an equal volume of DMSO. Each drug diluted in DMSO and PBS (1.16% v/v) was given 6 and 12 h  after ICH induction, followed by twice

Acknowledgments

This work was supported by NIH grants AI068730, GM069736 and NS040409.

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