Elsevier

Neuroscience Letters

Volume 542, 10 May 2013, Pages 92-96
Neuroscience Letters

TNF-alpha receptor antagonist, R-7050, improves neurological outcomes following intracerebral hemorrhage in mice

https://doi.org/10.1016/j.neulet.2013.02.051Get rights and content

Highlights

  • R-7050, a novel TNFR antagonist, reduces neurovascular injury after ICH.

  • Pharmacological inhibition of TNFR improves outcomes after ICH.

  • TNFR may represent a viable therapeutic target after ICH.

Abstract

Intracerebral hemorrhage (ICH), the most common form of hemorrhagic stroke, exhibits the highest acute mortality and the worst long-term prognosis of all stroke subtypes. Unfortunately, treatment options for ICH are lacking due in part to a lack of feasible therapeutic targets. Inflammatory activation is associated with neurological deficits in pre-clinical ICH models and with patient deterioration after clinical ICH. In the present study, we tested the hypothesis that R-7050, a novel cell permeable triazoloquinoxaline inhibitor of the tumor necrosis factor receptor (TNFR) complex, attenuates neurovascular injury after ICH in mice. Up to 2 h post-injury administration of R-7050 significantly reduced blood–brain barrier opening and attenuated edema development at 24 h post-ICH. Neurological outcomes were also improved over the first 3 days after injury. In contrast, R-7050 did not reduce hematoma volume, suggesting the beneficial effects of TNFR inhibition were downstream of clot formation/resolution. These data suggest a potential clinical utility for TNFR antagonists as an adjunct therapy to reduce neurological injury and improve patient outcomes after ICH.

Introduction

Spontaneous intracerebral hemorrhage (ICH), the most prevalent form of hemorrhagic stroke, induces one-year mortality rates >60% and induces the worst long-term neurological outcomes of all stroke subtypes [29]. ICH is caused by the rupture of small vessels damaged by chronic hypertension or amyloid angiopathy, inducing the formation of an intracranial space-occupying hematoma. Neurosurgical clot evacuation improves outcomes in some ICH patients, although many patients are not amenable to surgical intervention due to an inaccessible location or concurrent intraventricular hemorrhage [27]. As such, conservative management remains a clinical mainstay. The lack of efficacious therapeutic options reinforces the notion that ICH is the least treatable form of stroke and stresses the need for improved medical approaches.

Hemolysis promotes spontaneous hematoma resolution, although this process simultaneously induces the release of pro-inflammatory mediators adjacent to the hematoma. Inflammatory activation is associated with increased neurovascular damage, neurological deterioration, and a poor functional recovery after ICH [15], [22], [28], [37], although the precise mechanisms remain undefined. In particular, elevated plasma concentrations of the pro-inflammatory mediator, tumor necrosis factor-α (TNF-α), clinically correlated with acute hematoma enlargement, edema development, and poor patient outcome following ICH [3], [8], [9], [17], [35]. Similarly, increased TNF-α expression was observed in several different species and in multiple experimental models of ICH. Importantly, our laboratory and others reported a functional association between peri-hematomal TNF-α expression and the development brain edema and neurological injury after ICH [16], [19], [26], [34]. As a whole, these findings support the notion that TNF-α represents a rationale therapeutic target after ICH.

Although emerging pre-clinical and clinical evidence suggests a detrimental role, small molecule TNF-α pathway inhibitors remain largely unexplored in the context of a brain hemorrhage. TNF-α induces biological activity via stimulation of the tumor necrosis factor receptor (TNFR) [1], [23]. TNFR interacts with downstream adaptor proteins, including TRADD, TRAF and RIP1, providing specificity of the response toward a pro-inflammatory and/or a cell death response. R-7050 is a novel cell-permeable triazoloquinoxaline compound that selectively inhibited TNF-α induced cellular signaling using differential screening of a 300,000 compound library [14]. Unlike biologic TNF inhibitors (e.g. infliximab, etanercept and adalimumab) that directly bind TNF-α and function as decoy receptors, R-7050 does not affect binding of TNF-α to TNFR. In contrast, R-7050 selectively inhibits the association of TNFR with intracellular adaptor molecules (e.g. TRADD and RIP), limits receptor internalization, and prevents subsequent cellular responses after TNF-α binding [14]. In the present study, we tested the hypothesis that R-7050 reduces neurovascular injury after ICH.

Section snippets

ICH model

Animal studies were reviewed and approved by the Committee on Animal Use for Research and Education at Georgia Health Sciences University, in compliance with NIH guidelines. A mouse collagenase model of ICH was utilized for all studies, as detailed by our laboratory [19], [32]. Briefly, male CD-1 mice (8–10 weeks old; Charles River, Wilmington, MA, USA) were placed into a stereotactic frame and a 0.5 mm diameter burr hole was drilled over the parietal cortex, 2.2 mm lateral to the bregma. A

Results

R-7050 attenuates neurovascular injury after ICH. Blood–brain barrier opening contributes to the development of vasogenic edema, an important cause of neurological deterioration after ICH. Evans blue extravasation, a sensitive estimate of blood–brain barrier integrity, increased from 12.2 ± 1.5 μg Evans blue/g brain tissue in sham-operated mice to 47.2 ± 5.8 μg Evans blue/g brain tissue at 24 h post-ICH (p < 0.01 vs. sham) (Fig. 1). R-7050 (6 mg/kg) reduced Evans blue extravasation to 28.7 ± 5.9 μg and 30.3 ±

Discussion

ICH induces the highest mortality of all stroke subtypes and <20% ICH survivors recover functional independence after 6 months [13], [29]. Hematoma volume directly correlates with neurological deterioration and patient mortality and neurosurgical clot evacuation produces more favorable outcomes in subsets of ICH patients [4], [29]; however, many patients remain poor surgical candidates and the efficacy of surgical intervention for spontaneous supratentorial ICH remains controversial [2], [27].

Conflict of interest

The authors declare no competing interests or conflicts.

Acknowledgments

This work was supported in part by grants from the National Institute of Health (NS065172 and NS075774) and from the American Heart Association (BGIA2300135) to K.M.D. The funding agencies played no role in study design, data collection and interpretation, or publication.

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