TNF-alpha receptor antagonist, R-7050, improves neurological outcomes following intracerebral hemorrhage in mice
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.
References (37)
- et al.
RIP kinases at the crossroads of cell death and survival
Cell
(2009) - et al.
Inducible heat shock protein 70, interleukin-18, and tumor necrosis factor alpha correlate with outcomes in spontaneous intracerebral hemorrhage
J. Clin. Neurosci.
(2007) - et al.
Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production
Blood
(2012) - et al.
A class of small molecules that inhibit TNFalpha-induced survival and death pathways via prevention of interactions between TNFalphaRI, TRADD, and RIP1
Chem. Biol.
(2007) - et al.
Hemin-induced necroptosis involves glutathione depletion in mouse astrocytes
Free Radic. Biol. Med.
(2008) - et al.
The TNF and TNF receptor superfamilies: integrating mammalian biology
Cell
(2001) - et al.
Comparison Evans Blue injection routes: intravenous versus intraperitoneal for measurement of blood–brain barrier in a mice hemorrhage model
J. Neurosci. Methods
(2011) - et al.
Early surgery versus initial conservative treatment in patients with spontaneous supratentorial intracerebral haematomas in the International Surgical Trial in Intracerebral Haemorrhage (STICH): a randomised trial
Lancet
(2005) - et al.
Recognizing death: the phagocytosis of apoptotic cells
Trends Cell Biol.
(1998) - et al.
Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation
Neurobiol. Dis.
(2010)
Evolution of the inflammatory response in the brain following intracerebral hemorrhage and effects of delayed minocycline treatment
Brain Res.
Death receptors: signaling and modulation
Science
Guidelines for the management of spontaneous intracerebral hemorrhage in adults: 2007 update: a guideline from the American Heart Association/American Stroke Association Stroke Council, High Blood Pressure Research Council, and the Quality of Care and Outcomes in Research Interdisciplinary Working Group
Stroke J. Cereb. Circ.
Molecular signatures of brain injury after intracerebral hemorrhage
Neurology
Size matters: hemorrhage volume as an objective measure to define significant intracranial hemorrhage associated with thrombolysis
Stroke J. Cereb. Circ.
Citicoline treatment for experimental intracerebral hemorrhage in mice
Stroke J. Cereb. Circ.
Identification of RIP1 kinase as a specific cellular target of necrostatins
Nat. Chem. Biol.
Intracerebral hemorrhage triggers interleukin-6 and interleukin-10 release in blood
Stroke
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