Elsevier

Experimental Neurology

Volume 214, Issue 2, December 2008, Pages 160-167
Experimental Neurology

An integrin inhibiting molecule decreases oxidative damage and improves neurological function after spinal cord injury

https://doi.org/10.1016/j.expneurol.2008.09.006Get rights and content

Abstract

Our previous studies have shown that treatment with an α4β1 integrin blocking antibody after spinal cord injury (SCI) in rats decreases intraspinal inflammation and oxidative damage, improving neurological function. Here, we studied effects of a high affinity small molecule α4β1 inhibitor, BIO5192. First, rats were treated intravenously with BIO5192 (10 mg/kg) or with vehicle (controls) to assess effects of integrin blockade for 24 h or 72 h after thoracic clip-compression SCI. BIO5192 treatment significantly decreased the MPO enzymatic activity (neutrophil infiltration) and ED-1 expression (macrophage density) by 40% and 38% at 24 h and by 52% and 25% at 72 h post injury, respectively. In cord homogenates, BIO5192 treatment decreased expression of the oxidative enzymes gp91phox, inducible nitric oxide and cyclooxygenase-2 by ∼ 40% at both times of analysis. Free radical concentration decreased by 30% and lipid peroxidation decreased by 34% and 46%, respectively, at 24 h and 72 h after SCI. Next, after blockade by BIO5192 for 72 h, neurological outcomes were analyzed for 1–6 weeks after SCI. Motor function significantly improved when assessed by an open-field test. Treated rats planter placed their hind paws and/or dorsal stepped, with weight support, whereas controls only swept their hindlimbs. BIO5192 treatment also decreased mechanical allodynia elicited from the trunk and hind paw by up to 35%. This improved function correlated with decreased lesion size and spared myelin-containing white matter. The neurological improvement offered by this neuroprotective strategy supports the potential for an anti-integrin treatment for SCI.

Introduction

After spinal cord injury (SCI), progressive secondary tissue destruction is partly due to intraspinal inflammation, (Blight, 1985, Taoka and Okajima, 1998, Tator and Fehlings, 1991, Young, 1993) characterized by a large influx of neutrophils and monocyte/macrophages (Blight, 1992, Fleming et al., 2006, Saville et al., 2004). Neutrophils are major exogenous sources of cell-damaging free radicals (Bao et al., 2005, Fleming et al., 2006, Juurlink and Paterson, 1998, Lewen et al., 2000) and the initial infiltrating macrophages are also pro-inflammatory (Popovich et al., 1999), supporting the well-documented concept that early anti-inflammatory intervention is neuroprotective (Fleming et al., Fleming et al., 2006, Gonzalez et al., 2003, Gris et al., 2004, Taoka and Okajima, 1998).

The leukocyte integrin α4β1 that is expressed by rat and human neutrophils, monocyte/macrophages and lymphocytes (Hemler et al., 1987, Lobb and Hemler, 1994) plays an important role in regulating leukocyte trafficking. The α4β1 integrin participates in early tethering and rolling of leukocytes as well as firm adhesion, thereby contributing to the full process of leukocyte extravasation (Davenpeck et al., 1998, Nandi et al., 2004, Yednock et al., 1995). Therefore, blocking the α4β1 integrin is potentially a robust strategy for anti-inflammatory treatments.

Monoclonal antibodies (mAb) to the α4 subunit of the α4β1 integrin have been used to study the role of α4β1 integrins in models of inflammatory disease. Anti-α4 antibodies have been used to block integrin-mediated lymphocyte migration to inflammatory sites and homing to lymphoid tissues (Issekutz, 1991, Issekutz and Wykretowicz, 1991), decrease eosinophil and neutrophil migration into the pleural cavity and decrease late airway responses in ovalbumin-sensitized and challenged rats, (Taylor et al., 1997, Hojo et al., 1998, Schneider et al., 1999, Ramos-Barbón et al., 2001). These antibodies also have been shown to block neutrophil migration to the inflamed joint even after joint inflammation had developed, proving that an anti-α4β1 regimen is an effective arthritis treatment (Issekutz et al., 1996). Recently we used an anti-α4 mAb to reduce neutrophil and monocyte/macrophage infiltration into the injured rat spinal cord, reducing oxidative damage, and improving neurological function (Fleming et al., in press).

Although anti-α4 function-blocking antibodies can have robust treatment effects, they potentially may be associated with undesirable side effects (Bell and Issekutz, 1993, Lobb and Hemler, 1994). For example, antibody ligation to the α4β1 integrin on leukocytes can activate them, potentially inducing oxidative activity (Pereira et al., 2001) or inducing pro-inflammatory gene expression (Yurochko et al., 1992). A recent study in our laboratory revealed that incubation of neutrophils with the anti-α4 mAb, that was used in our studies of SCI, (Fleming et al., in press) increased oxidative activity in these cells if the exposure lasted longer than 1 h (J. C. Fleming, PhD dissertation, University of Western Ontario). As small molecule inhibitors can also have function-blocking effects, we took advantage of a potent small molecule blocker of the α4β1 integrin, BIO5192 [2(S)-{[1-(3,5-dichlorobenzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-otolyl-ureido)-phenyl]-acetyl}-mino) pentanoylamino]-butyric acid]. BIO5192 (KD < 10 pM) was of special interest because it is a highly selective inhibitor of both unactivated and activated forms of the human, mouse, and rat α4β1 integrin and it has a slow dissociation rate from the bound complex, with substantial binding remaining in the absence of circulating plasma levels of the compound (Leone et al., 2003). Our previous study demonstrated that the expression of α4β1 by neutrophils and monocytes renders this integrin an excellent target for reduction of acute inflammation after SCI (Fleming et al., in press). Therefore, we conducted this study to ascertain whether blockade by BIO5192 would also have neuroprotective effects on the injured spinal cord leading to improved neurological outcomes. We delivered BIO5192 intravenously to achieve integrin blockade for up to 72 h after clip-compression thoracic SCI and assessed effects on intraspinal inflammation and oxidative damage, locomotor recovery, neuropathic pain and lesion size.

Section snippets

Spinal cord injury and treatment paradigm

Twenty-seven female Wistar rats (Charles River, St. Constant, Quebec), weighing 200–220 g, were used to assess leukocyte infiltration and oxidative damage. Twenty-one male Wistar rats (250–320 g) were used to assess motor function and mechanical allodynia, a form of neuropathic pain. Males were used in these behavioural studies to avoid the confounding variable of hormonal cycles on the assessments of pain (Berkley, 1997, LaCroix-Fralish, 2008). Forty-eight Wistar rats (Charles River, St.

BIO5192 treatment decreases MPO activity and ED-1 expression at the spinal cord injury site

We assessed treatment effects on the inflammatory infiltrate in the injured rat cord at 24 h and 72 h after SCI by an MPO assay for neutrophils and by western blotting for ED-1 in homogenates of the lesion site and adjacent segments (T2–6). MPO enzymatic activity (8 ± 0.7 U/g tissue) was minimal in uninjured spinal cord (Fig. 1A). At 24 and 72 h after SCI, MPO activity of the vehicle control group increased to 62 ± 12 and 52 ± 5 U/g tissue, respectively; these values were significantly greater than

Discussion

The small molecule BIO5192 blocks leukocyte migration into tissues by inhibiting α4β1 integrin binding to VCAM-1 on endothelial cells (Alon et al., 1995, Issekutz et al., 1996, Schneider et al., 1999). BIO5192 treatment in the first 3 days after SCI decreased inflammatory cell influx into the spinal cord and the number of phagocytic macrophages, reducing oxidative damage and lesion size. This neuroprotective effect resulted in improved motor function and reduction of neuropathic pain. The

Acknowledgments

This research was supported by a grant from the Canadian Institutes of Health Research. K.S. received a John D. Schultz Summer Studentship from the Heart and Stroke Foundation of Ontario during the tenure of this research. We thank Dr. Jane K. Relton and BiogenIdec for the gift of BIO5192 and Dr. Canio Polosa for his constructive criticism of the research.

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