Transient blockade of the CD11d/CD18 integrin reduces secondary damage after spinal cord injury, improving sensory, autonomic, and motor function

Abstract

The early inflammatory response to spinal cord injury (SCI) causes significant secondary damage. Strategies that nonselectively suppress inflammation have not improved outcomes after SCI, perhaps because inflammation has both adverse and beneficial effects after SCI. We have shown that the selective, time-limited action of a monoclonal antibody (mAb) to the CD11d subunit of the CD11d/CD18 integrin, delivered intravenously during the first 48 hr after SCI in rats, markedly decreases the infiltration of neutrophils and delays the entry of hematogenous monocyte-macrophages into the injured cord. We hypothesized that this targeted strategy would lead to neuroprotection and improved neurological outcomes. In this study the development of chronic pain was detected in rats by assessing mechanical allodynia on the trunk and hindpaws 2 weeks to 3 months after a clinically relevant clip-compression SCI at the twelfth thoracic segment. The anti-CD11d mAb treatment reduced this pain by half. Motor performance also improved as rats were able to plantar-place their hindpaws and use them for weight support instead of sweeping movements only. Improved cardiovascular outcome was shown after SCI at the fourth thoracic segment by significant decreases in autonomic dysreflexia. Locomotor performance was also improved. These functional changes correlated with significantly greater amounts and increased organization of myelin and neurofilament near the lesion. The improved neurological recovery after the specific reduction of early inflammation after SCI demonstrates that this selective strategy increases tissue at the injury site and improves its functional capacity. This early neuroprotective treatment would be an ideal foundation for building later cell-based therapies.

Figure 1.

Anti-CD11d mAb treatment improves open field locomotor scores (BBB) (a) and inclined plane test scores (c) for 12 weeks after SCI at T12 and for 6 weeks after SCI at T4 (b). BBB scores of 1-7 indicate increasing movement of the three hindlimb joints, 8 indicates sweeping of the hind limbs with no weight support, and 10 indicates development of more complex motor control, including consistent weight-supported plantar stepping. The inclined plane test (Rivlin and Tator, 1977) determines the ability of the rat to hold its position on an inclined plane with forelimbs and hindlimbs. □, Control rats (a, c, n = 6); ▪, mAb-treated rats (a, c, n = 9). b, Control 1 and 2 weeks, n = 20; 3-6 weeks, n = 6; mAb 1 and 2 weeks, n = 19; 3-6 weeks, n = 7. Decreased number at 3 weeks in c is caused by autonomic testing and perfusion of rats at 2 weeks after SCI. ^*p < 0.05 compared with control rats.

Figure 2.

Anti-CD11d mAb treatment decreases mechanical allodynia for 12 weeks after SCI at T12. Avoidance responses were elicited by stimulating the trunk (a) and the hindpaw (b) in controlrats (□, a, n = 7; b, n = 6) and in mAb-treated rats (▪, a, n = 8; b, n = 7).Scores from 2 weeks of testing for allodynia on the trunk were averaged at each time point plotted. ^*p < 0.05 compared with control rats.

Figure 3.

Anti-CD11d mAb treatment reduces the magnitude of autonomic dysreflexia. Changes in pulsatile arterial pressure (AP), mean arterial pressure (MAP), and heart rate (HR) are shown during a 2 ml balloon distension of the colon in control (a, d) and mAb-treated rats (b, e) 2 weeks (top row) and 6 weeks (bottom row) after SCI at T4. The large increase in pressure evoked at 6 weeks was often associated with arrhythmias (d). Average change in MAP was assessed at 2 (c) and 6 (f) weeks after SCI in control rats (□, c, n = 14; f, n = 6) and mAb-treated rats (▪, c, n = 10; f, n = 7). The dotted lines on the bar graphs show the approximate change in pressure that would be elicited by such stimulation in a rat with an intact spinal cord (Maiorov et al., 1997). bpm, beats per minute; ^*p < 0.05 compared with control rats.

Figure 4.

Morphological and cellular characteristics of the lesion site at T4 (6 weeks) and T12 (12 weeks) in control and mAb-treated rats. Low-power photomicrographs of sections stained with hematoxylin and eosin show the general appearance of the lesion at T4 in control (a) and mAb-treated (b) rats and at T12 in control (c) and mAb-treated rats (d). Asterisks on panels indicate areas containing inflammatory cells. Large arrows on a and b indicate bands of fibrovascular tissue. Plus signs (+) indicate areas of necrotic debris. e and f illustrate examples of foamy macrophages (arrowhead) and lymphocytes (small arrow) present in these lesions. An area of necrotic debris is indicated by the + in f. g and h show examples of arterioles within bundles of fibrous tissue. Scale bars: (in a) a-d, 100 μm; (in e) e-h,= 5.0 μm.

Figure 5.

Anti-CD11d mAb treatment increases myelin in the injured cord after SCI. Solochrome cyanin-stained sections taken at 6 weeks after T4 SCI are shown at the lesion epicenter and at 2.0 mm caudal to the epicenter in control and mAb-treated rats (a). Arrows on photomicrographs at the epicenter indicate patches of intact dark blue compact myelin. Note the more intact neuropil at 2 mm caudal to the injury site in the rat treated with anti-CD11d mAb, reflected by more abundant compact myelin and a smaller cavitation than in the control rat. Scale bar, 200 μm (applies to all photomicrographs). Treatment effects on normalized areas of compact myelin are illustrated after SCI at T4 (b, 2 weeks; c, 6 weeks) and at T12 (d, 12 weeks). Myelin was stained with solochrome cyanin in c and d and with luxol fast blue in b. □, Control rats (b, n = 10; c, n = 5; d, n = 5). ▪, mAb-treated rats (b, n = 8; c, n = 7; d, n = 6). ^*p < 0.05 compared with control rats. +Shortest distance where area is larger than area at epicenter, p < 0.05.

Figure 6.

Anti-CD11d mAb treatment increases neurofilament in the injured cord after SCI. Neurofilament-stained sections taken at 6 weeks after T4 SCI are illustrated at low and higher (inset) power at the lesion epicenter and at 2.0 mm caudal to the epicenter in control and mAb-treated rats (a). These sections are within 40 μm of those stained for myelin illustrated in Figure 5a. Within the lesion, neurofilament fibers were mostly arrayed in disorganized bundles in control and mAb-treated rats. At 2.0 mm caudal to the lesion, the neurofilament was more intact throughout the gray and white matter, and the greater integrity of the neuropil after anti-CD11d mAb treatment is again reflected by the smaller cavitation in the section. White matter axon bundles were aligned more normally at 2 mm from the lesion site, and greater numbers were visualized in cross-section in the treated rats (arrowheads in insets). ^*Indicates area from which high-power inset was taken. Scale bar is 200 μm for the low-power photomicrographs and 10 μm for the insets. Treatment effects on normalized areas of neurofilament are illustrated after SCI at T4 (b,6 weeks) and T12 (c,12 weeks). □, Control rats (b, c, n = 5). ▪, mAb-treated rats (b, n = 7; c, n = 6). ^*p < 0.05 compared with control rats. +Shortest distance where area is larger than area at epicenter, p < 0.05.

Figure 7.

Anti-CD11d mAb-treatment increases neurofilament in white matter and gray matter at 2 mm rostral and caudal to the lesion site. The normalized total areas of neurofilament sampled at 2 mm from the T4 and T12 lesions were compartmentalized according to their locations in white or gray matter. Treatment effects on the white matter and gray matter neurofilament are illustrated after SCI at T4 (a, 6 weeks) and T12 (b, 12 weeks). □, Control rats (a, b, n = 5). ▪, mAb-treated rats (a, n = 7; b, n = 6). ^*p < 0.05 compared with control rats.