Inhibition of vasospasm with lymphocyte function-associated antigen—1 monoclonal antibody in a femoral artery model in rats

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Object. The authors have previously shown that a monoclonal antibody (mAb) that recognizes intercellular adhesion molecule—1 (ICAM-1), also known as CD54, when administered systemically inhibits experimental vasospasm in a rat femoral artery model, suggesting that ICAM-1 and leukocyte-endothelial adhesion play a crucial role in the molecular chain of events leading to posthemorrhagic vasospasm. In this report the authors confirm this hypothesis with mAbs directed against lymphocyte function-associated antigen—1 ([LFA-1] CD11a/CD18), the molecule on the surface of leukocytes that interacts with ICAM-1.

Methods. Femoral arteries in 38 Sprague—Dawley rats were isolated and exposed to autologous blood. Twenty-nine animals were then randomized into three groups and received intraperitoneal injections of anti—LFA-1 mAb (10 rats), anti—ICAM-1 mAb (10 rats), or an isotype-matched control mAb (nine rats). Injections were administered at 3 hours and 3, 6, and 9 days after surgery. Before their deaths, six animals underwent spleen harvest, and splenocytes were used in fluorescence-activated cell sorter (FACS) analysis to verify saturation of appropriate binding sites. Animals were killed at 12 days and vessels were harvested for histological study and measurement of the luminal cross-sectional area. Nine animals were randomized as earlier, killed 24 hours after a single injection of mAb, and evaluated for periadventitial infiltration of granulocytes and macrophages. Results of FACS analysis demonstrated saturation of both LFA-1 and ICAM-1 binding sites in animals treated with the respective mAb. The mean ratios of blood-exposed to saline-exposed luminal cross-sectional areas (expressed as the percentage of lumen patency) were 90.1 ± 5.8% (mean ± standard error of the mean) for animals treated with the anti—LFA-1 mAb (p = 0.0218), 94.2 ± 3.3% for animals treated with the anti-ICAM-1 mAb (p = 0.0067), and 62 ± 7.4% for animals treated with the isotype-matched control mAb. Macrophage and granulocyte counts in the periadventitial region were 39.5 ± 3.2/hpf for animals treated with anti—LFA-1 mAb (p = 0.001), 42 ± 3.7/hpf for animals treated with anti—ICAM-1 mAb (p = 0.003), and 72.2 ± 6.2/hpf for control animals.

Conclusions. The systemic administration of anti—LFA-1 or anti—ICAM-1 mAb initiated 3 hours after exposure to autologous blood inhibits the development of delayed chronic vasospasm at 12 days in a rat femoral artery model and leads to a significant reduction in periadventitial inflammatory cells at 24 hours. The authors conclude that blocking the migration of inflammatory cells across the endothelial surface of an artery after adventitial exposure to blood prevents the initiation of biological cascades necessary for the subsequent development of chronic vasospasm.

Article Information

Address reprint requests to: Rafael J. Tamargo, M.D., Department of Neurological Surgery, The Johns Hopkins Hospital, Meyer 7–113, 600 North Wolfe Street, Baltimore, Maryland 21287. email: rtamarg@jhmi.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Representative histograms obtained during FACS analysis of saturation of ICAM-1 and LFA-1 binding sites. Splenocytes were harvested from animals treated with systemic administration of anti—ICAM-1 mAb, anti—LFA-1 mAb, or nonspecific control mAb and stained with anti—mouse IgG alone (dotted lines) or after exposure to additional anti—ICAM-1 or anti—LFA-1 mAb prior to anti—mouse IgG staining (solid lines). The absence of a shift in the FACS histogram obtained in animals treated with anti—ICAM-1 (A) and anti—LFA-1 (B) mAbs indicates saturation of the respective binding sites. Splenocytes from control animals demonstrated a shift in the FACS histograms on exposure to additional anti—ICAM-1 (C) or anti—LFA-1 (D) mAbs, thus indicating open binding sites for the respective target molecule. This experiment was repeated twice with similar results.

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    Bar graph demonstrating arterial lumen patency 12 days after exposure to autologous blood. Values represent the mean ratios of luminal cross-sectional areas of blood-exposed to saline-exposed femoral arteries, expressed as the percentage of lumen patency for anti—LFA-1 mAb treatment (10 animals), anti—ICAM-1 mAb treatment (10 animals), and isotype-matched control mAb treatment (nine animals). Note that the y-axis scale starts at 50% to emphasize the observed difference. Error bars represent the SEMs, with an asterisk denoting a statistically significant difference between a treatment group mean compared with the mean in control animals.

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    Bar graph demonstrating mean macrophage and granulocyte counts per high-power field 24 hours after blood exposure. Immunohistochemical staining for macrophages and granulocytes was performed and positive staining cells per representative high-power field were counted. Values represent the mean objective cell counts for anti—LFA-1 mAb treatment (11 vessel sections, three animals), anti—ICAM-1 mAb treatment (11 vessel sections, three animals), and isotype-matched control mAb treatment (11 vessel sections, three animals). Error bars represent SEMs, with an asterisk denoting a statistically significant difference between a treatment group mean compared with the mean in control animals.

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    Artist's rendering of the molecular cascades leading to delayed cerebral vasospasm. Rupture of a cerebral aneurysm and the release of blood into the subarachnoid space at arterial pressure leads to an as yet unidentified initial signal. This signal initiates a cascade of molecular events leading to an upregulation of ICAM-1 (green rectangular receptors) on endothelial cells. Leukocytes, which are already tethered to and rolling along the endothelium through the interactions of their sialylated carbohydrates (pink triangles) and endothelial selectins (purple triangular receptors), experience adhesion and arrest when LFA-1 (green rectangles) on leukocytes interacts with ICAM-1 on the endothelium. Following arrest, leukocytes migrate through the vessel wall, releasing endothelins that induce contraction of the vascular smooth muscle and oxygen free radicals (O·) that inactivate nitric oxide (NO). Both result in contraction of the vessel wall and the resultant vasospasm. Although a leukocyte in the periadventitial space is shown releasing these mediators of vascular tone for illustrative purposes, it is more likely that leukocytes migrating through the vessel wall closer to the endothelium and underlying smooth muscle are responsible for the molecular events leading to vasospasm.

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