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Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion

Departments of ¹Molecular and Cellular Physiology and ²Pathology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, 71130; and ³Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

Submitted 14 June 2004 ; accepted in final form 9 August 2004

	ABSTRACT

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Although the ₂-integrins have been implicated in the pathogenesis of cerebral ischemia-reperfusion (I/R) injury, the relative contributions of the -subunits to the pathogenesis of ischemic stroke remains unclear. The objective of this study was to determine whether and how genetic deficiency of either lymphocyte function-associated antigen-1 (LFA-1) or macrophage-1 (Mac-1) alters the blood cell-endothelial cell interactions, tissue injury, and organ dysfunction in the mouse brain exposed to focal I/R. Middle cerebral artery occlusion was induced for 1 h (followed by either 4 or 24 h of reperfusion) in wild-type mice and in mice with null mutations for either LFA-1 or Mac-1. Neurological deficit and infarct volume were monitored for 24 h after reperfusion. Platelet- and leukocyte-vessel wall adhesive interactions were monitored in cortical venules by intravital microscopy. Mice with null mutations for LFA-1 or Mac-1 exhibited significant reductions in infarct volume. This was associated with a significant improvement in the I/R-induced neurological deficit. Leukocyte adhesion in cerebral venules did not differ between wild-type and mutant mice at 4 h after reperfusion. However, after 24 h of reperfusion, leukocyte adhesion was reduced in both LFA-1- and Mac-1-deficient mice compared with their wild-type counterparts. Platelet adhesion was also reduced at both 4 and 24 h after reperfusion in the LFA-1- and Mac-1-deficient mice. These findings indicate that both -subunits of the ₂-integrins contribute to the brain injury and blood cell-vessel wall interactions that are associated with transient focal cerebral ischemia.

lymphocyte function-associated antigen-1; macrophage-1; ischemic stroke; ₂-integrins; platelet

The ₂-integrins are heterodimeric proteins that consist of noncovalently associated - (e.g., CD11a, CD11b) and - (CD18) subunits. The expression of ₂-integrins is largely restricted to leukocytes (25), although there is limited evidence for expression on platelets (26). Peripheral blood lymphocytes express primarily LFA-1 (CD11a/CD18), whereas Mac-1 (CD11b/CD18) neutrophils, monocytes, and natural killer cells express both LFA-1 and Mac-1. Although LFA-1 is constitutively expressed on the surface of leukocytes, Mac-1 is normally stored in intracellular granules where it can be rapidly mobilized to the cell surface upon leukocyte stimulation. Both LFA-1 and Mac-1 mediate the firm adhesion of leukocytes to (and transmigration across) endothelial cells with intercellular adhesion molecule-1 (ICAM-1) serving as the counterreceptor for ₂-integrins (16, 25).

Several lines of evidence implicate the ₂-integrins in ischemic stroke. Pretreatment with blocking antibodies directed against either LFA-1 or CD18 reduces infarct size and neutrophil accumulation after cerebral ischemia-reperfusion (I/R; Refs. 24, 34). Similarly, mice that are genetically deficient in either CD18 or Mac-1 are protected against ischemic brain injury (27, 30). However, little is known about the relative contributions of CD11a and CD11b to the adhesion of leukocytes and platelets in the cerebral microcirculation and the relevance of these ₂-integrin-mediated blood cell-vascular wall interactions to the tissue injury and brain dysfunction caused by focal I/R.

The objectives of this study were 1) to define the contributions of LFA-1 and Mac-1 to the leukocyte and platelet vessel-wall interactions elicited in cerebral venules by focal I/R and 2) to determine whether the consequences of LFA-1 or Mac-1 deficiency on these blood cell-vessel wall interactions include improvements in neurological behavior, infarct volume, and mortality.

	MATERIALS AND METHODS

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Animal preparation. Experiments were carried out on wild-type (WT) C57BL/6J mice obtained from Jackson Laboratories (Bar Harbor, ME) and mice with null mutations for either LFA-1 or Mac-1 on a C57BL/6J background (10, 23) that weighed 25–30 g. The mice were anesthetized by administration of ketamine hydrochloride (50 mg/kg ip) and pentobarbitol sodium (50 mg/kg ip). The right jugular vein was cannulated with a polyethylene-10 catheter for intravenous administration of the labeled platelets and rhodamine 6G. The femoral artery was cannulated with a polyethylene-10 catheter (Intramedic; Clay Adams) to monitor mean arterial blood pressure (BP-1; World Precision Instruments) and to sample arterial blood for blood gas analysis (OMNI Modular System).

Middle cerebral artery occlusion. After induction of anesthesia, the middle cerebral artery (MCA) was occluded using the intraluminal filament (6-0) method as previously described (18). Briefly, after mice received a midline neck incision, the left external carotid and pterygopalatine arteries were isolated and ligated with 5-0 silk thread. The internal carotid artery was occluded at the peripheral site of the bifurcation of the internal carotid artery (ICA) and the pterygopalatine artery with a small clip, and the common carotid artery (CCA) was ligated with 5-0 silk thread. The external carotid artery (ECA) was cut, and a 6-0 nylon monofilament with a tip that was blunted (0.2–0.22 mm) with a coagulator (Geigen) was inserted into the ECA. After the clip at the ICA was removed, the nylon thread was advanced until light resistance was felt. The nylon thread and the CCA ligature were removed after the 1-h occlusion period. In the sham group, these arteries were visualized but not disturbed. After the 1-h occlusion period, the cerebral cortex was reperfused for either 4 or 24 h.

In a separate set of experiments (n = 3–5 mice), anesthetized animals from all groups underwent cerebral blood flow (CBF) measurements using a Vasamedics laser Doppler perfusion monitor. All CBF measurements were conducted with the mouse fixed in a plastic frame with the probe placed at the level of the dura directly above the major infarcted region after MCA occlusion (MCAO). The CBF measurements were obtained solely for the purpose of determining whether adhesion molecule deficiency alters the magnitude of the ischemia induced by MCAO. No other data were collected from these mice.

Intravital fluorescence microscopy. After a 4- or 24-h reperfusion period, the head of each mouse was fixed in a plastic frame with the animal in the sphinx position. All mice were tracheostomized and mechanically ventilated (model 683 rodent ventilator; Harvard Apparatus; South Natick, MA) with room air. The left parietal bone was exposed by a midline skin incision followed by a craniectomy using a high-speed microdrill (Fine Science Tools; Foster City, CA) at 1 mm posterior from the bregma and 4 mm lateral from the midline. Artificial cerebrospinal fluid (CSF, which contained 147.8 meq/l Na⁺, 3.0 meq/l K⁺, 2.3 meq/l Mg²⁺, 2.3 meq/l Ca²⁺, 135.2 meq/l Cl^–, 19.61 meq/l HCO₃^–, 1.67 meq/l lactate, 1.1 mmol/l phosphate, and 3.9 mmol/l glucose) was placed over the exposed brain tissue. The observation area represents the major infarcted region after MCAO (18). The dura mater was not cut, because the fluorescently labeled platelets and leukocytes are readily visualized, and intracranial pressure is well maintained in the absence of this procedure (17). A 12-mm glass coverslip was placed over the craniectomy, and the space between the glass and dura mater was filled with artificial CSF. The coverslip was not sealed to prevent an elevation in intracranial pressure. These experimental procedures were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee.

Neurological assessment. The functional consequences of cerebral I/R injury were evaluated by using a five-point neurological deficit score (0, no deficit; 1, failure to extend right paw; 2, circling to the right; 3, falling to the right; and 4, unable to walk spontaneously; Ref. 5) and were assessed in a blinded fashion.

Detection and quantification of cerebral infarction. After a 24-h reperfusion period, the mice were killed with a lethal dose of pentobarbital sodium (150 mg/kg ip). The brains were immediately removed and placed into 4°C PBS for 15 min, and 2-mm coronal sections were cut with a tissue cutter. The brain sections were stained with 2% 2,3,5-triphenyltetrazolium chloride in phosphate buffer at 37°C for 15 min (4). The stained sections were photographed, and the digitized images of each brain section (and the infarcted area) were quantified using a computerized image-analysis program (NIH Image). To minimize the effect of brain edema, calculation of the infarcted volume was indirectly determined as previously described (22), and infarct volume was expressed as a percentage of the ipsilateral hemisphere.

Circulating cell-endothelial cell interactions. Monitoring the activity of circulating cells with the endothelium in the cerebral microcirculation required fluorescent labeling of cells: leukocytes were labeled in vivo with rhodamine 6G (0.02% in 100 µl), and platelets were labeled ex vivo with 5-(6)-carboxyfluorescein diacetate succinimidyl ester (90 µM; Molecular Probes) as previously described (17). The platelet isolation procedure does not alter the expression of P-selectin on the platelet surface, which suggests the absence of platelet activation (32). Platelets were derived from WT mice or mice with null mutations for either CD11a or CD11b. In each mouse, platelets (100 x 10⁶) were infused over 5 min using an infusion pump (Harvard Apparatus) to yield 10% of the total platelet count. The platelets were allowed to circulate for a period of 5 min before images of the microcirculation were recorded.

Intravital fluorescence microscopy and video analysis. An upright Nikon microscope equipped with a silicon-intensified target camera (model C2400-08; Hamamatsu Photonics) and a mercury lamp were used to observe the cerebral microcirculation. The microscopic images were received by a charge-couple device video camera and were recorded on a video recorder (Sony) equipped with a video timer (model WJ-810 time-date generator; Panasonic). Five randomly selected venular segments were evaluated in each preparation for platelet and/or leukocyte adhesion. The pial venules under study were 30–60 µm in diameter and at least 100 µm in length. Venules within the dura mater were not included in this analysis. Adherent platelets and leukocytes were defined as cells remaining stationary for >30 s on the venular wall. Adherent platelets and leukocytes were expressed as the number of cells per square millimeter of venular surface and were calculated from diameter and length values assuming the vessel was cylindrical in shape.

Statistics. All experimental results are expressed as means ± SE. Statistical comparisons were made using ANOVA followed by Newman-Keuls comparison test analysis. The ²- and log-rank Mantel-Haenszel tests were used to determine significance for mortality rates. Statistical significance was assessed at P < 0.05.

	RESULTS

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Deficiency of LFA-1 or Mac-1 protects against focal cerebral I/R injury. Blood gas analysis and blood pressure measurements revealed no statistically significant differences between WT mice and mice with null mutations for either LFA-1 (LFA-1^–/–) or Mac-1 (Mac-1^–/–). The CBF measurements obtained immediately before and after MCAO indicate that blood flow in the MCAO infarct region was reduced to the same extent (90%) in all animal groups. Mortality rate, represented as the percentage of death during the 25-h experimental period (Fig. 1), was significantly higher (20.6%) in WT mice (n = 34) than in either LFA-1^–/– (0%, n = 20) or Mac-1^–/– (4.3%, n = 23) mice. The neurological scores (Fig. 2) were similarly attenuated both in the LFA-1 (0.47 ± 0.2, n = 20) and Mac-1 (1.2 ± 0.3, n = 22) mutant mice compared with their WT counterparts (2.3 ± 0.3, n = 27). In WT mice, an infarct volume of 26.5 ± 8% (n = 10) was detected, whereas LFA-1^–/– (9.4 ± 3%, n = 10) and Mac-1^–/– (10.2 ± 5%, n = 11) mice exhibited significantly smaller ischemic infarcts (Fig. 3). Sham-operated WT mice did not show neurological deficit or infarcted tissue.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Mortality of wild-type (WT) lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 (Mac-1) mice exposed to 1 h of middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. In WT mice, 7 of 34 mice died within the 24-h reperfusion period. No deaths were recorded in sham control (0 of 5) or LFA-1-null mutant (LFA-1–/–; 0 of 20) mice. Only 1 of 23 mice died in the Mac-1-null mutant (Mac-1–/–) group. *P < 0.05 vs. sham mice; +P < 0.05 vs. WT ischemia-reperfusion (I/R) mice.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effects of genetic deficiency of either LFA-1 or Mac-1 on the neurological deficit induced by 1 h of MCAO and 24 h of reperfusion. A five-point neurological score was applied to the WT sham (n = 5), WT I/R (n = 27), LFA-1–/–-I/R (n = 20), and Mac-1–/–-I/R (n = 22) mice. *P < 0.05 vs. sham mice; +P < 0.05 vs. WT I/R mice.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Ischemic infarct volume in LFA-1–/– and Mac-1–/– mice. WT I/R (n = 10) but not LFA-1–/– (n = 10) or Mac-1–/– (n = 11) mice exhibited a significant (P < 0.05) increase in infarct volume compared with WT sham (n = 5) animals. *P < 0.05 vs. WT sham mice.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Adherent leukocytes in cerebral venules after MCAO followed by either 4 h (A) or 24 h (B) of reperfusion. Both LFA-1–/– and Mac-1–/– mice exhibited a blunted adhesion response at 24 h but not at 4 h of reperfusion. *P < 0.05 vs. WT sham mice; +P < 0.05 vs. WT I/R mice; n = 4–6 mice/group.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Adherent platelets in cerebral venules after MCAO followed by either 4 h (A) or 24 h (B) of reperfusion. I/R-induced platelet-recruitment responses were significantly blunted in LFA-1–/– and Mac-1–/– mice at both 4 and 24 h of reperfusion. *P < 0.05 vs. WT sham mice; +P < 0.05 vs. WT I/R mice; n = 4–6 mice/group.

	DISCUSSION

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Previous studies have demonstrated a role for the -subunits of the ₂-integrins in the pathogenesis of cerebral I/R (12, 27, 34). However, this is the first study to show that a genetic deficiency of LFA-1 improves the pathogenic outcome of ischemic stroke. Although LFA-1 can play a major role in modulating the accumulation of leukocytes at sites of inflammation, there is a limited amount of direct evidence to implicate LFA-1 in modulating cell migration in the extravascular space surrounding cerebral venules. For example, it has been shown that defective expression of LFA-1 eliminates the capacity of microglial cells to migrate toward injured neurons (33). Mac-1-positive microglial cells have also been identified after permanent MCAO (14). However, it remains unclear whether microglial ₂-integrins actually contribute to the tissue injury and organ dysfunction induced by transient MCAO.

Our findings also support previously published reports (12, 17, 19, 28) that the postischemic brain microvasculature assumes an inflammatory phenotype that is characterized by the recruitment-adherent leukocytes. As noted in several other vascular beds, I/R-induced leukocyte-endothelial cell interactions occur exclusively in the postcapillary segment (venules) of the microcirculation (7, 9, 31, 32), which is likely explained by the preferential expression of leukocyte adhesion receptors such as P-selectin and ICAM-1 in venules (15, 21). On the basis of the results of the present study, we are unable to implicate a role for either LFA-1 or Mac-1 in the leukocyte adhesion observed at 4 h after reperfusion in the MCAO-reperfusion model. However, genetic deficiency of either LFA-1 or Mac-1 was highly effective in blunting leukocyte adhesion after 24 h of reperfusion, which suggests that both adhesion molecules contribute to the later and more intense recruitment of inflammatory cells in the postischemic brain.

Although a definitive explanation for the differential roles of LFA-1 and Mac-1 at 4 vs. 24 h of reperfusion is not readily available, there are some possibilities that warrant consideration. One possibility is that LFA-1 and Mac-1 expressed on microglial cells play a role in the activation of these macrophage-like cells, which may contribute to a time-dependent amplification of the inflammatory response elicited by I/R. A second possibility is that different leukocyte populations are recruited into the cerebral venules at 4 vs. 24 h after reperfusion. This is consistent with reports that show the early accumulation of neutrophils after I/R and a later, more prolonged accumulation of mononuclear cells in the postischemic brain (3, 11). Finally, the possibility also exists that the reduction in platelet adhesion observed at 4 h of reperfusion may lead to the subsequent reduction in leukocyte adhesion observed at 24 h. Salter et al. (29) have previously demonstrated that recruitment of neutrophils in the postischemic intestinal microvasculature is dependent on the adhesion of platelets.

Another important response of the cerebral microcirculation to I/R is the adhesion of platelets in postcapillary venules (17). We have previously shown that 1 h of global ischemia in mouse brain followed by 4 h of reperfusion promotes P-selectin- and ICAM-1-dependent recruitment of adherent platelets in venules. Our findings implicate a role for LFA-1 and Mac-1 in the recruitment of platelets in postischemic cerebral venules after either 4 or 24 h of reperfusion. Because neither LFA-1 nor Mac-1 deficiency was associated with a reduction in leukocyte recruitment at 4 h of reperfusion, it appears less likely that the LFA-1- and Mac-1-dependent accumulation of platelets at 4 h of reperfusion involves the binding of platelets to adherent leukocytes. It is possible that although leukocytes deficient in either LFA-1 or Mac-1 are still capable of binding to venular endothelium at 4 h of reperfusion, the level of activation of these cells may be significantly reduced. Because superoxide is known to modulate platelet adhesion in postcapillary venules (7), diminished production of this reactive oxygen specie by adherent LFA-1- or Mac-1-deficient leukocytes could account for the blunted platelet adhesion observed at 4 h after reperfusion. Another possible explanation for the reduced I/R-induced platelet adhesion in LFA-1^–/– and Mac-1^–/– mice may relate to previous studies that demonstrated the expression of CD11/CD18 on platelets (26) as well as a functional role for platelet-associated ₂-integrins in interacting with endothelial ICAM-1 during pulmonary fibrosis (13).

In summary, our findings indicate that the ₂-integrins LFA-1 and Mac-1 play a major role in mediating the tissue injury, organ dysfunction, and mortality associated with transient focal cerebral ischemia. The present study also implicates the recruitment and possible activation of adherent leukocytes and platelets as a mechanism that underlies the involvement of ₂-integrins in the pathogenesis of ischemic stroke. These observations lend additional support for the therapeutic potential of anti-adhesion strategies for cerebrovascular diseases such as ischemic stroke.

	GRANTS

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This study was supported by National Heart, Lung, and Blood Institute Grant HL-26441.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. N. Granger, Dept. of Molecular and Cellular Physiology, Louisiana State Univ. Health Sciences Center, 1500 Kings Highway, Shreveport, LA 71130 (E-mail: dgrang{at}lsuhsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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