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(Stroke. 1996;27:951-956.)
© 1996 American Heart Association, Inc.

Articles

Treatment of Focal Cerebral Ischemia With Synthetic Oligopeptide Corresponding to Lectin Domain of Selectin

Eiharu Morikawa, MD, DMSc; Shi-Ming Zhang, MD; Yoshinori Seko, MD, DMSc; Tomikatsu Toyoda, MD Takaaki Kirino, MD, DMSc

From the Department of Neurosurgery (E.M., S.-M.Z., T.T., T.K.) and the Third Department of Internal Medicine (Y.S.), University of Tokyo Faculty of Medicine, Bunkyo-ku, Tokyo, Japan.

Correspondence to Eiharu Morikawa, MD, DMSc, Department of Neurosurgery, Saitama Medical School/Center, 1981 Kamoda, Kawagoe, Saitama 350, Japan.

	Abstract

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Background and Purpose Synthetic oligopeptides with amino acid sequences of the lectin domain of selectin block selectin-mediated cell adhesion in vitro, which may be applied to a therapeutic intervention to attenuate acute inflammatory reactions. To evaluate the efficacy of such treatment against ischemic brain injury, the effects of administering a selectin oligopeptide that selectively blocks selectin-mediated cell adhesion on histological outcome and on cerebral blood flow (CBF) were studied in models of rodent focal cerebral ischemia.

Methods Spontaneously hypertensive rats were anesthetized with halothane. Permanent focal cerebral ischemia was induced by tandem left middle cerebral artery (MCA) and common carotid artery (CCA) occlusion. Focal cerebral ischemia with partial reperfusion was introduced by reperfusing the CCA after 2 hours of tandem MCA/CCA occlusion. A synthetic oligopeptide (amino acid residues 23-30 from N terminal) of E-selectin was dissolved in physiological saline and was injected intravenously at a dosage of 2 mg/kg or 10 mg/kg before artery occlusion. Control animals received equivalent volumes of physiological saline or 10 mg/kg of synthetic oligopeptide with a scrambled amino acid sequence. Twenty-four hours after the occlusion, seven coronal brain slices were stained with 2,3,5-triphenyltetrazolium chloride, and the volume of ischemic injury was calculated. In a separate set of animals, regional CBF was monitored with laser-Doppler flowmetry at the dorsolateral cerebral cortex during 2-hour ischemia and 30 minutes after partial reperfusion.

Results The volume of ischemic injury did not differ among groups in permanent ischemia. In ischemia with partial reperfusion, 10 mg/kg selectin oligopeptide, but not the same dosage of scrambled oligopeptide, significantly reduced the volume of ischemic injury (95±13, 73±11, 55±6, and 111±14 mm³ for saline [n=11]; 2 mg/kg [n=10] and 10 mg/kg [n=16] selectin oligopeptide and 10 mg/kg scrambled oligopeptide [n=6], respectively; P<.01 by one-way ANOVA followed by the Tukey test). Laser-Doppler flowmetry demonstrated a larger increase in CBF after reperfusion of the CCA in the 10-mg/kg selectin oligopeptide group.

Conclusions Our data demonstrate that administration of a synthetic oligopeptide corresponding to the lectin domain of selectin decreases the size of ischemic injury after transient, but not after permanent, focal cerebral ischemia as evaluated at 24 hours after onset of ischemia. These effects were associated with an improved CBF at the dorsolateral cerebral cortex after partial reperfusion.

Key Words: cerebral ischemia, focal • leukocytes • reperfusion injury • rats

	Introduction

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Brain injury after focal cerebral ischemia is associated with acute inflammatory reactions that are evidenced by leukocyte recruitment occurring as early as 30 minutes after arterial occlusion.¹ Accumulation of polymorphonuclear leukocytes may exacerbate brain damage by a "no-reflow" phenomenon due to microvascular plugging² or by injurious mediators released from leukocytes.³ Thus, depletion of circulating neutrophils has been shown to attenuate brain damage after focal cerebral ischemia.⁴

The cell surface glycoproteins called adhesion molecules have been shown to mediate leukocyte adhesion to endothelium after ischemia/reperfusion.⁵ Administration of monoclonal antibodies against CD11b/18 integrin,⁶ an adhesion molecule expressed on leukocytes, and against ICAM-1,⁷ which is expressed on the endothelium, thus attenuated leukocyte accumulation and cerebral damage after transient focal cerebral ischemia.

The selectins are a recently identified family of adhesion molecules that regulates the early step of leukocyte adhesion to endothelium, causing slowing down or "rolling" of leukocytes on the endothelia that promotes the firm attachment by integrins and ICAMs (for review, see Bevilacqua and Nelson⁸ and Lefer et al⁹ ).

There are three selectins identified. L-selectin is a constitutive surface molecule on certain subsets of leukocytes, including neutrophils; E-selectin is an inducible molecule on vascular endothelium synthesized de novo after stimulation by cytokines and bacterial endotoxin; and P-selectin is in the storage granules of platelets and endothelial cells and translocates itself to the cell surface shortly after activation.

The selectins have a calcium-dependent lectin domain and bind to carbohydrate ligands such as sialyl-Lewis^x. This selectin-carbohydrate interaction can be blocked in vitro by synthetic oligopeptides corresponding to the lectin domain of selectins.¹⁰ Therefore, the present study was undertaken to elucidate the effects of administration of an LDO, an octapeptide with the amino acid sequence of E-selectin, on the histological outcome and on CBF in models of focal cerebral ischemia. An amino acid sequence homology analysis revealed that this octapeptide sequence is not shared by any known adhesion molecules other than selectins or by any membrane-related proteins.

To select a proper model of transient focal cerebral ischemia, a pilot study was performed in which the volumes of ischemic injury were measured after tandem CCA/MCA occlusion with or without CCA reperfusion. Tandem CCA/MCA occlusion with CCA reperfusion at 1 hour resulted in a marked reduction in the volume of injury (approximately 30 mm³), whereas reperfusion at 2 hours yielded volumes of ischemic injury equivalent to permanent CCA/MCA occlusion (approximately 100 mm³). To detect possible protective effects of the treatment against ischemic brain injury, 2-hour CCA reperfusion was chosen in the present study.

	Materials and Methods

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Normally fed male spontaneously hypertensive rats (Charles River Japan Inc) weighing between 280 and 320 g were used in this study. The procedures followed were in accordance with institutional guidelines. Anesthesia was induced and maintained by halothane (3% and 1%, respectively) plus 70% nitrous oxide and the balance of oxygen. Rats were intubated (PE-240 polyethylene tubing) and mechanically ventilated (model 131, PMI). The right femoral artery was cannulated (PE-50 polyethylene tubing) for continuous arterial blood pressure monitoring (RMP-6004, Nihon Koden). Arterial blood was sampled intermittently for pH, PaCO₂, and PaO₂ (170 pH/Blood Gas Analyzer, CIBA-Corning). Respiratory parameters were adjusted as needed to maintain normal arterial blood gases. Rectal temperature was monitored throughout the experiment, and normothermia was maintained with an animal blanket controller (ATB-1100, Nihon Koden) preset to 37°C.

The distal segment of MCA crossing over the rhinal fissure was exposed as described previously.¹¹ Briefly, a 1.5-cm skin incision was placed approximately midway between the left outer canthus and anterior pinna. The temporalis muscle was incised and retracted to expose the squamous portion of the temporal bone. With the use of a surgical microscope (Zeis), a craniotomy (3 mm in diameter) was created at the juncture of the zygomatic process and the temporal bone. The dura mater was opened with a fine curved needle to expose the MCA.

In experiment 1 (permanent ischemia), the left CCA was ligated with 4-0 silk followed by electrocauterization of a 2-mm segment of MCA. The coagulated MCA segment was then transected with microscissors. In experiment 2 (ischemia with partial reperfusion), the left CCA was occluded with a metal clip (Sugita temporary mini-clip, Mizuho Ikakogyo) followed by left MCA occlusion, as in experiment 1. The clip on the left CCA was removed 2 hours later under light halothane anesthesia, when reperfusion of the artery was visually confirmed. The rats were returned to their cages after surgery and allowed free access to food and water. A single dose of antibiotics (Viccillin S, 30 mg IM, Meiji Seika) was injected.

The 23-30 amino acid sequence of the lectin domain of E-selectin (YTHLVAIQ) was synthesized and purified by high-performance liquid chromatography, and its composition was confirmed by amino acid analysis. This oligopeptide was easily dissolved in saline at concentrations of 1 and 5 mg/mL. The solution was injected at doses of 2 mg/kg and 10 mg/kg body wt 5 to 10 minutes before induction of ischemia. Control animals received equivalent volumes of physiological saline. The effect of an oligopeptide that has a scrambled amino acid sequence (LQTAYDVI) that does not block selectin-mediated cell adhesion¹⁰ was also evaluated in experiment 2.

Twenty-four hours after the vessel occlusion, rats were anesthetized with pentobarbital (65 mg/kg IP) and decapitated. To measure the volume of ischemic injury, brains were removed and placed in ice-cold saline for 10 minutes and sectioned coronally into seven 2-mm slices in a rodent brain matrix (Rat Brain Slicer, Medical Agent Co Ltd). Slices were placed in 2% 2,3,5-triphenyltetrazolium chloride monohydrate (Sigma) at room temperature for 30 minutes followed by 10% formalin overnight.¹² Brain slices were directly scanned on an image scanner (JX-320M, Sharp). The area of each hemisphere and that of the ischemic injury, which is outlined in white, was measured on the posterior surface of each section (NIH IMAGE 1.51). The volumes were calculated by numeric integration of the sequential areas. The magnitude of brain edema in the ischemic hemisphere was estimated according to Kaplan et al.¹³ Analysis of the volumes of ischemic injury were performed not only on raw measurements but also on the data corrected for brain edema.

With laser-Doppler flowmetry (LBF-IIIF, Biomedical Science), rCBF was monitored at the dorsolateral cerebral cortex in a separate set of animals during 2-hour ischemia and 30-minute partial reperfusion.¹⁴ The head of the rat was fixed in a stereotaxic frame (Narishige), and a second craniotomy, covering 4 to 6 mm lateral to the midline and from 1 mm rostral to 2 mm caudal to the bregma, was made for the placement of the laser-Doppler flowmetry probe (0.8 mm in diameter). This region of the brain has been shown to represent ischemic penumbra by autoradiography¹⁵ and by electrophysiology¹⁶ in this ischemia model. A paper-thin layer of the skull was left intact, thus enabling the tip of the probe to remain dry throughout the measurement. Steady-state baseline measurements were obtained before CCA/MCA occlusion. CCA occlusion was performed using a 4-0 monofilament nylon snare ligature according to the method reported by Tone et al.¹⁷ The MCA was occluded by electrocoagulation and transection of the artery after CCA occlusion. Reperfusion of the CCA was accomplished by cutting and removing the nylon ligature. The patency of the CCA was confirmed after the experiment.

Data are presented as mean±SEM. One-way ANOVA followed by a post hoc Tukey test was used for the comparison of data among groups. For the analysis of injured areas, a two-way ANOVA was applied first for overall significance. One-way ANOVA followed by the Tukey test was applied for each slice if it proved significant. A value of P<.05 was taken as statistically significant.

	Results

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Table 1 summarizes the preischemic physiological parameters obtained before the treatment in each group. There were no significant intergroup differences in mean arterial pressure, blood gases, and body temperature in these experiments.

View this table: [in this window] [in a new window]   Table 1. Physiological Parameters Before Induction of Cerebral Ischemia

The hemispheric volumes and the volumes of ischemic injury are summarized in Table 2. There were no significant intergroup differences in magnitude of brain edema in these experiments. Thus, the correction for brain edema did not affect statistical analysis of the volumes of injury. The volumes of ischemic injury in permanent focal ischemia (experiment 1) did not differ among groups (103±10, 106±7, and 93±7 mm³ for saline [n=10], 2 mg/kg LDO [n=10], and 10 mg/kg LDO [n=9] groups, respectively). In focal cerebral ischemia with partial reperfusion (experiment 2), however, the treatment with 10 mg/kg LDO reduced the volume of ischemic injury compared with saline or 10 mg/kg scrambled LDO (95±13, 73±11, 55±6, and 111±14 mm³ for saline [n=11], 2 mg/kg LDO [n=10], 10 mg/kg LDO [n=16], and 10 mg/kg scrambled LDO [n=6], respectively). Thus, LDO reduced the volumes of ischemic injury in a dose-dependent and sequence-specific manner as evaluated at 24 hours after focal cerebral ischemia/reperfusion. Fig 1 shows the areas of ischemic injury on each brain slice. A significant overall difference was detected with a two-way ANOVA (F=80.3 and P=.0001 for slice; F=16.43 and P=.0001 for group). A post hoc Tukey test demonstrated a significant reduction of the areas of ischemic injury with 10 mg/kg LDO treatment on slices 4 and 5 compared with the saline group and on slices 2, 4, and 5 compared with scrambled LDO.

View this table: [in this window] [in a new window]   Table 2. Volumes of Injury After Focal Cerebral Ischemia With and Without Correction for Brain Edema

View larger version (27K): [in this window] [in a new window]   Figure 1. Areas of injury on coronal brain slices after focal cerebral ischemia with partial reperfusion. Treatment with 10 mg/kg LDO significantly decreased the areas on slices 4 and 5 in comparison with saline treatment and on slices 2, 4, and 5 in comparison with the same dosage of scrambled LDO treatment. Error bars denote SEM. **P<.01 in comparison with saline group; +P<.05 and ++P<.01 in comparison with scrambled LDO group.

The rCBF profiles at the dorsolateral cerebral cortex after CCA/MCA occlusion followed by CCA reperfusion are shown in Fig 2. The mean rCBF decreased to 46% to 65% of the preischemic baseline after CCA occlusion alone and further decreased to 26% to 37% of the baseline when MCA was occluded. These blood flow reductions after tandem CCA/MCA occlusion are comparable to those shown in the data published in the literature.¹⁸ The mean cortical rCBF at the end of 2-hour ischemia was unchanged during ischemia at 25% to 32% of the baseline. Thirty minutes after CCA reperfusion, the mean rCBF was 23% to 45% of the baseline.

View larger version (19K): [in this window] [in a new window]   Figure 2. rCBF at the dorsolateral cortex as measured by laser-Doppler flowmetry. MCA occlusion combined with CCA occlusion (CCAO) reduced rCBF to approximately 30% of the preischemic baseline. Reperfusion of CCA (CCAR) resulted in a trend of increased rCBF in the 10-mg/kg LDO group but not in the saline or scrambled LDO group. MCAO indicates MCA occlusion.

The difference between rCBF measurements at 120 and 150 minutes, which corresponds to the rCBF change induced by CCA reperfusion, was significant among the three groups, and a significantly large increase in rCBF was observed in animals treated with LDO before ischemia compared with those treated with saline or scrambled LDO (4.1±3%, 12.4±2%, and -6.7±3% for saline, LDO, and scrambled LDO groups, respectively).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present data demonstrate a significant reduction, by a treatment aimed at inhibiting selectin-mediated cell adhesion, in the volumes of injury as evaluated at 24 hours after focal cerebral ischemia with reperfusion. The effects were not observed in permanent focal ischemia. Although there is a possibility that this treatment simply delayed the maturation of the infarct, these results indicate injurious mechanisms associated with reperfusion¹⁹ that are amenable to anti–leukocyte adhesion therapy.

Focal cerebral ischemia produces a smaller cerebral infarct if reperfusion is instituted within a certain limit of time.²⁰ Reperfusion after this time window results in a cerebral infarct as large as but not larger than those obtained after permanent focal ischemia. In our pilot study, CCA reperfusion at 2 hours yielded volumes of cerebral ischemic injury equivalent to permanent CCA/MCA occlusion, although CCA reperfusion at 1 hour markedly reduced the size of the injury. The timing and the magnitude of reperfusion used in the present study were thus selected to detect therapeutic efficacy against brain injury.

The brain region salvaged by a timely reperfusion after focal cerebral ischemia includes a moderately ischemic brain region called the ischemic penumbra,²¹ from which rCBF was monitored in the present study. In control animals, the rCBF at the penumbra did not show any increase after reperfusion of the CCA. Treatment with LDO, however, significantly increased penumbral rCBF after CCA reperfusion in comparison with the controls. Treatment with LDO thus resulted in an acute hemodynamic improvement at the penumbra followed by the diminished expansion of ischemic cerebral injury into the same region at 24 hours. Because the rCBF threshold for infarct formation is approximately 30% to 40% of baseline,²² given these data it is reasonable to attribute the beneficial effects of LDO to the increased rCBF possibly achieved by the reduced microvascular plugging and by the attenuated release of the vasoconstrictor agents. The present data did not show any differences in the magnitude of brain edema 24 hours after the vessel occlusion. It is possible, however, that the evolution of brain edema was acutely different among the groups and affected the penumbral rCBF at reperfusion.

Direct consequences of attenuated leukocyte recruitment, such as decreased release of injurious and proinflammatory mediators, may also underlie the beneficial effects of LDO treatment. In this regard, it is demonstrated that the soluble form of P-selectin not only inhibits neutrophil adhesion to endothelium but also inhibits superoxide anion release by neutrophils and serves as a regulator for the inadvertent activation of neutrophils in the circulation.^{23 24} Thus, the effects of selectin oligopeptide administration can be due to attenuated neutrophil activation as well as accumulation. Direct vasodilatory effects of this compound, which may increase penumbral rCBF, cannot be ruled out.

In the present study, spontaneously hypertensive rats were used to reduce data variability. However, significantly higher levels of ICAM-1 expression were observed on the endothelial cells from these rats as opposed to the normotensive strain when cultured with suboptimal cytokine concentrations. Furthermore, it has been recently reported that a significantly higher level of messenger RNA for a leukocyte chemoattractant factor is expressed in spontaneously hypertensive compared with normotensive rats after focal cerebral ischemia.²⁵ Therefore, leukocyte-mediated reperfusion injury may be more dominant in the hypertensive than normotensive strain.²⁶

Although all the three selectins are implicated in the pathophysiology of ischemia/reperfusion injury,²⁷ acute rCBF consequences shown in the present data favor the possibility that LDO blocked leukocyte adhesion mediated by L-selectin or P-selectin because the expression of E-selectin takes several hours after the stimuli. The expression of P-selectin on the endothelial cells has been reported after 2 hours of focal cerebral ischemia.²⁸ P-selectin is also expressed on the surface of platelets mediating platelet-leukocyte adhesion and fibrin deposition.²⁹ Therefore, inhibition of P-selectin–mediated adhesion reduces thrombus formation in the vasculature, indicating an alternative mechanism by which this oligopeptide treatment increased reperfusion rCBF at the penumbra. The role of E-selectin in the later events of acute cerebral ischemia, however, is not ruled out because induction of messenger RNA of E-selectin is reported in the focal cerebral ischemia of rats³⁰ and a transient increase of soluble E-selectin is observed in patients after stroke.³¹

The 23-30 lectin-domain peptide of E-selectin was shown to inhibit neutrophil adhesion to both E-selectin and P-selectin,¹⁰ and the sequences of 23-30 peptide of P-selectin and L-selectin are identical. Therefore, this oligopeptide blocks cell adhesion that is mediated by all three selectins. It seems that oligopeptide therapy has certain advantages over monoclonal antibody therapy against cell-adhesion molecules because there is no risk of serum sickness. Recently, in vivo administration of oligosaccharides containing sialyl-Lewis^x, which is the ligand for selectins, was shown to be effective in reducing myocardial reperfusion injury.³² Thus, blockage of selectin-mediated neutrophil adhesion can be achieved by monoclonal antibodies, free soluble selectins, lectin-domain oligopeptides of selectins, and soluble carbohydrates mimicking ligands to the selectins. Further studies are needed to elucidate an ideal therapeutic compound to alleviate leukocyte-mediated brain injury after transient focal cerebral ischemia.

	Selected Abbreviations and Acronyms

 

CBF = cerebral blood flow CCA = common carotid artery ICAM-1 = intercellular adhesion molecule-1 LDO = lectin-domain oligopeptide MCA = middle cerebral artery rCBF = regional cerebral blood flow

	Acknowledgments

 
This study was supported by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan. The authors thank Dr Akira Tamura, Professor and Chairman, Department of Neurosurgery, Teikyo University, for generously allowing us to use some of the equipment for rat focal cerebral ischemia in the present study. Dr Chikuma Hamada helped with statistical analysis of the data.

Received September 8, 1995; revision received January 29, 1996; accepted January 29, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Garcia JH, Liu KF, Yoshida Y, Lian J, Chen S, del Zoppo GJ. Influx of leukocytes and platelets in an evolving brain infarct (Wistar rat). Am J Pathol. 1994;144:188-199. [Abstract]

2. del Zoppo GJ, Schmid-Schönbein GW, Mori E, Copeland BR, Chang C-M. Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons. Stroke. 1991;22:1276-1283. [Abstract/Free Full Text]

3. Lucchesi BR. Complement activation, neutrophils, and oxygen radicals in reperfusion injury. Stroke. 1993;24(suppl I):I-41-I-49.

4. Matsuo Y, Onodera H, Shiga Y, Nakamura M, Ninomiya M, Kihara T, Kogure K. Correlation between myeloperoxidase-quantified neutrophil accumulation and ischemic brain injury in the rat: effect of neutrophil depletion. Stroke. 1994;25:1469-1475. [Abstract]

5. Kurose I, Anderson DC, Miyasaka M, Tamatani T, Paulson JC, Todd RF, Rusche JR, Granger DN. Molecular determinants of reperfusion-induced leukocyte adhesion and vascular protein leakage. Circ Res. 1994;74:336-343. [Abstract/Free Full Text]

6. Chopp M, Zhang RL, Chen H, Li Y, Jiang N, Rusche JR. Postischemic administration of an anti-Mac-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in rats. Stroke. 1994;25:869-876. [Abstract]

7. Zhang RL, Chopp M, Li Y, Zaloga C, Jiang N, Jones ML, Miyasaka M, Ward PA. Anti-ICAM-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in the rat. Neurology. 1994;44:1747-1751. [Abstract/Free Full Text]

8. Bevilacqua MP, Nelson RM. Selectins. J Clin Invest. 1993;91:379-387.

9. Lefer AM, Weyrich AS, Buerke M. Role of selectins, a new family of adhesion molecules, in ischemia-reperfusion injury. Cardiovasc Res. 1994;28:289-294. [Free Full Text]

10. Geng JG, Heavner GA, McEver RP. Lectin domain peptides from selectins interact with both cell surface ligands and Ca²⁺ ions. J Biol Chem. 1992;267:19846-19853. [Abstract/Free Full Text]

11. Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W. Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J Cereb Blood Flow Metab. 1988;8:474-485. [Medline] [Order article via Infotrieve]

12. Kano M, Moskowitz MA, Yokota M. Parasympathetic denervation of rat pial vessels significantly increases infarction volume following middle cerebral artery occlusion. J Cereb Blood Flow Metab. 1991;11:628-637. [Medline] [Order article via Infotrieve]

13. Kaplan B, Brint S, Tanabe J, Jacewicz M, Wang XJ, Pulsinelli W. Temporal thresholds for neocortical infarction in rats subjected to reversible focal cerebral ischemia. Stroke. 1991;22:1032-1039. [Abstract/Free Full Text]

14. Dirnagl U, Kaplan B, Jacewicz M, Pulsinelli W. Continuous measurement of cerebral cortical blood flow by laser-Doppler flowmetry in a rat stroke model. J Cereb Blood Flow Metab. 1989;9:589-596. [Medline] [Order article via Infotrieve]

15. Jacewicz M, Brint S, Tanabe J, Wang X, Pulsinelli W. Nimodipine pretreatment improves cerebral blood flow and reduces brain edema in conscious rats subjected to focal cerebral ischemia. J Cereb Blood Flow Metab. 1990;10:903-913. [Medline] [Order article via Infotrieve]

16. Dalkara T, Morikawa E, Panahian N, Moskowitz MA. Blood flow-dependent functional recovery in a rat model of focal cerebral ischemia. Am J Physiol. 1994;267:H678-H683. [Abstract/Free Full Text]

17. Tone O, Miller JC, Bell JM, Rapoport SI. Regional cerebral palmitate incorporation following transient bilateral carotid occlusion in awake gerbils. Stroke. 1987;18:1120-1127. [Abstract/Free Full Text]

18. Morikawa E, Rosenblatt S, Moskowitz MA. L-Arginine dilates rat pial arterioles by nitric oxide-dependent mechanisms and increases blood flow during focal cerebral ischaemia. Br J Pharmacol. 1992;107:905-907. [Medline] [Order article via Infotrieve]

19. Hallenbeck JM, Dutka AJ. Background review and current concepts of reperfusion injury. Arch Neurol. 1991;47:1245-1254.

20. Memezawa H, Smith ML, Siejo BK. Penumbral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats. Stroke. 1992;23:552-559. [Abstract/Free Full Text]

21. Siejo BK. Pathophysiology and treatment of focal cerebral ischemia, I: pathophysiology. J Neurosurg. 1992;77:169-184. [Medline] [Order article via Infotrieve]

22. Jacewicz M, Tanabe J, Pulsinelli WA. The CBF threshold and dynamics for focal cerebral infarction in spontaneously hypertensive rats. J Cereb Blood Flow Metab. 1992;12:359-370. [Medline] [Order article via Infotrieve]

23. Gamble JR, Skinner MP, Berndt MC, Vadas MA. Prevention of activated neutrophil adhesion to endothelium by soluble adhesion protein GMP140. Science. 1990;249:414-417. [Abstract/Free Full Text]

24. Wong CS, Gamble JR, Skinner MP, Lucas CM, Berndt MC, Vadas MA. Adhesion protein GMP-140 inhibits superoxide anion release by human neutrophils. Proc Natl Acad Sci U S A. 1991;88:2397-2401. [Abstract/Free Full Text]

25. Wang X, Yue T-L, Barone FC, Feuerstein GZ. Monocyte chemoattractant protein-1 messenger RNA expression in rat ischemic cortex. Stroke. 1995;26:661-666. [Abstract/Free Full Text]

26. McCarron RM, Wang L, Siren A-L, Spatz M, Hallenbeck JM. Adhesion molecules on normotensive and hypertensive rat brain endothelial cells. Proc Soc Exp Biol Med. 1993;205:257-262. [Medline] [Order article via Infotrieve]

27. Seekamp A, Till GO, Mulligan MS, Paulson JC, Anderson DC, Miyasaka M, Ward PA. Role of selectins in local and remote tissue injury following ischemia and reperfusion. Am J Pathol. 1994;144:592-598. [Abstract]

28. Okada Y, Copeland BR, Mori E, Tung M-M, Thomas WS, del Zoppo GJ. P-Selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion. Stroke. 1994;25:202-211. [Abstract]

29. Palabrica T, Lobb R, Furie BC, Aronovitz M, Benjamin C, Hsu Y-M, Sajer SA, Furie B. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets. Nature. 1992;359:848-851.[Medline] [Order article via Infotrieve]

30. Zhang RL, Chopp M, Zhang ZG, Gautam SC, Anderson DC, Manning AM. Induction of E-selectin mRNA in the brain after transient focal cerebral ischemia in rat. J Cereb Blood Flow Metab. 1995;15(suppl 1):S113.

31. Fassbender K, Mossner R, Motsch L, Kischka U, Grau A, Hennerici M. Circulating selectin- and immunoglobulin-type adhesion molecules in acute ischemic stroke. Stroke. 1995;26:1361-1364. [Abstract/Free Full Text]

32. Buerke M, Weyrich AS, Zheng Z, Gaeta FCA, Forrest MJ, Lefer AM. Sialyl Lewis^x-containing oligosaccharide attenuates myocardial reperfusion injury in cats. J Clin Invest. 1994;93:1140-1148.

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