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(Stroke. 1995;26:1665-1669.)
© 1995 American Heart Association, Inc.

Articles

Demonstration of Increased Endothelial-Leukocyte Adhesion Molecule–1 mRNA Expression in Rat Ischemic Cortex

Xinkang Wang, PhD; Tian-Li Yue, PhD; Frank C. Barone, PhD Giora Z. Feuerstein, MD

From the Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.

Correspondence to Xinkang Wang, PhD, Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, PO Box 1539, UW2511, King of Prussia, PA 19406.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose Leukocyte infiltration from circulating blood into ischemic brain tissue contributes significantly to ischemic injury. The role of adhesion molecules in leukocyte attachment and infiltration in ischemic tissue has been emphasized. The aim of the present study was to evaluate whether endothelial-leukocyte adhesion molecule–1 (ELAM-1 or E-selectin) mRNA expression is upregulated in focal brain ischemia.

Methods Northern blot analysis with the use of poly(A) RNA isolated from the ischemic and nonischemic rat cortex at 2 and 12 hours after permanent occlusion of the middle cerebral artery (PMCAO) was used to examine ELAM-1 mRNA expression. The temporal expression profile of ELAM-1 mRNA in the ischemic cortex was further evaluated with the use of a quantitative reverse transcription and polymerase chain reaction technique.

Results A very low level of ELAM-1 mRNA was detected in the sham-operated cortex or in the nonischemic cortex. The expression of ELAM-1 mRNA in the focal ischemic cortex was significantly induced by PMCAO, reaching a peak level at 12 hours (6.9-fold increase compared with sham surgery cortical samples, P<.01) and remained elevated for up to 2 days (3.3-fold increase, P<.01) after PMCAO.

Conclusions The demonstration of upregulated ELAM-1 mRNA expression after focal stroke suggests that ELAM-1 may play an important role in leukocyte infiltration into the ischemic brain and that ELAM-1 may provide a potential therapeutic target in ischemic stroke. However, the demonstration of translated ELAM-1 and its cellular localization in the ischemic tissue is required when specific antibodies become available.

Key Words: cerebral ischemia, focal • endothelial • leukocytes • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The infiltration of PMNs into ischemic brain tissue has been demonstrated in several animal models of stroke.^{1 2 3 4} The recruitment of circulating PMNs into ischemic tissue initially requires the interaction of microvascular endothelial cells with the inflammatory cells via specific adhesion molecules, including ICAM-1, ICAM-2, vascular cell adhesion molecule–1, ELAM-1 (or E-selectin), and P-selectin on endothelial cells, and their counterparts such as integrin molecules on leukocytes.^{5 6} However, the specific adhesion molecules on brain capillaries responsible for leukocyte adhesion have not been clearly defined. In a previous study we demonstrated upregulation of ICAM-1 mRNA and protein in rat focal ischemic cortex after PMCAO or temporary MCAO with reperfusion.⁷ Furthermore, Okada et al⁸ reported that focal cerebral ischemia and reperfusion in the baboon stimulated the expression of endothelial P-selectin and ICAM-1 peptides. The functional significance of the upregulation of ICAM-1 has been suggested by studies demonstrating that monoclonal antibodies against ICAM-1 significantly reduce ischemic injury.^{9 10 11}

ELAM-1 is a single-chain glycoprotein belonging to the selectin family. The expression of ELAM-1 has been found exclusively in vascular endothelial cells after stimulation with inflammatory cytokines (eg, IL-1 and TNF-) and bacterial endotoxin.^{5 6 12} Intravital microscopic studies have revealed that ELAM-1 mediates leukocyte rolling in microvessels rather than firm adhesion.¹³ However, rolling leukocytes frequently lead to firm adhesion to the endothelial cells in inflamed tissue.¹³ The ligand for ELAM-1 on leukocytes has been identified as the blood group antigen sialyl-Lewis x.⁶ Since TNF- and IL-1ß are upregulated in the ischemic cortex after PMCAO^{14 15} and/or MCAO with reperfusion¹⁶ and increased infiltration of PMNs into the focal ischemic tissue is observed under these conditions,^{1 2 3 4} we postulated that ELAM-1 is also induced in response to this ischemic brain injury. In the present report we describe the use of both Northern analysis and a sensitive quantitative RT-PCR technique to demonstrate that ELAM-1 mRNA expression increases in ischemic cortex after PMCAO in the rat.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare [Department of Health and Human Services] publication No. [NIH] 85-23, revised 1985, Office of Science and Health Reports, Division of Research Resources/National Institutes of Health, Bethesda, Md). Procedures using laboratory animals were approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals.

Cerebral focal ischemia or sham surgery was performed in male SHR (Taconic Farms, Germantown, NY) or normotensive rats (WKY rats; Charles River, Danvers, Mass) at 16 to 18 weeks of age (weight, 250 to 330 g) by PMCAO, as described in detail previously.^{1 17} Briefly, the MCA was occluded and cut dorsal to the lateral olfactory tract at the level of the inferior cerebral vein with the use of electrocoagulation (Force 2 Electrosurgical Generator, Valley Lab Inc). In sham-operated rats the dura was opened over the MCA, but the artery was not occluded. Rats were later overdosed with pentobarbital, and forebrains were removed for cortical dissection at various times after PMCAO. The ischemic frontoparietal cortex (ie, the cortex ipsilateral to surgery) was dissected from the ipsilateral hemisphere. The contralateral cortex was dissected as the nonischemic control from the same rat.¹ The cortical samples were immediately frozen in liquid nitrogen and stored at -80°C.

Total cellular RNA was prepared from cortical samples as previously described.^{7 18} As a routine procedure of Northern analysis, RNA samples (40 µg per lane) isolated from cortex at various time points after PMCAO were resolved by electrophoresis, transferred to a nylon membrane, and hybridized to an ELAM-1 cDNA probe (generated by RT-PCR; see below). However, under these conditions no ELAM-1 mRNA signal was detected. Therefore, total cellular RNA was isolated from ischemic and nonischemic cortex of 50 animals at either 2 or 12 hours after PMCAO, and the RNA was subjected to poly(A) RNA isolation with a standard procedure.¹⁹ Poly(A) RNA (10 µg per lane) was used for Northern analysis, as described previously.^{7 20} ELAM-1, ICAM-1,⁷ c-fos,²¹ and rpL32⁷ cDNA probes were hybridized to the same membrane sequentially. A probe was stripped from the membrane before rehybridization with the next probe, as described previously.^{7 20}

RT-PCR was used to study the extended temporal expression of ELAM-1 in the ischemic cortex. Total cellular RNA (5 µg) isolated from the cortical samples at the indicated time points after PMCAO was reverse transcribed with 200 U of RNase H^- SuperScript II reverse transcriptase (GIBCO BRL) for 60 minutes at 37°C primed with 1 µg of oligo(dT)_12-18 (GIBCO BRL) at conditions recommended by the manufacturer. The RT products were extracted with phenol/chloroform, precipitated with ethanol, and the products were resuspended in 20 µL of a mixture of 10 mmol/L Tris and 1 mmol/L EDTA (pH 7.5) and stored at -20°C. PCR primers used for amplification of ELAM-1 and rpL32 were synthesized according to published sequences (Table). The rpL32 mRNA expression has been shown previously to be constant throughout the time course after PMCAO,⁷ and therefore it provided an ideal internal PCR control for normalizing the degree of expression in a quantitative manner. To ensure that the sensitivity of the amplification was in a linear range, thus validating the quantitation of signals, we performed the following control experiments. First, dose-dependent amplification of the RT-PCR was tested with 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 µg RNA isolated from the rat ischemic cortex at 12 hours after PMCAO to ensure that the amplification for ELAM-1 and rpL32 mRNAs was in a linear range. Second, different cycles (15, 20, 25, 30, 35, and 40 cycles) were compared to identify an optimal number of amplification cycles for both genes; 25 to 30 PCR cycles were found to be in the linear portion of the amplification for both genes. Finally, the optimal amounts of ³²P-labeled primers for both genes were added to adjust the relative signal intensity for the coamplification. Based on these results, we selected standard conditions for our experiments as follows: RT products from 0.1 µg RNA, 28 cycles in a total 50 µL reaction mixture containing 1x10⁶ cpm (2 ng) and 6x10⁴ cpm (0.12 ng) ³²P-labeled antisense primers for ELAM-1 and rpL32, respectively, together with 100 ng of each nonradioactive sense and antisense primer for both genes (Table). The amplification was performed with 2.5 U of TaqAmpli polymerase (Perkin-Elmer Cetus) in a thermocycler (Perkin-Elmer Cetus) according to the conditions described previously²⁰ : initial denaturation, 3 minutes at 94°C; initial annealing, 1 minute at 54°C; initial extension, 3 minutes at 72°C. The subsequent 27 cycles were as follows: denaturation, 15 seconds at 94°C; annealing, 20 seconds at 54°C; extension, 1 minute at 72°C. Ten microliters of the PCR product was electrophoresed through a 6% native polyacrylamide gel. The gel was dried and subjected to autoradiography at room temperature. The identity of the amplified DNA bands for ELAM-1 and rpL32 was confirmed by Southern blot and/or DNA sequencing, and the DNA fragment was prepared and used for Northern analysis.

View this table: [in this window] [in a new window]   Table 1. Oligonucleotide Primers Used for Quantitative PCR1

PHOSPHORIMAGER (Molecular Dynamics) was used to quantitate the band intensities of the PCR products (or Northern blots), and IMAGEQUANT software version 3.0 (Molecular Dynamics) was used to analyze the results. The relative ELAM-1 mRNA level was determined by the ratio of ELAM-1 to rpL32 signal in each coamplification sample and illustrated the sum of the ratios for the full time course of animals to be 100%.

Statistical evaluation was performed with five complete sets of cortical samples from each time point with the use of one-way ANOVA followed by a post hoc t test. The results are expressed as mean±SE. Significance was accepted for P<.05 by comparing the relative mRNA levels in the ischemic cortex with sham-operated cortex.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fig 1 shows the Northern hybridization of ELAM-1 mRNA expression in the ischemic and nonischemic cortex at 2 and 12 hours after PMCAO in SHR. Almost no ELAM-1 mRNA signal was detected in the ischemic cortex at 2 hours (lane 1) after MCAO or in the nonischemic cortex (lanes 2 and 4). The 3.8-kb ELAM-1 mRNA was markedly upregulated in the ischemic cortex at 12 hours (lane 3) after MCAO. As a control, the same membrane was hybridized to ICAM-1 cDNA probe, which showed a significant level of expression in the ischemic cortex at 12 hours after PMCAO, as demonstrated previously,⁷ and to c-fos cDNA probe, which showed an early induction profile in the same focal ischemia model.²¹ The amounts of mRNA loaded in each lane were examined by hybridizing to an rpL32 cDNA probe that was not subject to any change in the ischemic cortex after MCAO, as demonstrated previously.^{7 16 21}

View larger version (64K): [in this window] [in a new window]   Figure 1. Northern blot analysis of ELAM-1 mRNA expression in rat cortex after PMCAO. Poly(A) RNA (10 µg per lane from a pool of 50 animals) was resolved by electrophoresis, transferred to a nylon membrane, and sequentially hybridized to ELAM-1, ICAM-1, c-fos, and rpL32 cDNA probes. The mRNA sizes were determined by comparing them with the migration of RNA ladder (GIBCO BRL), and the number of kilobases was marked on the right. Lane 1, 2 hours after PMCAO, ischemic (ipsilateral) cortex; lane 2, 2 hours after PMCAO, nonischemic (contralateral) cortex; lane 3, 12 hours after PMCAO, ischemic cortex; and lane 4, 12 hours after PMCAO, nonischemic cortex.

Fig 2 illustrates a representative autoradiograph of an RT-PCR experiment to detect ELAM-1 mRNA expression in the focal ischemic and nonischemic cortex and in sham-operated cortical samples in SHR. The quantitative data for ELAM-1 mRNA (n=5), after normalizing to rpL32 mRNA by calculating the ratios of ELAM-1 to rpL32 in each sample, are summarized graphically in Fig. 3. Only a low, basal level of ELAM-1 mRNA was detected in the sham-operated animals or in the contralateral (nonischemic) cortex, as well as in the early time points of the ipsilateral (ischemic) cortical samples. The level of ELAM-1 mRNA was increased in the ischemic cortex at 6 hours after PMCAO, reached a peak level at 12 hours (6.9-fold increase compared with the sham samples; P<.01; n=5), and maintained an elevated level up to 2 days (3.3-fold increase; P<.01) after PMCAO (Figs 2 and 3).

View larger version (30K): [in this window] [in a new window]   Figure 2. RT-PCR analysis of ELAM-1 mRNA induction in ischemic cortex after PMCAO. The PCR was performed according to standard conditions as described in detail in "Materials and Methods." The PCR products were resolved by electrophoresis in a 6% polyacrylamide gel, dried, and autoradiographed. Ipsilateral and contralateral cortex samples (denoted by +) from individual rats after sham surgery (S; 12 hours after surgery) or after 1, 3, 6, 12, or 24 hours or 2 or 5 days of PMCAO are depicted.

View larger version (13K): [in this window] [in a new window]   Figure 3. Bar graph shows time course of the relative ELAM-1 mRNA levels quantitated by RT-PCR analysis after PMCAO. The PCR-amplified DNA bands of ELAM-1 and rpL32 were quantitated by PHOSPHORIMAGER analysis. The ratios of ELAM-1 to rpL32 were calculated from the coamplified samples, and relative levels of ELAM-1 mRNA were determined based on the differences of these ratios. Data are mean±SE of five separate experiments in SHR (n=5) for each time point. **P<.01 compared with sham-operated cortex samples.

In addition, the ischemia-induced expression of ELAM-1 mRNA was examined in a normotensive rat strain (WKY rats) 12 hours after PMCAO with the use of quantitative RT-PCR. The results demonstrated a significant upregulation of ELAM-1 mRNA in the ischemic cortex at 12 hours after PMCAO (6.7-fold increase compared with sham-operated samples; P<.01; n=4) in WKY rats (data not shown), which is strikingly similar to that observed in SHR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study we demonstrated a significant increase in ELAM-1 mRNA expression in the rat ischemic cortex after PMCAO using both Northern blot analysis and quantitative RT-PCR. This represents the first demonstration that altered expression of this critical adhesion molecule occurs after brain injury. The temporal expression profile of ELAM-1 mRNA is strikingly parallel to that of PMN infiltration into the ischemic tissue (ie, it also occurs at 12 hours after PMCAO in this model).^{1 2} Moreover, the significantly elevated expression of ELAM-1 mRNA at 1 to 2 days after PMCAO appears temporally related to the onset of monocyte recruitment in ischemic tissue.^{1 2} These data suggest that ELAM-1 may play a significant role in leukocyte recruitment during focal brain ischemia. Since similar increases in ELAM-1 mRNA expression were observed in both SHR and WKY rats, the molecule may be important for leukocyte infiltration that has been shown to occur in both strains after focal stroke. In addition, the time course for ELAM-1 mRNA expression is also remarkably similar to that of ICAM-1 (although ICAM-1 mRNA appears to have a relatively higher basal expression) (Fig 1 and Reference 7) and the proinflammatory cytokines, including TNF- and IL-1ß,^{14 15} in the same focal ischemia model. The coordinated expression of inflammatory cytokines and adhesion molecules before and during the infiltration of PMNs into ischemic tissue suggests that a coordinated molecular mechanism initiates and develops the inflammatory response in focal stroke. For example, the increased expression of inflammatory cytokines may be responsible for the upregulation of adhesion molecules including ICAM-1 and ELAM-1, which in turn contributes to the increased recruitment of leukocytes into the ischemic tissue and results in increased tissue injury. In addition, other factors may contribute to the increased expression of these adhesion molecules after focal brain ischemia. For example, ICAM-1 upregulation has been recently demonstrated in human brain microvascular cells in vitro following reoxygenation after exposure to low oxygen tensions.²⁴ However, the relation of the degree of ELAM-1 protein levels to the degree of the mRNA expression induced by focal ischemia remains to be further examined when antibodies against rat ELAM-1 become available. Also, the cellular sources of ELAM-1 expression in the ischemic cortex also need to be confirmed, although in previous studies the expression of this molecule has been found exclusively on vascular endothelial cells.^{5 6 12}

ELAM-1 mRNA appears to be a rare message upregulated in focal cerebral ischemia, as we failed to detect any signal using standard Northern blot methodology (40 µg total RNA per lane). We were able to successfully detect ELAM-1 mRNA by using either a large amount of RNA for Northern blot [10 µg poly(A) RNA per lane] or the highly sensitive quantitative RT-PCR method. The relatively rare message of ELAM-1 may reflect a limited cellular source for this gene expression (ie, the vascular endothelium). In addition to the techniques used in this report, other methods such as ribonuclease protection assay or nuclear run-on analysis may also be useful to detect rare messages. Comparatively, RT-PCR–based quantitation appears to be more attractive because of its sensitivity and simplicity and the lower amount of RNA required. In addition to the quantitative RT-PCR method described in this report and introduced previously by Chelly et al,²⁵ which uses a housekeeping gene as an internal control for the coamplification, a "mimic" PCR technique can also be used.^{26 27} When all these techniques used for the detection of mRNA levels are compared, however, Northern blot analysis should be the first choice, since it is simple, reliable, and directly assesses the amount of message present.

In conclusion, the present data demonstrated a kinetic induction profile for ELAM-1 mRNA in the ischemic cortex after focal ischemia. The data are consistent with the possibility that ELAM-1 is expressed and plays a role in the inflammatory response to cerebral ischemic tissue injury.

	Selected Abbreviations and Acronyms

 

ELAM-1 = endothelial-leukocyte adhesion molecule–1 ICAM = intercellular adhesion molecule IL = interleukin MCA = middle cerebral artery MCAO = middle cerebral artery occlusion PMCAO = permanent middle cerebral artery occlusion PMN(s) = polymorphonuclear leukocyte(s) rpL32 = ribosomal protein L32 RT-PCR = reverse transcription and polymerase chain reaction SHR = spontaneously hypertensive rats TNF- = tumor necrosis factor– WKY = Wistar-Kyoto

	Acknowledgments

 
The authors wish to acknowledge the excellent technical assistance of Raymond F. White in generating the focal ischemic animals.

Received December 8, 1994; revision received May 23, 1995; accepted May 25, 1995.

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