This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, X. Articles by Feuerstein, G. Z. Search for Related Content PubMed PubMed Citation Articles by Wang, X. Articles by Feuerstein, G. Z.

(Stroke. 1995;26:661-666.)
© 1995 American Heart Association, Inc.

Articles

Monocyte Chemoattractant Protein–1 Messenger RNA 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 Giora Z. Feuerstein, MD, Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, PO Box 1539, King of Prussia, PA 19406.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose Previously we demonstrated that focal cerebral ischemia results in an increased expression of several cytokines/chemokines that precede the infiltration of leukocytes into the ischemic cortex after focal stroke induced by occlusion of the middle cerebral artery (MCAO). Monocyte chemoattractant protein–1 (MCP-1) is a potent chemoattractant specific for monocytes. The aim of the present study was to examine whether MCP-1 messenger RNA (mRNA) is expressed in ischemic brain tissue after MCAO.

Methods The expression of MCP-1 mRNA in the ischemic cortex was first identified by means of a sensitive reverse transcription and polymerase chain reaction technique. The time course of expression of MCP-1 mRNA in the ischemic and nonischemic cerebral cortex after both permanent MCAO and temporary MCAO (160 minutes) with reperfusion was then examined by means of Northern blot analysis.

Results Almost no expression of MCP-1 mRNA was found in the sham-operated or nonischemic (contralateral) cortex. A significant increase in MCP-1 mRNA expression in the ischemic cortex was observed after either permanent or temporary MCAO. MCP-1 mRNA was elevated at 6 hours (4.4-fold increase over sham; n=4), reached its highest expression from 12 hours to 2 days (22.7-fold at the peak level; P<.01), and remained elevated up to 5 days (5.6-fold; P<.01) after permanent MCAO. The profile of MCP-1 mRNA expression in the ischemic cortex after MCAO with reperfusion was similar to that of permanent MCAO except that MCP-1 mRNA was increased earlier (ie, 12.5-fold increase at 3 hours; n=4; P<.01). Also, MCP-1 mRNA expression in the ischemic cortex after permanent MCAO was significantly greater in hypertensive rats than in two normotensive rats (n=4; P<.05).

Conclusions The demonstration of induced MCP-1 mRNA expression early after focal ischemia suggests that MCP-1 may represent a locally expressed monocyte chemoattractant that plays an important role in monocyte infiltration into ischemic tissue and therefore may contribute to the tissue injury in ischemic stroke. Further studies must concentrate on identifying the induced expression of MCP-1 and its cellular localization in the ischemic brain when the appropriate antibodies become available.

Key Words: cerebral ischemia, focal • cytokines • proteins • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monocyte chemoattractant protein–1 (MCP-1) was initially discovered as a rapidly induced gene in platelet-derived growth factor (PDGF)–stimulated murine fibroblasts and designated as JE.^{1 2} The mature MCP-1 secreted product contains 76 amino acids and belongs to the chemokine ß superfamily (CC chemokines). Similar to other family members, MCP-1 functions as a chemoattractant specific for monocytes and basophils but not neutrophils.³ MCP-1 also induces adhesion molecule expression and cytokine production in monocytes.^{4 5} MCP-1 is secreted by many cell types in response to lipopolysaccharide, interleukin (IL)-1, IL-4, tumor necrosis factor– (TNF-), interferon gamma, and PDGF.⁵

Permanent occlusion of the middle cerebral artery (PMCAO) and temporary occlusion of the middle cerebral artery (TMCAO) with reperfusion in rats are the most commonly used animal models for ischemic stroke⁶ and resemble the focal stroke that occurs in humans as a result of thromboembolic events. One well-characterized consequence of cerebral ischemia relevant to humans⁷ is the inflammatory reaction that occurs in the ischemic brain. This reaction involves the infiltration of leukocytes, which is led by polymorphonuclear cells and followed by mononuclear cells (for review, see Reference 8⁸ ). Systematic histological studies of the development of cerebral ischemia in spontaneously hypertensive rats (SHR) after both PMCAO and TMCAO have been described in detail.^{9 10} After focal ischemia, monocyte infiltration and macrophage activation occur just after the initial neutrophil recruitment. Specifically, monocytes or macrophages can be identified in ischemic cortical tissue by 2 to 3 days after PMCAO or 1 day after TMCAO.^{9 10} After 5 days of MCAO, macrophages are the predominant inflammatory cells located in the infarct zone and appear to play a significant role in phagocytosis of necrotic tissue.

The chemotactic mediators involved in monocyte recruitment and activation after brain ischemia are not clear. Recently we demonstrated that cytokines (TNF-, IL-1ß, IL-6) and chemokines (cytokine-induced neutrophil chemoattractant [CINC]/KC) are upregulated early in the ischemic brain after both PMCAO^{11 12 13 14} and TMCAO with reperfusion.¹⁵ Since TNF- and IL-1ß are known to stimulate MCP-1 gene expression in vitro,^{16 17} we postulated that MCP-1 might also be induced in focal ischemia and could play a role in monocyte recruitment under these conditions. Therefore, in the present study we evaluated the time course of MCP-1 messenger RNA (mRNA) expression after both PMCAO and TMCAO with reperfusion in rats.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Focal Brain Ischemia
Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (Bethesda, Md: Office of Science and Health Reports, Division of Research Resources/National Institutes of Health; 1985. US Dept of Health, Education, and Welfare [Dept of Health and Human Services] publication [NIH] 85-23). Procedures in which laboratory animals were used were approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals.

Cerebral focal ischemia or sham surgery was carried out in male SHR (Taconic Farms, Germantown, NY) and in three normotensive rat strains (Fisher-344, Wistar-Kyoto, or Sprague-Dawley; Charles River, Danvers, Mass) (age, 18 weeks; weight, 250 to 330 g) by PMCAO or TMCAO, as described in detail previously.^{18 19 20} Briefly, for PMCAO, the middle cerebral artery (MCA) was permanently occluded and cut dorsal to the lateral olfactory tract at the level of the inferior cerebral vein by means of electrocoagulation (Force 2 Electrosurgical Generator, Valley Lab Inc). For TMCAO with reperfusion, the MCA was lifted from the brain surface to occlude blood flow for 160 minutes and then reperfused, as described in detail previously.^{19 20} In sham-operated rats the dura was opened over the MCA, but the artery was not occluded. Rats were overdosed with pentobarbital, and forebrains were removed and dissected at various times after PMCAO or after MCAO with reperfusion and at 12 hours after sham surgery. The ischemic cortex (ie, the cortex ipsilateral to surgery) was dissected from the ipsilateral frontoparietal cortical hemisphere; the contralateral cortex was dissected as the nonischemic control from the same rat.^{18 20} The cortical samples were immediately frozen in liquid nitrogen and stored at -80°C.

Reverse Transcription and Polymerase Chain Reaction
Total cellular RNA was prepared from cortical samples by means of the acid guanidinium thiocyanate, phenol, and chloroform extraction procedure.²¹ To evaluate the expression of MCP-1 mRNA in the ischemic cortex, we initially used a highly sensitive reverse transcription and polymerase chain reaction (RT/PCR) technique. Two amplifying primers for rat MCP-1 mRNA (5' sense primer, 5'-CAGGTCTCTGTCACGCTTCT-3', from bases 4 to 23; 3' antisense primer, 5'-AGTATTCATGGAAGGGAATAG-3', from bases 510 to 530)²² and the amplifying primer pairs for rat ribosomal protein L32 (rpL32) (5' sense primer, 5'-GTGAAGCCCAAGATCGTC-3', from bases 28 to 45; 3' antisense primer, 5'-GAACACAAAACAGGCACAC-3', from bases 422 to 440)²³ were synthesized according to the published sequences. RNA isolated from the cortical samples at 1 hour, 6 hours, 12 hours, and 5 days after PMCAO was reverse transcribed with RNase H^- reverse transcriptase (GIBCO BRL) for 60 minutes at 37°C in the presence of oligo(dT)_12-18 primer (GIBCO BRL) according to the manufacturer's specification. After phenol/chloroform extraction and ethanol precipitation, the RT products (0.1 µg RNA per sample) were subjected to coamplification in the presence of primers for MCP-1 and rpL32 at conditions described in detail previously.^{24 25} In this study rpL32 was used as an internal control for the coamplification to normalize any difference of the samples because a constant expression of rpL32 mRNA in the cortical samples after MCAO has been observed.²⁶

The identities of the amplified complementary DNA (cDNA) for MCP-1 (527 base pairs) and rpL32 (412 base pairs) were confirmed by DNA sequencing. These DNA fragments were also used as probes for Northern analysis, as described below.

Northern Hybridization Analysis
RNA samples (40 µg per lane) were electrophoresed through formaldehyde-agarose slab gels²⁷ and transferred to GeneScreen Plus membranes (Du Pont–New England Nuclear). For Northern blot analysis, MCP-1 cDNA or rpL32 cDNA was uniformly labeled with [-³²P]dATP (3000 Ci/mmol, Amersham Corp) with the use of a random-priming DNA labeling kit (Boehringer Mannheim). Hybridization was carried out overnight with 1x10⁶ cpm/mL of probe at 42°C in 5x SSPE (750 mmol/L NaCl, 50 mmol/L NaH₂PO₄ [pH 7.6], 5 mmol/L ethylenediaminetetraacetic acid [EDTA]), 50% formamide, 5x Denhardt's solution, 2% sodium dodecyl sulfate (SDS), and 200 µg/mL boiled salmon sperm DNA. The membranes were washed in 2x SSPE, 2% SDS at 65°C for 1 to 2 hours with a change for every 30 minutes, then autoradiographed at -80°C with a Cronex Lightning Plus intensifying screen for various times, depending on the signal intensity. A probe was stripped from the membranes by heating at 95°C in a solution containing 10 mmol/L tris(hydroxymethyl)aminomethane (pH 7.5), 1 mmol/L EDTA (pH 8.0), and 1% SDS for 20 minutes before rehybridization with the other probe.

PhosphorImager (Molecular Dynamics) was used to quantitate the band intensities of the Northern blots, and IMAGEQUANT-TM software version 3.0 (Molecular Dynamics) was used to analyze the results. The relative mRNA was calculated by normalizing to the rpL32 signal in each sample.^{24 26}

Statistical Analysis
Statistical evaluation was performed on four complete sets of cortical samples at various times with the use of both Student's t test and one-way ANOVA followed by a post hoc t test. Since the two methods provided identical results, we illustrated the data according to Student's 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 the sham-operated cortex.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Semiquantitative RT/PCR (with rpL32 as internal control for the coamplification) was initially used to detect the expression of MCP-1 mRNA in the ischemic cortex at selected time points (see "Materials and Methods") after PMCAO. Marked increase of MCP-1 mRNA expression in the ischemic cortex was observed (data not shown). Northern analysis was then used to measure the MCP-1 mRNA expression in the extended time course after MCAO. Fig 1A illustrates a representative Northern blot for MCP-1 mRNA expression in the ipsilateral (ischemic) and contralateral (nonischemic) cortical samples after PMCAO. Quantitative Northern blot data (n=4), after normalizing to an rpL32 probe, are illustrated graphically in Fig 1B. A low level of MCP-1 mRNA was detected in sham-operated animals, and almost no signal was detected in the cortical samples from normal or contralateral cortical samples. The absence of MCP-1 mRNA expression in normal or nonischemic cortex was also confirmed even when the highly sensitive RT/PCR technique was used (data not shown). MCP-1 mRNA began to be induced in the ischemic cortex after 6 hours (4.4-fold increase compared with sham; n=4), reached its largest expression level between 12 hours and 2 days (22.7-fold increase at peak level, ie, at 24 hours; P<.01), and remained elevated up to 5 days (5.6-fold; P<.01) after PMCAO (Fig 1).

View larger version (0K): [in this window] [in a new window]   Figure 1. Northern blot analysis of monocyte chemoattractant protein–1 (MCP-1) messenger RNA (mRNA) expression in rat ischemic cortex after permanent middle cerebral artery occlusion (PMCAO). A, Representative Northern blot for MCP-1 and ribosomal protein (rp) L32 complementary DNA probes to the samples isolated at various times and conditions from spontaneously hypertensive rats (SHR) subjected to PMCAO. Total cellular RNA (40 µg per lane) was resolved by electrophoresis, transferred to a nylon membrane, and hybridized to an indicated complementary DNA probe, as detailed in "Materials and Methods." The mRNA size was determined by comparison with the migration of RNA ladder (GIBCO BRL) and the number of kilobases (marked on the right). Ipsilateral and contralateral cortex samples (denoted by +) from individual rats at 12 hours after sham surgery (S) or after 1, 3, 6, 12, 24, 48, and 120 hours of PMCAO are depicted. B, Bar graph shows quantitative Northern blot signals for relative MCP-1 mRNA levels. The quantitation was carried out by means of PhosphorImager analysis. The samples loaded in each lane were normalized with the values of rpL32 mRNA signals. The normalized values for each probe were displayed graphically with a sum of 100% total. Data are presented as the mean values of four separate experiments in SHR (n=4) for each time point. Solid bar indicates ischemic or surgery/ipsilateral cortex; open bar, nonischemic or control/contralateral cortex. Because the MCP-1 mRNA level in the contralateral cortex is so low, the open bar next to each solid bar may not be seen. **P<.01 compared with sham-operated animals as determined with Student's t test.

MCP-1 mRNA expression in the ischemic cortex after TMCAO (160 minutes) with reperfusion exhibited a temporal profile similar to that of PMCAO. Fig 2A shows a representative Northern blot for the increase in MCP-1 mRNA levels after TMCAO with reperfusion. Quantitative data (n=4) are depicted in Fig 2B. MCP-1 mRNA after TMCAO with reperfusion was upregulated somewhat earlier, ie, beginning at 3 hours after reperfusion (12.5-fold increase compared with sham; n=4; P<.01), and the elevated MCP-1 mRNA returned to baseline earlier, ie, there was no significant increase at day 5 (4.3-fold increase) (Fig 2).

View larger version (0K): [in this window] [in a new window]   Figure 2. Northern blot analysis of monocyte chemoattractant protein–1 (MCP-1) messenger RNA (mRNA) expression in rat ischemic cortex after temporary middle cerebral artery occlusion (MCAO) with reperfusion. The figure is illustrated as described in Fig 1 legend except that temporary occlusion (160 minutes) of the middle cerebral artery was used. The time points indicated refer to the time of reperfusion. rp indicates ribosomal protein.

MCP-1 mRNA expression in the ischemic cortex was relatively higher in SHR compared with three normotensive rat strains (Fisher-344, Wistar-Kyoto, and Sprague-Dawley) at 12 hours after PMCAO. A representative comparative Northern blot for MCP-1 mRNA expression in these rat strains is shown in Fig 3A, and the quantitative data (n=4) are illustrated in Fig 3B. Northern blot analysis revealed that although MCAO induced a high level of MCP-1 mRNA expression in the ipsilateral (ischemic) cortex of all four rat strains (P<.01), the MCP-1 mRNA expression follows an order, ie, SHR>Fisher-344>Sprague-Dawley>Wistar-Kyoto (Fig 3). The elevated MCP-1 mRNA level was significantly lower in Wistar-Kyoto rats (0.36-fold of the mean value; P<.01) and Sprague-Dawley rats (0.5-fold; P<.05) than in SHR.

View larger version (0K): [in this window] [in a new window]   Figure 3. Northern blot analysis of monocyte chemoattractant protein–1 (MCP-1) messenger RNA (mRNA) expression in spontaneously hypertensive rats (SHR) and normotensive rats at 12 hours after permanent middle cerebral artery occlusion (MCAO). A, Total cellular RNA was isolated from the ischemic (ipsilateral) and nonischemic (contralateral) cortex of hypertensive (SHR) and normotensive (Wistar-Kyoto [WKY], Sprague-Dawley [SD], and Fisher-344 [F344]) rats at 12 hours after permanent MCAO. This representative Northern blot was carried out with the use of an MCP-1 complementary DNA probe, as described in "Materials and Methods" and in the legend to Fig 1. B, Bar graph shows quantitative analysis of MCP-1 mRNA expression in hypertensive (SHR) and normotensive (WKY, SD, and F344) rats at 12 hours after permanent MCAO, as described in the legend to Fig 1B. Ribosomal protein (rp) L32 mRNA was used to normalize the samples loaded in each lane. Data are presented as the mean values of four animals (n=4) from each strain. A significant induction for MCP-1 mRNA expression was observed in the ischemic (ipsilateral) cortex (solid bar) compared with the nonischemic (contralateral) cortex (open bar, next to the solid bar) of all four strains (n=4; P<.01). *P<.05, **P<.01 compared with SHR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Focal brain ischemia produces an inflammatory reaction that is characterized by the infiltration and accumulation of leukocytes. This phenomenon has been demonstrated repeatedly in animal models of stroke^{9 10 18 20 28 29} and has also been observed in humans.⁷ Of these inflammatory cells present in the ischemic tissue, monocytes/macrophages are the predominant cells present after early polymorphonuclear cell infiltration into the ischemic lesions has occurred.^{9 10} The chemotactic mediators involved in neutrophil infiltration after MCAO are thought to include CINC/KC, a chemokine specific for neutrophils, as demonstrated previously.¹¹ However, the chemokines that mediate monocyte/macrophage recruitment after brain ischemia are not known. The present study represents the first demonstration that MCP-1 (a chemotactic cytokine for monocytes) mRNA expression is induced in response to cerebral ischemia. The increased MCP-1 mRNA expression clearly precedes the influx of monocytes/macrophages in both focal ischemia models.^{9 10} If MCP-1 synthesis parallels the induced mRNA expression, MCP-1 could be the initiating factor in this more chronic inflammatory process. However, additional studies will be necessary to demonstrate the time course for translated MCP-1 under these conditions when appropriate antibodies are available.

It is of interest that the temporal expression of MCP-1 mRNA follows that of TNF- and IL-1ß mRNA expression after PMCAO^{12 13} or TMCAO with reperfusion.¹⁵ Since TNF- and IL-1ß are known to be able to induce MCP-1 mRNA expression in a number of cell lines in vitro,^{5 16} it is possible that MCP-1 mRNA is induced by these two earlier expressed inflammatory cytokines after focal stroke. Additional time-course studies also revealed that the induced expression of chemokines for neutrophils (eg, CINC/KC) precedes that for monocytes (eg, MCP-1), which is well in accord with the temporal profile for neutrophil and monocyte/macrophage infiltration in this focal stroke model.^{9 10}

It is also of interest to note that the elevated level of MCP-1 mRNA expression in SHR is higher than that of normotensive rats (Fig 3). Increased mRNA expression in SHR has also been demonstrated for TNF-, IL-1ß, and intercellular adhesion molecule–1^{13 14 26} and parallels the increase in neurological deficits and the degree of cortical infarction that occur when hypertensive rats are compared with normotensive strains after focal ischemia.¹⁹ Hypertensive rats are known to carry an increased risk factor for stroke and are more vulnerable to focal stroke than normotensive rats.⁶ This appears to be due to increased release of prothrombotic and inflammatory mediators from brain endothelium in the higher-risk, hypertensive animals.^{30 31} The data presented in this report also demonstrate that increased MCP-1 mRNA expression after focal ischemia, although greater in hypertensive rats, is not specific to the SHR strain, because significant postischemia expression was also observed in the three normotensive rat strains (Fig 3). Whether the degree of MCP-1 protein level is reflected by the degree of MCP-1 mRNA expression induced in these rat strains after focal ischemia will need to be determined in the future.

The cellular sources of MCP-1 mRNA in the ischemic cortex are not known. Different cellular sources could include monocytes/macrophages, fibroblasts, B lymphocytes, endothelial cells, smooth muscle cells, and glioblastoma cells, which are all able to express MCP-1.^{4 5} It is important to note that normal brain tissue does not express MCP-1, as reflected in the contralateral cortical samples. After MCAO, MCP-1 mRNA expression begins at about the same time as neutrophils begin to enter the ischemic tissue and increased glial activation and cell death are observed.^{9 10} The presence of mononuclear cells in infarcted tissue begins after 1 to 3 days and peaks at 5 days after ischemia, which is delayed relative to MCP-1 mRNA expression. Therefore, the cellular sources of MCP-1 mRNA expression can be infiltrating leukocytes and/or cells endogenous to the brain. Evidence for the source of MCP-1 mRNA expression will require in situ hybridization studies.

The MCP-1 receptor has recently been cloned from human monocytes and is almost nondetectable in normal brain tissue.³² Therefore, it is possible that induced MCP-1 in focal stroke may be specific for attracting circulating monocytes into ischemic tissue. However, it will be interesting to determine whether brain microglia can be activated by MCP-1 to transform into macrophages after MCAO. The potential importance of cytokines in leukocyte infiltration into the ischemic brain has been described previously.^{33 34} Also, the potentially positive and negative effects of brain- and blood-derived macrophages have been discussed in detail.^{35 36 37}

In summary, the present study describes the temporal induction profile for MCP-1 mRNA in the ischemic cortex after focal ischemia. The early induction of MCP-1 mRNA expression in ischemic brain tissue suggests that this chemokine may be responsible for the recruitment of circulating monocytes into the ischemic tissue and/or for the activation of local microglia to become macrophages after focal stroke. However, future studies must demonstrate that the induced expression of MCP-1 mRNA is translated in the ischemic lesion. This will be possible when the appropriate antibodies to this peptide become available.

	Acknowledgments

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

Received August 24, 1994; revision received November 7, 1994; accepted December 29, 1994.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Cochran B, Reffel A, Stiles C. Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell. 1983;33:939-947. [Medline] [Order article via Infotrieve]
2. Rollins B, Morrison E, Stiles C. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Proc Natl Acad Sci U S A. 1988;85:3738-3742. [Abstract/Free Full Text]
3. Bischoff SC, Krieger M, Brunner T, Dahinden CA. Monocyte chemotactic protein 1 is a potent activator of human basophils. J Exp Med. 1992;175:1271-1275. [Abstract/Free Full Text]
4. Miller MD, Krangel MS. Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines. Crit Rev Immunol. 1992;12:17-46. [Medline] [Order article via Infotrieve]
5. Tani M, Ransohoff RM. Do chemokines mediate inflammatory cell invasion of the central nervous system parenchyma? Brain Pathol. 1994;4:135-143. [Medline] [Order article via Infotrieve]
6. Ginsberg MD, Busto R. Rodent models of cerebral ischemia. Stroke. 1989;20:1627-1642. [Abstract/Free Full Text]
7. Wang PY, Kao CH, Mui MY, Wang SJ. Leukocyte infiltration in acute hemispheric ischemic stroke. Stroke. 1991;229:175-179.
8. Kochanek PM, Hallenbeck JM. Polymorphonuclear leukocytes and monocytes/macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke. 1992;23:1367-1379. [Abstract/Free Full Text]
9. Clark RK, Lee EV, Fish CJ, White RF, Price WJ, Jonak ZL, Feuerstein GZ, Barone FC. Development of tissue damage, inflammation and resolution following stroke: an immunohistochemical and quantitative planimetric study. Brain Res Bull. 1993;31:565-572. [Medline] [Order article via Infotrieve]
10. Clark RK, Lee EV, White RF, Jonak ZL, Feuerstein GZ, Barone FC. Reperfusion following focal stroke hastens inflammation and resolution of ischemic injured tissue. Brain Res Bull. 1994;35:387-391. [Medline] [Order article via Infotrieve]
11. Liu T, Young PR, McDonnell PC, White RF, Barone FC, Feuerstein GZ. Cytokine-induced neutrophil chemoattractant mRNA expressed in cerebral ischemia. Neurosci Lett. 1993;164:125-128. [Medline] [Order article via Infotrieve]
12. Liu T, McDonnell PC, Young PR, White RF, Siren AL, Hallenbeck JM, Barone FC, Feuerstein GZ. Interleukin-1ß mRNA expression in ischemic rat cortex. Stroke. 1993;24:1746-1751. [Abstract/Free Full Text]
13. Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GZ. Tumor necrosis factor- expression in ischemic neurons. Stroke. 1994;25:1481-1488. [Abstract]
14. Wang XK, Yue T-L, Young PR, Barone FR, Feuerstein GZ. Expression of interleukin-6, c-fos and zif268 mRNAs in rat ischemic cortex. J Cereb Blood Flow Metab. 1995;15:166-171. [Medline] [Order article via Infotrieve]
15. Wang XK, Yue T-L, Barone FR, White RF, Feuerstein GZ. Concomitant cortical expression of TNF- and IL-1ß mRNAs follows early response gene expression in transient focal ischemia. Mol Chem Neuropathol. 1994;23:103-114. [Medline] [Order article via Infotrieve]
16. Sica A, Wang JM, Colotta F, Dejana E, Mantovani A, Oppenheim JJ, Larsen CG, Zachariae COC, Matsushima K. Monocyte chemotactic and activating factor gene expression induced in endothelial cells by IL-1 and tumor necrosis factor. J Immunol. 1990;144:3034-3038. [Abstract]
17. Nelken NA, Coughlin SR, Gordon D, Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest. 1991;88:1121-1127.
18. Barone FC, Hillegass LM, Price WJ, White RF, Lee EV, Feuerstein GZ, Sarau HM, Clark RK, Griswold DE. Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: myeloperoxidase activity assay and histological verification. J Neurosci Res. 1991;29:336-345. [Medline] [Order article via Infotrieve]
19. Barone FC, Price WJ, White RF, Willette RN, Feuerstein GZ. Genetic hypertension and increased susceptibility to cerebral ischemia. Neurosci Biobehav Rev. 1992;16:219-233. [Medline] [Order article via Infotrieve]
20. Barone FC, Schmidt DB, Hillegass LM, Price WJ, White RF, Feuerstein GZ, Clark RK, Lee EV, Griswold DE, Sarau HM. Reperfusion increases neutrophils and LTB4 receptor binding in rat focal ischemia. Stroke. 1992;23:1337-1348. [Abstract/Free Full Text]
21. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]
22. Yoshimura T, Takeya M, Takahashi K. Molecular cloning of rat monocyte chemoattractant protein-1 (MCP-1) and its expression in rat spleen cells and tumor cell lines. Biochem Biophy Res Commun. 1991;174:504-509. [Medline] [Order article via Infotrieve]
23. Rajchel A, Chan Y-L, Wool IG. The primary structure of rat ribosomal protein L32. Nucleic Acids Res. 1988;16:2347. [Free Full Text]
24. Wang XK, Lee G, Liebhaber SA, Cooke NE. Human cysteine-rich protein: a member of the LIM/double-finger family displaying coordinate serum induction with c-myc. J Biol Chem. 1992;267:9176-9184.
25. Wang XK, Yue T-L, Barone FR, Feuerstein GZ. Demonstration of increased endothelial-leukocyte adhesion molecule 1 (ELAM-1) mRNA expression in rat ischemic cortex using quantitative reverse transcription and polymerase chain reaction. Stroke. In press.
26. Wang XK, Siren A-L, Liu Y, Yue T-L, Barone FC, Feuerstein GZ. Upregulation of intercellular adhesion molecule 1 (ICAM-1) on brain microvascular endothelial cells in rat ischemic cortex. Mol Brain Res. 1994;26:61-68. [Medline] [Order article via Infotrieve]
27. Lehrach H, Diamond D, Wozney JM, Boedtder H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions: a critical reexamination. Biochemistry. 1977;16:4743-4751. [Medline] [Order article via Infotrieve]
28. Hallenbeck JM, Dutka AJ, Tanishima T, Kochanek PM, Kumaroo KK, Thompson CB, Obrenovitch TP, Contreras TJ. Polymorphonuclear leukocyte accumulation in brain region with low blood flow during the early postischemic period. Stroke. 1986;17:246-253. [Abstract/Free Full Text]
29. 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]
30. Siren AL, Heldman E, Doron D, Lysko PG, Yue TL, Liu Y, Feuerstein GZ, Hallenbeck JM. Release of proinflammatory and prothrombotic mediators in the brain and peripheral circulation in spontaneously hypertensive and normotensive Wistar-Kyoto rats. Stroke. 1992;23:1643-1651. [Abstract/Free Full Text]
31. Doron DA, McCarron RM, Heldmon E, Esiren AL, Spatz M. Comparison of stimulated tissue factor expression by brain microvascular endothelial cells from normotensive (WKY) and hypertensive (SHR) rats. Brain Res. 1992;597:346-349. [Medline] [Order article via Infotrieve]
32. Yamagami S, Tokuda Y, Ishii K, Tanaka H, Endo N. cDNA cloning and functional expression of a human monocyte chemoattractant protein 1 receptor. Biochem Biophys Res Commun. 1994;202:1156-1162. [Medline] [Order article via Infotrieve]
33. Feuerstein GZ, Wang XK, Yue TL, Barone FC. Inflammatory cytokines and stroke: emerging new strategies for stroke therapeutics. Stroke. In press.
34. Feuerstein GZ, Liu T, Barone FC. Cytokines, inflammation and brain injury: the role of tumor necrosis factor . Cerebrovasc Brain Metab Rev. 1994;6:341-360. [Medline] [Order article via Infotrieve]
35. Piani D, Constram DB, Frei K, Fortana A. Macrophages in the brain: friends or enemies? News Physiol Sci. 1994;9:80-84. [Abstract/Free Full Text]
36. Guilian D. Microglia, cytokines, and cytotoxins: modulators of cellular responses after injury to the central nervous system. J Immunol Immunopharmacol. 1990;10:10-21.
37. Chamak B, Morandi V, Mallat M. Brain macrophages stimulate meurite growth and regeneration by secreting thrombospondin. J Neurosci Res. 1994;38:221-233.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				H. Offner, S. Subramanian, S. M. Parker, C. Wang, M. E. Afentoulis, A. Lewis, A. A. Vandenbark, and P. D. Hurn Splenic Atrophy in Experimental Stroke Is Accompanied by Increased Regulatory T Cells and Circulating Macrophages. J. Immunol., June 1, 2006; 176(11): 6523 - 6531. [Abstract] [Full Text] [PDF]

				C. Zhang, B. Lei, T. T. Lam, F. Yang, D. Sinha, and M. O. M. Tso Neuroprotection of Photoreceptors by Minocycline in Light-Induced Retinal Degeneration Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2753 - 2759. [Abstract] [Full Text] [PDF]

				C J S Price, E A Warburton, and D K Menon Human cellular inflammation in the pathology of acute cerebral ischaemia J. Neurol. Neurosurg. Psychiatry, November 1, 2003; 74(11): 1476 - 1484. [Abstract] [Full Text] [PDF]

				D. Huang, M. Tani, J. Wang, Y. Han, T. T. He, J. Weaver, I. F. Charo, V. K. Tuohy, B. J. Rollins, and R. M. Ransohoff Pertussis Toxin-Induced Reversible Encephalopathy Dependent on Monocyte Chemoattractant Protein-1 Overexpression in Mice J. Neurosci., December 15, 2002; 22(24): 10633 - 10642. [Abstract] [Full Text] [PDF]

				W. Panenka, H. Jijon, L. M. Herx, J. N. Armstrong, D. Feighan, T. Wei, V. W. Yong, R. M. Ransohoff, and B. A. MacVicar P2X7-Like Receptor Activation in Astrocytes Increases Chemokine Monocyte Chemoattractant Protein-1 Expression via Mitogen-Activated Protein Kinase J. Neurosci., September 15, 2001; 21(18): 7135 - 7142. [Abstract] [Full Text] [PDF]

				L. Pang, W. Ye, X.-M. Che, B. J. Roessler, A. L. Betz, and G.-Y. Yang Reduction of Inflammatory Response in the Mouse Brain With Adenoviral-Mediated Transforming Growth Factor-{beta}1 Expression Stroke, February 1, 2001; 32(2): 544 - 552. [Abstract] [Full Text] [PDF]

				J. Huang, T. F. Choudhri, C. J. Winfree, R. A. McTaggart, S. Kiss, J. Mocco, L. J. Kim, T. S. Protopsaltis, Y. Zhang, D. J. Pinsky, et al. Postischemic Cerebrovascular E-Selectin Expression Mediates Tissue Injury in Murine Stroke Editorial Comment Stroke, December 1, 2000; 31(12): 3047 - 3053. [Abstract] [Full Text] [PDF]

				T. Mabuchi, K. Kitagawa, T. Ohtsuki, K. Kuwabara, Y. Yagita, T. Yanagihara, M. Hori, M. Matsumoto, D.-I. Chang, and G. J. del Zoppo Contribution of Microglia/Macrophages to Expansion of Infarction and Response of Oligodendrocytes After Focal Cerebral Ischemia in Rats Editorial Comment Stroke, July 1, 2000; 31(7): 1735 - 1743. [Abstract] [Full Text] [PDF]

				J. M. Galasso, J. K. Harrison, and F. S. Silverstein Excitotoxic Brain Injury Stimulates Expression of the Chemokine Receptor CCR5 in Neonatal Rats Am. J. Pathol., November 1, 1998; 153(5): 1631 - 1640. [Abstract] [Full Text] [PDF]

				L. Pantoni, C. Sarti, and D. Inzitari Cytokines and Cell Adhesion Molecules in Cerebral Ischemia : Experimental Bases and Therapeutic Perspectives Arterioscler. Thromb. Vasc. Biol., April 1, 1998; 18(4): 503 - 513. [Abstract] [Full Text] [PDF]

				A. Martin-Ancel, A. Garcia-Alix, D. Pascual-Salcedo, F. Cabanas, M. Valcarce, and J. Quero Interleukin-6 in the Cerebrospinal Fluid After Perinatal Asphyxia Is Related to Early and Late Neurological Manifestations Pediatrics, November 1, 1997; 100(5): 789 - 794. [Abstract] [Full Text] [PDF]

				X. Wang, F. C. Barone, N. V. Aiyar, G. Z. Feuerstein, and G. J. del Zoppo Interleukin-1 Receptor and Receptor Antagonist Gene Expression After Focal Stroke in Rats Stroke, January 1, 1997; 28(1): 155 - 162. [Abstract] [Full Text]

				X. Wang, T.-L. Yue, E. H. Ohlstein, C.-P. Sung, and G. Z. Feuerstein Interferon-inducible Protein-10 Involves Vascular Smooth Muscle Cell Migration, Proliferation, and Inflammatory Response J. Biol. Chem., September 27, 1996; 271(39): 24286 - 24293. [Abstract] [Full Text] [PDF]

				J. S. Kim, S. S. Yoon, Y. H. Kim, and J. S. Ryu Serial Measurement of Interleukin-6, Transforming Growth Factor-ß, and S-100 Protein in Patients With Acute Stroke Stroke, September 1, 1996; 27(9): 1553 - 1557. [Abstract] [Full Text]

				C. Iadecola, F. Zhang, R. Casey, H. B. Clark, M. E. Ross, and R. C Kukreja Inducible Nitric Oxide Synthase Gene Expression in Vascular Cells After Transient Focal Cerebral Ischemia Stroke, August 1, 1996; 27(8): 1373 - 1380. [Abstract] [Full Text]

				E. Morikawa, S.-M. Zhang, Y. Seko, T. Toyoda, and T. Kirino Treatment of Focal Cerebral Ischemia With Synthetic Oligopeptide Corresponding to Lectin Domain of Selectin Stroke, May 1, 1996; 27(5): 951 - 956. [Abstract] [Full Text]

				A. Turler, N. T. Schwarz, E. Turler, J. C. Kalff, and A. J. Bauer MCP-1 causes leukocyte recruitment and subsequently endotoxemic ileus in rat Am J Physiol Gastrointest Liver Physiol, January 1, 2002; 282(1): G145 - G155. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, X. Articles by Feuerstein, G. Z. Search for Related Content PubMed PubMed Citation Articles by Wang, X. Articles by Feuerstein, G. Z.