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(Stroke. 1998;29:516-520.)
© 1998 American Heart Association, Inc.

Original Contributions

Subtractive Cloning Identifies Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) Increased Gene Expression Following Focal Stroke

Xinkang Wang, PhD; Frank C. Barone, PhD; Raymond F. White, MS; 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. E-mail Xinkang_Wang-1{at}SBPHRD.COM

	Abstract

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Background and Purpose—Differential gene expression has been reported following the onset of focal stroke. To identify de novo expression of ischemia-induced genes, we applied subtractive cDNA library strategy to identify the genes that are selectively upregulated by focal stroke.

Methods—Spontaneously hypertensive rats were subjected to permanent occlusion of the middle cerebral artery (MCAO). mRNAs prepared from ischemic and nonischemic cortex 2 and 12 hours after MCAO were subtracted, and a subtractive cDNA library was constructed. A cDNA that encodes for tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) was identified in the subtractive cDNA library. The temporal expression of cortical TIMP-1 mRNA was further characterized in ischemic cortex subjected to permanent or temporary (160-minute) MCAO.

Results—A panel of genes isolated from the subtractive cDNA library was subjected to Southern analysis to confirm ischemia-induced gene expression. TIMP-1 demonstrated robust induction after ischemic injury. Time-course studies revealed that TIMP-1 mRNA was induced threefold over controls at 12 hours (P<.001, n=4 animals) and reached a peak level at 2 days after permanent MCAO (sevenfold increase, P<.001). Similar induction profile of TIMP-1 mRNA was observed in the ischemic cortex after temporary MCAO followed by reperfusion.

Conclusions—This work demonstrated the utility of subtractive cDNA library strategy for discovery of genes differentially expressed in focal stroke. Furthermore, our data implicate TIMP-1 in ischemia-induced brain injury.

Key Words: cerebral ischemia, focal • gene expression • rats

	Introduction

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Differential gene expression occurs in a variety of pathophysiological conditions of the central nervous system, including neurodegeneration, neurotrauma, and ischemia. Various mRNAs are expressed in a given cell at any given time, and changes in relative mRNAs and their levels may have important implications in the development of pathophysiology. Therefore, elucidation of differentially expressed genes is critical for understanding of the molecular mechanisms involved in normal and pathological states as well as providing for new molecular targets that can be pharmacologically manipulated for drug development.

Focal brain ischemia represents a pathophysiological condition that modulates gene expression and function in the brain. A number of genes have been identified for their increased expression in focal stroke, including early response genes (peak induction at 1 to 3 hours) such as c-fos and zif268,¹ intermediate response genes (peak induction at 6 to 12 hours) such as TNF- and interleukin-1ß,^{2 3 4} and delayed response genes (induced after 2 days) such as TGF-ß and interleukin-1 receptor II,^{5 6} by means of Northern blot analysis, quantitative reverse transcription–polymerase chain reaction, RNase protection assays, and in situ hybridization/immunohistochemical studies, all these techniques for the investigation of known genes. Identification of these changes has helped to provide an increased understanding of ischemic brain injury. However, more information is required. To better understand and further extend knowledge on de novo gene expression induced by ischemia, other techniques, including differential hybridization, subtractive library screening, and mRNA differential display, are required, especially for novel gene discovery. In fact, the mRNA differential display technique has been successfully applied in novel gene discovery in focal stroke.⁷ However, mRNA differential display is primer dependent and may have difficulty providing an overall gene expression profile. Comparatively, other techniques, such as subtractive cDNA library screening, are likely to reveal overall altered gene expression. Moreover, subtractive cDNA library screening has been proved to be a powerful method for identification of differentially expressed genes,⁸ although it has not yet been applied to stroke research. Therefore, in the present study we applied modified cDNA subtraction procedure⁹ and constructed a subtractive cDNA library of ischemic and nonischemic mRNA obtained 2 and 12 hours after permanent MCAO in rats. As illustrated in this work, a cDNA clone encoding TIMP-1 was identified with use of this subtractive cDNA library approach. Since TIMP-1 is known to be involved in remodeling of extracellular matrix by preferential inhibition of MMPs in diverse conditions such as wound healing/scar formation, angiogenesis, and cancer metastasis, the induced expression of TIMP-1 mRNA in ischemic cortex suggests a role for TIMP-1 in the brain's response to ischemia.

	Materials and Methods

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Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (DHEW [DHHS] Publication No. [NIH] 85–23, revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, Md). Procedures using laboratory animals were approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals.

Focal Brain Ischemia
Cerebral focal ischemia or sham surgery was carried out by MCAO in male spontaneously hypertensive rats (Taconic Farms, Germantown, NY) 16 to 18 weeks of age and weighing 250 to 330 g, as described in detail previously.¹⁰ Briefly, the MCA was permanently occluded and cut dorsal to the lateral olfactory tract at the level of the inferior cerebral vein by use of electrocoagulation (Force 2 Electrosurgical Generator, Valley Laboratory Inc). For temporary MCAO 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.¹¹ In sham-operated rats the dura was opened over the MCA, but the artery was not occluded. Rats were overdosed with pentobarbital, and their forebrains were removed and dissected at various times after permanent MCAO or after temporary MCAO with reperfusion and at 12 hours and 5 days after sham surgery. The ischemic frontoparietal cortex was dissected from the ipsilateral hemisphere. The contralateral cortex was dissected as the nonischemic control from the same rat.¹⁰ The cortical samples were immediately frozen in liquid nitrogen and stored at -80°C.

RNA Preparation
Total cellular RNA was prepared by homogenizing brain cortical samples in an acid guanidinium thiocyanate solution and extracted with phenol and chloroform as previously described.¹² For subtractive cDNA library construction, poly(A)+ mRNA was extracted with an oligo(dT) cellulose column from total cellular RNA isolated from ischemic and nonischemic cortexes of 50 animals at 2 and 12 hours after permanent MCAO.

Subtractive cDNA Library Construction
The procedure for subtractive cDNA library construction, basically following one previously reported⁹ with specific modifications, is illustrated schematically in Fig 1. Briefly, 20 µg poly(A)+ RNA isolated from 2 and 12 hours nonischemic cortex (10 µg each) in 75 µL water was mixed with 25 µL 10% (wt/vol) oligotex-dT (QIAGEN) and heated at 70°C for 5 minutes, followed by rapid cooling on ice. After addition of 100 µL 2x TMK buffer (100 mmol/L Tris-HCl, pH 8.3; 200 mmol/L KCl; and 20 mmol/L MgC_l2), the mixture was incubated at 37°C for 20 minutes and then microcentrifuged for 10 minutes at room temperature. The precipitate containing mRNA–oligotex-dT complex was dissolved in 400 µL RT buffer (80 µl 5x first strand buffer [Gibco BRL], 40 µL 0.1 mol/L DTT, 2 µL each dNTPs [0.5 mmol/L], 1 µL 1:20 diluted ³²[P]--dATP [Amersham], 300 U RNase inhibitor [Boehringer Mannheim], 10,000 U SuperScript II RNase H^- reverse transcriptase [Gibco BRL]) and incubated at 37°C for 90 minutes. The reaction mixture was then heated at 90°C for 3 minutes and rapidly cooled on ice. The RNA dissociated from the cDNA–oligotex-dT was removed by microcentrifugation. The precipitate was washed with 400 µL 10 mmol Tris-HCl, pH 8.0, 1 mmol EDTA (TE), centrifuged, and dissolved in 100 µL TE containing 100 µg (dA)₃₀(dG)₁₀ oligodeoxynucleotide. The suspension was heated at 65°C for 5 minutes, then 20 µL 3 mol/L NaCl was added, followed by incubation at 37°C for 10 minutes to cover the free oligo(dT) residues on the oligotex-dT. The excess (dA)₃₀(dG)₁₀ was removed by centrifugation and saved for later use. The precipitate was dissolved in 200 µL 1.25x hybridization buffer (12.5 mmol/L Tris-HCl, pH 7.5; 125 mmol/L NaCl; 1.25 mmol/L EDTA; 0.125% SDS; 2 µg oligo(dT)_12–18); 4 µg poly(A)+ RNA isolated from 2- and 12-hour ischemic cortex in 50 µL water was then added and the mixture hybridized at 55°C for 20 minutes. The reaction mixture was centrifuged at room temperature for 10 minutes, and the supernatant (containing the subtracted mRNA) was saved at 4°C. The precipitate was dissolved in 400 µL TE, heated at 90°C for 3 minutes and cooled on ice, then centrifuged at 4°C for 10 minutes to removed the RNA. The cDNA–oligotex-dT precipitate was dissolved again in TEcontaining (dA)₃₀(dG)₁₀, and the subtractive hybridization was repeated an additional six times.

View larger version (30K): [in this window] [in a new window]   Figure 1. Schematic of the subtractive procedure for the library construction. The mRNA isolated from ischemic cortex at 2 and 12 hours aftert ischemic injury was subtracted from the mRNA of nonischemic cortex using oligo(dT)30-latex. See text for details.

The subtractive mRNA was used for cDNA library construction. The first and second strand of cDNA was synthesized with use of a cDNA cycle kit for RT-PCR (Invitrogen) according to the manufacturer's specifications. An EcoRI adapter was ligated onto the cDNA, and fractions of the cDNA larger than 400 bp were collected, digested with EcoRI enzyme, and ligated into ZAP vector (EcoRI sites) (Stratagen) according to the manufacturer's specifications.

Differential Southern Blot Analysis
The expression sequence tags of the subtractive cDNA library was determined robotically by Human Genome Sciences, Inc, and a panel of samples from this library was further analyzed in the present report. The differential expression of these cDNAs in the ischemic cortex was confirmed by Southern blot analysis. Briefly, miniprep DNA was digested with EcoRI to release the cDNA insert from the plasmid and analyzed by agarose gel electrophoresis. Southern hybridization was carried out as described in detail previously,¹³ except that the probe was generated by reverse transcription reaction using poly(A)+ mRNA isolated from either 2- and 12-hour ischemic cortex (for the ischemic probe) or the nonischemic cortex (for the nonischemic probe). SuperScript II RNase H^- reverse transcriptase was used for this labeling reaction in the presence of both ³²[P]--dATP and ³²[P]--dCTP (Amersham) according to the manufacturer's specifications.

Northern Blot Analysis
For Northern blot analysis, 30 µg/lane total cellular RNA was electrophoresed through formaldehyde agarose gel and transferred to a GeneScreen Plus membrane (Du Pont–New England Nuclear). cDNA fragment for TIMP-1 was released from the plasmid and gel purified. rpL32 cDNA was generated by RT-PCR as described previously.⁶ The cDNA probes were uniformly labeled with [-³²P]dATP (3000 Ci/mmol, Amersham Corp) using a random-priming DNA labeling kit (Boehringer Mannheim). Hybridization and washing were performed as described in detail previously.¹³

Statistical Analysis
Statistical evaluation was performed with four complete sets of cortical samples from each time point by use of one-way ANOVA followed by a Fisher protected 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 Introduction  References

 
DNA sequencing analysis was used to identify clones present in the subtractive cDNA library. The expression sequence tag was determined for each clone and subjected to a computer database search. A panel of clones was selected for further analyses. The plasmid DNA of these clones was isolated and digested with restriction enzyme (EcoRI) to release the cDNA insert. Comparative Southern hybridization (Fig 2), using probes generated at 2 and 12 hours after ischemic and nonischemic injury, revealed that TIMP-1 and c-fos were markedly induced in the ischemic condition compared with the nonischemic. The increased expression of c-fos mRNA in the ischemic cortex has been demonstrated previously,¹ and the present work is in agreement with previous data. Actually, the positive signal for c-fos mRNA expression represents its induced expression at 2 hours after MCAO,¹ whereas the signal for TIMP-1 is for 12 hours after stroke (see below). The induced expression of TIMP-1 has not been previously reported. Therefore, the full-length DNA sequence for TIMP-1 cDNA was carried out and confirmed its sequence identity.

View larger version (60K): [in this window] [in a new window]   Figure 2. Southern blot analysis of cDNA clones identified by the subtractive cDNA library. DNA sequencing analysis was carried out and used for computer database search against GenBank. Plasmid DNA was prepared and digested with EcoRI restriction enzyme (unless specified for some clones), and then analyzed by gel electrophoresis, transferred to a nylon membrane, and subjected to Southern blot analysis. A, Ethedium bromide staining of the agarose gel. B, Southern blot hybridization of the membrane with a probe generated from 2- and 12-hour ischemic cortex (see "Materials and Methods" for details). C, Southern blot analysis of the same membrane with a probe generated from 2- and 12-hour nonischemic cortex. Lane 1, c-fos, undigested plasmid DNA; lane 2, undigested bluescript plasmid DNA; lane 3, mitochondria DNA; lane 4, chloride channel protein; lane 5, ATPase inhibitor protein; lane 6, unknown; lane 7, unknown; lane 8, major synaptic vesicle protein; lane 9, proline-rich protein; lane 10, P-19 protein tyrosin phosphatase; lane 11, unknown; lane 12, neuronal olfactomedin-related glycoprotein (U03415); lane 13, tissue inhibitor of metalloproteinase-1; and lane 14, DNA marker ( DNA digested with BstEII). Note that the band indicated with an arrow in B refers to the cDNA insert of TIMP-1, which was specifically hybridized to ischemic probe.

The temporal expression of TIMP-1 mRNA in ischemic cortex was examined. A representative autoradiograph of Northern blotting for TIMP-1 mRNA expression in the focal ischemic and nonischemic cortex and in sham-operated cortical samples is illustrated in Fig 3A. The quantitative data for TIMP-1 mRNA (n=4), after normalizing to a housekeeping gene, rpL32, are summarized graphically in Fig 3B. Sham-operated samples were taken at 12 hours after surgery. As shown in Fig 3, only a low level of TIMP-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 TIMP-1 mRNA was significantly elevated at 12 hours (2.9-fold increase over control; P<.001) and reached a peak level at 2 days (7.3-fold increase; P<.001) in the ischemic cortex after permanent MCAO (Fig 3). The expression profile of TIMP-1 mRNA revealed a similar pattern in the ischemic cortex after temporary MCAO with reperfusion (Fig 4). A significant induction of TIMP-1 mRNA was not observed until 12 hours after reperfusion (a 2.5-fold increase; P<.001, n=4) and was sustained up to 2 days (3.0-fold increase; P<.001).

View larger version (42K): [in this window] [in a new window]   Figure 3. Temporal expression of TIMP-1 mRNA in ischemic cortex after permanent MCAO in rats. A, Representative autoradiograph to show the TIMP-1 mRNA expression after MCAO. Total cellular RNA (40 µg/lane) was resolved by electrophoresis, transferred to a nylon membrane, and hybridized to the indicated cDNA probe. Ipsilateral and contralateral cortex samples (denoted by +) from individual rats after sham surgery (S; 12 hours) or after 1, 3, 6, 12, and 24 hours and 2, 5, 10, and 15 days of permanent MCAO are depicted. B, Quantitative Northern blot data (n=4) for TIMP-1 mRNA expression after focal stroke. The data were generated through PhosphorImager analysis and displayed graphically after normalization with rpL32 mRNA signals. ***P<.001 compared with sham-operated rats.

View larger version (47K): [in this window] [in a new window]   Figure 4. Northern blot analysis of TIMP-1 mRNA expression in rat ischemic cortex after temporary MCAO with reperfusion. The figure is illustrated as described in Fig 3, except that temporary occlusion (160 min) of the MCA was used. The time points indicated refer to the time of reperfusion.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
The present work illustrates the application of the subtractive cDNA library screening strategy for the cloning of altered gene expression after focal stroke. The identification of TIMP-1 expression in focal stroke represents the first successful application of this methodology, as previously demonstrated in other systems,⁸ for the discovery of differential gene expression in stroke. Compared with other techniques, such as mRNA differential display¹⁴ and representational difference analysis,^{15 16} the major advantage of subtractive cDNA library screening is its reproducibility for identifying differentially expressed genes, whereas its disadvantage is the bias on these of abundantly expressed messages. To minimize this potential problem, multiple cycles of subtractions were applied in the present work, which allowed us to enrich some of the low abundant mRNAs. Moreover, the subtraction may be combined with PCR for the identification of differentially expressed genes, such as representational difference analysis.^{15 16} Also, mRNA differential display, another PCR-based technique, may be used to isolate abundant and rare messages that are differentially expressed.

TIMP-1 is a specific inhibitor for a group of zinc-dependent proteolytic enzymes known as MMPs, particularly for MMP-1, MMP-2, MMP-3, and MMP-9. MMPs and TIMPs have been widely implicated in the process of tissue remodeling under pathological conditions such as wound healing/scar formation, angiogenesis, and cancer metastasis. Focal brain ischemia elicits a robust inflammatory reaction marked by significant leukocyte infiltration, along with disruption and reconstruction of the extracellular brain matrix.^{17 18 19} The induced expression of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) and their increased proteolytic activities have been demonstrated previously in focal stroke.²⁰ Maximal induction of MMP-9 and its activity were observed at 12 hours to 5 days after ischemic injury, whereas MMP-2 remained the same in both ischemic and nonischemic tissue until 5 days after injury.²⁰ Interestingly, the present demonstration of the induction of TIMP-1 mRNA after focal stroke is remarkably parallel to that of MMP-9.²⁰ Therefore, the induced expression of TIMP-1 at the precise time frame of MMP-9 may serve to inhibit or attenuate this MMP action. This possibility, however, awaits further clarification when reagents capable to selectively inhibit TIMP-1 become available. The factors that regulate TIMP-1 gene expression in focal stroke are as yet unknown. However, previous in vitro studies have demonstrated that the synthesis of TIMP-1 is regulated by cytokines and growth factors, most notably IL-1, IL-6, IL-10, TNF, and TGFß.^{21 22 23 24 25 26} It is interesting to note that the expression of these cytokines after ischemia is concomitant with the upregulation of TIMP-1 after ischemic injury.^{1 2 3 4 5}

In conclusion, the present study demonstrated a successful application of subtractive cDNA library strategy for discovery of altered gene expression in focal stroke. The elevated expression of TIMP-1 along with MMPs after ischemic injury may suggest that MMP inhibitors play a role in matrix remodeling at specific time points after brain injury.

	Selected Abbreviations and Acronyms

 

MCA(O) = middle cerebral artery (occlusion) MMP = matrix metalloproteinases PCR = polymerase chain reaction RT = reverse transcription TIMP-1 = tissue inhibitor of metalloproteinase-1

Received August 14, 1997; revision received October 30, 1997; accepted October 30, 1997.

	References

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2. 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]

3. 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]

4. 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]

5. Wang XK, Yue TL, White RF, Barone FC, Feuerstein GZ. Transforming growth factor-ß1 exhibits delayed gene expression following focal cerebral ischemia. Brain Res Bull. 1995b;36:607–609.

6. Wang XK, Barone FC, Aiyar NV, Feuerstein GZ. Interleukin-1 receptor and receptor antagonist gene expression after focal stroke in rats. Stroke. 1997;28:155–162.[Abstract/Free Full Text]

7. Wang XK, Yue TL, Barone FC, White RF, Clark RK, Willette RN, Sulpizio AC, Aiyar NV, Ruffolo RR Jr, Feuerstein GZ. Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage. Proc Natl Acad Sci U S A. 1995;92:11480–11484.[Abstract/Free Full Text]

8. Hedrick SM, Cohen DI, Nielsen EA, Davis MM. Isolation of cDNA clones encoding T-cell specific membrane-associated proteins. Nature. 1984;308:149–153.[Medline] [Order article via Infotrieve]

9. Hara E, Kato T, Nakada S, Sekiya S, Oda K. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells. Nucleic Acids Res. 1991;19:7097–7104.[Abstract/Free Full Text]

10. Barone FC, Hillegass LM, Tzimas MN, Schmidt DB, Foley JJ, White RF, Price WJ, Feuerstein GZ, Clark RK, Griswold DE, Sarau HM. Time-related changes in myeloperoxidase activity and leukoterine by receptor binding reflect leukocyte influx in cerebral focal stroke. Mol Chem Neuropath. 1995;24:13–30.[Medline] [Order article via Infotrieve]

11. 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]

12. 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]

13. 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.[Abstract/Free Full Text]

14. Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992;257:967–971.[Abstract/Free Full Text]

15. Lisitsyn N, Lisitsyn N, Wigler M. Cloning the differences between two complex genomes. Science. 1993;259:946–951.[Abstract]

16. Hubank M, Schatz DG. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 1994;22:5640–5648.[Abstract/Free Full Text]

17. Hallenbeck JM, Dutka AJ, Tanishima T, Kochanek PM, Kumaroo KK, Thompson CB, Obrenovitch TP, Contreras TJ. Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period. Stroke. 1986;17:246–253.[Abstract/Free Full Text]

18. 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]

19. 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]

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Editorial Comment

Ted M. Dawson, MD, PhD, Guest Editor

Department of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
A variety of molecular approaches have been developed for determining the differential expression of genes in many pathologic processes. Discovering potential changes in gene expression may yield valuable insight into disease mechanisms, as a number of the induced genes may play significant roles in the disease process. Wang and colleagues have made a significant advance in the characterization of genes that are increased after focal permanent occlusion of the middle cerebral artery. Using subtractive hybridization techniques comparing ischemic and nonischemic cortex 2 and 12 hours after occlusion, they identified a cDNA that encodes for tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as a major induced gene following permanent focal ischemia. Time-course studies indicate that TIMP-1 is induced three-fold over control and reaches a peak at 2 days after occlusion. Similar results were found in the temporary reperfusion model. The authors present data on 11 of the differentially expressed clones. TIMP-1 and cFOS are the most robust clones identified whose expression patterns change after focal occlusion; however, clone No. 6, which is novel, also appears to change. The significance of this change is not known. Identification of genes like this may yield novel targets for potential therapeutic intervention if the upregulated or downregulated proteins are found to play a role in the neuronal damage accompanying stroke.

What might be the therapeutic implications of this work? TIMP-1 is a specific inhibitor of a group of zinc-dependent proteolytic enzymes designated the metalloproteinases (MMP).¹ MMP-2 and MMP-9 are upregulated after focal stroke and are thought to play a role in blood-brain–barrier disruption, thus facilitating the inflammatory reaction and leukocyte infiltration that accompanies stroke.² Upregulation of TIMP-1 may act to modulate the function of MMP-2 and MMP-9. Augmentation of the function or expression of TIMP-1 might reduce the breakdown of the blood-brain barrier and diminish the secondary wave of neuronal injury that follows the initial insult of occlusion.

Exciting times are ahead as new genes are identified, whose levels go up and down after stroke. It will be a major challenge, but also a significant opportunity, to identify the key players in the hopes of identifying potential targets that might be useful therapeutically.

	Selected Abbreviations and Acronyms

 

MCA(O) = middle cerebral artery (occlusion) MMP = matrix metalloproteinases PCR = polymerase chain reaction RT = reverse transcription TIMP-1 = tissue inhibitor of metalloproteinase-1

Received August 14, 1997; revision received October 30, 1997; accepted October 30, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Romanic AM, Madri JA. Extracellular matrix–degrading proteinases in the nervous system. Brain Pathol.. 1994;4:145–146.[Medline] [Order article via Infotrieve]

2. Rosenberg GA, Navratil M, Barone F, Feuerstein G. Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J Cereb Blood Flow Metab.. 1996;16:306–366.

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