Genomics of the Periinfarction Cortex after Focal Cerebral Ischemia

Abstract

Understanding transcriptional changes in brain after ischemia may provide therapeutic targets for treating stroke and promoting recovery. To study these changes on a genomic scale, oligonucleotide arrays were used to assess RNA samples from periinfarction cortex of adult Sprague-Dawley rats 24 h after permanent middle cerebral artery occlusions. Of the 328 regulated transcripts in ischemia compared with sham-operated animals, 264 were upregulated, 64 were downregulated, and 163 (49.7%) had not been reported in stroke. Of the functional groups modulated by ischemia: G-protein–related genes were the least reported; and cytokines, chemokines, stress proteins, and cell adhesion and immune molecules were the most highly expressed. Quantitative reverse transcription polymerase chain reaction of 20 selected genes at 2, 4, and 24 h after ischemia showed early upregulated genes (2 h) including Narp, Rad, G33A, HYCP2, Pim-3, Cpg21, JAK2, CELF, Tenascin, and DAF. Late upregulated genes (24 h) included Cathepsin C, Cip-26, Cystatin B, PHAS-I, TBFII, Spr, PRG1, and LPS-binding protein. Glycerol 3-phosphate dehydrogenase, which is involved in mitochondrial reoxidation of glycolysis derived NADH, was regulated more than 60-fold. Plasticity-related transcripts were regulated, including Narp, agrin, and Cpg21. A newly reported lung pathway was also regulated in ischemic brain: C/EBP induction of Egr-1 (NGFI-A) with downstream induction of PAI-1, VEGF, ICAM, IL1, and MIP1. Genes regulated acutely after stroke may modulate cell survival and death; also, late regulated genes may be related to tissue repair and functional recovery.

Recent clinical trials of neuroprotective drugs for the acute treatment of stroke have failed. These included trials of sodium and calcium channel antagonists, N-methyl-D-aspartate receptor antagonists, γ-amino-butyric acid agonists, free radical scavengers, nitric oxide pathway modulators, blockers of adhesion molecules, and other drug classes (De Keyser et al., 1999; Barber et al., 2001; Albers et al., 2001; Fisher and Schaebitz, 2000). In spite of these failures, there is still optimism that pharmacologic approaches can be developed to treat acute stroke or enhance recovery (White et al., 2000). One new approach to search for targets is to perform genomic studies at different times after stroke to try to identify time-specific gene pathways or gene clusters related to specific injury or recovery processes after stroke.

DNA microarrays can assay thousands of transcripts in a single sample (Noordewier and Warren, 2001). The first brain ischemia study used custom-designed 750 gene arrays to examine RNA changes in cortex and striatum 3 h after focal ischemia (Soriano et al., 2000). Of the 24 genes regulated more than twofold, most were immediate early genes such as c-fos, NGFI-A, NGFI-C, Krox-20, and Arc (Soriano et al., 2000). Subsequent studies used oligodeoxynucleotide-based or complementary DNA (cDNA) microarrays to study RNA expression in hippocampus of rats subjected to transient global ischemia (Jin et al., 2001) and in cortex of rats at 6 h or 10 days after focal ischemia (Kim et al., 2002; Keyvani et al., 2002). A recent study combined cDNA array analysis of 74 genes with brain metabolic status studied using positron emission tomography scanning in a baboon focal cerebral ischemia model. A change in the pattern of gene expression when the cerebral metabolic rate for oxygen was reduced by 48% to 66% was suggested to serve as a molecular definition of the penumbra (Chuquet et al., 2002).

The present study used rat Affymetrix U34A oligonucleotide arrays to assess 8,740 transcripts in the periinfarction cerebral cortex at 24 h after permanent middle cerebral artery (MCA) occlusions in adult rats. Using very strict criteria, less than 4% of these transcripts were regulated, and 49.7% of these had not been reported previously. Real time reverse transcription polymerase chain reaction (RT-PCR) confirmed the expression of 20 of these genes and showed two general classes: those induced by 2 h, and hence might be targets for acute stroke therapy; and those induced at later times that could be targets for tissue repair and plasticity.

MATERIALS AND METHODS

Animal protocols were approved by the University of Cincinnati animal care committee and conform to the National Institutes of Health Guide for Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighed approximately 300 g to 350 g, had unrestricted access to food and water, and were housed two per cage with a 12-h light–dark cycle.

Stroke model

The left MCA was occluded using the intraluminal filament technique (Rajdev et al., 2000; Schwarz et al., 2002). Adult Sprague-Dawley rats (n = 3) were anesthetized with isoflurane. During anesthesia, rectal temperature was monitored and body temperature was maintained at 37 ± 0.2°C with a heating blanket. The left common carotid artery, external carotid artery, and internal carotid artery were isolated via a ventral midline incision. To occlude the MCA, a 3–0 monofilament nylon suture was inserted into the external carotid artery and advanced into the internal carotid artery approximately 20 mm from the carotid bifurcation until mild resistance was felt. The wound was closed. Once animals recovered, they were returned to their home cages with food and water available ad libitum. One day later (24 h), rats were reanesthetized and killed. Sham-operated animals (n = 3) were treated like ischemic animals except that no suture was inserted into the carotid.

RNA preparation

At 24 h after cerebral ischemia, rats were reanesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) and killed. The periinfarction cortex was dissected according to a published method in rat filament model of unilateral proximal MCA occlusion (Ashwal et al., 1998; Schwarz et al., 2002; see these articles for diagram of dissected brain region). The brain was quickly removed and cut coronally into three slices beginning 3 mm from the anterior tip of the frontal lobe in a brain matrix in a cold room. A longitudinal cut approximately 2 mm from the midline through left hemisphere in the sections was made to avoid medial hemispheric structures, which are supplied primarily by the anterior cerebral artery. Then, a transverse diagonal cut was made at approximately the “2 o'clock” position—avoiding obvious areas of infarction. The left parietal, periinfarction cortex was dissected. The parietal cerebral cortexes in ischemic 2- or 4-h rats from the same location were also dissected. The dissected brain tissues were homogenized in a Teflon–glass homogenizer with TRIzol Total RNA Isolation Reagent (Life Technology, Rockville, MD, U.S.A.). Total RNA was isolated according the manufacturer's instructions. Briefly, the brain homogenate was treated with chloroform; RNA was precipitated using isopropyl alcohol and cleaned using a RNAeasy mini kit (Qiagen Inc., Valencia, CA, U.S.A.).

GeneChip expression analysis and database search

GeneChip expression analysis was performed according to the Affymetrix expression analysis technical manual. Briefly, double-stranded cDNA was synthesized from total RNA with a high-performance liquid chromatography–purified oligo-dT primer. Biotin-labeled complementary RNA (cRNA) was synthesized from cDNA using T7 RNA polymerase and biotin-labeled ribonucleotides. The quality of the cRNA was assessed using gel electrophoresis. The cRNA was hybridized to Affymetrix U34A rat arrays (Affymetrix, Santa Clara, CA, U.S.A.). The U34A microarray was scanned with the GeneChip scanner.

The data were analyzed using Affymetrix GeneChip expression analysis software according to the Affymetrix GeneChip Analysis Suite (Tang et al., 2001). An absolute analysis reported the hybridization intensity data (average difference) and whether transcripts were present, absent, or marginal in the target from each probe array. Then, a comparison analysis was run. The patterns of change of the whole probe set were used to make a qualitative call of “Increase,” “Decrease,” “Marginal increase,” “Marginal decrease,” or “No change.” Three chips were used for each group (sham-operation and ischemia). The cross-comparisons were made between sham-operation and ischemia groups. Genes were included in the analysis only if they met all of the following criteria: they were present in all three sham or all three ischemia samples; all three ischemia samples for each gene showed either an “Increase or Decrease” when compared with all three sham samples for each gene; and the fold change in each of the individual comparisons between ischemia and sham had to be at least 1.7-fold (Jin et al., 2001). These are stringent criteria that probably eliminated many genes that were actually regulated by ischemia.

Functional information for the regulated genes was obtained using LocusLink, OMIM, GeneCards, PubMed, and referencing gene ontology (Ashburner et al., 2000). By searching Uni-Gene and doing Blast analyses of the GeneseqN database, we determined the similarity of the expressed sequence tags (ESTs) on the microarrays with known genes. For genes that were represented several times on an array, the alternate gene names are given, and the expression values for that gene were averaged. If the ESTs represented known or at least highly homologous genes, the known gene name is provided in the tables in the unigene/blast columns (Tables 1–14). Sometimes one EST may blast to short fragments of genes or blast to several genes, making these ESTs more difficult to interpret. A draft rat genome covering more than 90% of the rat genome is available. Most of the ESTs had known homologues (Butler, 2002).

TABLE 1.

Enzymes and inhibitors

Category	Gene name	Function summary	Fold change	Gene bank #	RNA change in chip	RNA change in paper	Unigene/blast	Percent identity, aligned region
Oxidoreductase	EST220459	Oxidoreductase, drug metabolism and synthesis of cholesterol, steroids, and other lipids. Eye morphogenesis	7.8	AI176856	I	I(CYP2E)	CP1B rat cytochrome P450 1B1	100% 542 aa
	Cytochrome P450 (CYP1B1)	Oxidoreductase, drug metabolism and synthesis of cholesterol, steroids, and other lipids. Eye morphogenesis	6.4	U09540	I	I(CYP2E)
Cysteine proteases and inhibitor	Calpain II 80 kDa subunit	Calcium-activated neutral proteases, nonlysosomal, intracellular cysteine proteases, catalyzing limited proteolysis of substrates involved in cytoskeletal remodeling and signal tranduction.	1.9	L09120	I	I
	Cyclic Protein-2 = cathepsin L proenzyme	Lysosomal cysteine (thiol) protease	1.8	S85184	I	I
	EST	Lysosome cysteine-type peptidase	2.8	AA925246	I	X	Cathepsin K precursor	100% 328 aa
	Cathepsin C	Lysosomal cysteine (thiol) protease	3.2	D90404	I	I
	ICE-like cysteine protease	Cysteine (thiol) protease, induction of apoptosis	3.8	U49930	I	I
	EST203339	Cysteine protease inhibitor, inhibits papain (cathepsins L, h and b)	2.1	AI008888	I	I	Cystatin B	100% 97 aa
	Major acute phase alpha-1	Inhibitors of thiol proteases, releasing bradykinin	3.8	K02814	I	X
Serine protease and inhibitor	Tissue-type plasminogen activator (t-PA)	Serine protease, converts inactive plasminogen to plasmin	2.6	M23697	I	I
	Plasminogen activator inhibitor-1 (PAI-1)	Member of the serpin family of serine protease inhibitors, “bait” for tissue plasminogen activator, urokinase, and protein c, regulation of fibrinolysis	17	M24067	I	I
	EST189815	Serine protease inhibitor, inhibition of complement activation	2.1	AA800318	I	X	Complement C1 inhibitor precursor	81% 171 aa
	Contrapsin-like protease inhibitor related protein (CPi-26)	Serine protease inhibitor, inhibiting neutrophil cathepsin g and mast cell chymase	∼71.6	D00753	I	I
Metalloproteinase and inhibitor	EST202002	Component of the neutrophil gelatinase complex, modulator of inflammation, apoptosis	∼21	AA94650	I	I	NGAL rat neutrophil gelatinase-associated lipocalin precursor	100% 197 aa
	Gelatinase B (GelB)	Collagenase, degrades type IV and V collagens	∼17.4	U24441	I	I
	EST215162	Inhibitors of the matrix metalloproteinases, known to act on mmp-1, mmp-2, mmp-3, mmp-7, mmp-8, mmp-9, mmp-10, mmp-11, mmp-12, mmp-13 and mmp-16. Does not act on mmp-14.	∼21	AI169327	I	I	TIM1 rat metalloproteinase inhibitor 1 precursor	100% 216 aa
Phospholipase and inhibitor	PS-PLA1	Phospholipase A1, phosphatidylserine metabolism	∼13.1	D88666	I	X
	EST217956	Ca2+-dependent phospholipid-binding protein, inhibiting phospholipase A2 and antiinflammation	4.3	AI171962	I	I	Annexin I	100% 345 aa
	Annexin II	Calcium-dependent phospholipid-binding protein, regulation of cellular growth, and signal transduction	5.1	L13039	I	I
RNA helicase	Nuclear RNA helicase	ATP dependent RNA helicase, down-regulation of acute phase cytokine production (TNFalpha, IL-1, and IL-6)	2.7	AF063447	I	X
Ribonuclease inhibitor	Ribonuclease inhibitor	Ribonuclease inhibitor	2.6	X62528	I	X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 2.

Genes related to metabolism 1

Category	Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
Carbohydrate metabolism	GLUT1 = glucose transporter 1, Rat brain glucose-transporter	Glucose transporter, transport of dehydroascorbic acid	2.9	S68135, M1 3979	I	I
	EST189687	Glycogen catabolism	−2.1	AA800190	D	X	Glycogen phosphorylase, brain form	99%, 137 aa
	HK2 gene for type II hexokinase	Phosphorylating glucose to produce glucose-6-phosphate, glycolysis	∼11	D26393	I	X
	6-phosphofructo-2-kinase/fructose-2,6 bisphosphatase	Regulation of the concentration of fructose-2,6-bisphosphate, glycolysis	∼8.2	AB006710	I	X
	Glycerol 3-phosphate dehydrogenase	Glycolysis, oxidoreductase	∼61.7	AB002558	I	X
Amino acid, protein metabolism	EST226923	Serine synthesis	−2.1	AI230228	D	X	Human phosphoserine transaminase	54%, 108 aa
	EST212157	Serine synthesis	−2.5	AI102868	D	X	Phosphoserine transaminase	54%, 108 aa
	Cystathionine gamma-lyase	Cysteine metabolism	∼3.1	D17370	I	X
	L-cysteine oxygen oxidoreductase	Degradation of cysteine, Taurine and hypotaurine metabolism, electron transporter	2.9	E03229	I	X
	EST198184	Degradation of cysteine, Taurine and hypotaurine metabolism, electron transporter	2.5	AA942685	I	X	CYDX rat cysteine dioxygenase	100%, 199 aa
	S-adenosyl-methionine synthetase	Amino acid metabolism, biosynthesis of polyamines	2	J05571	I	X
	Ornithine decarboxylase (ODC)	Rate-limiting enzyme of the polyamine biosynthesis	2.8	J04791, J04792, X07944	I	I
	EST	Mitochondrial ribosome 28S subunit protein	2.1	AI639387	I	X	Mitochondrial ribosomal protein S6 (MRPS6)
	Ribosomal protein S27	Cytosolic small ribosomal (40S)-subunit	2.7	X59375	I	I
	PHAS-I	Binding EIF4E, translational negative regulation	∼10.1	U05014	I	X
	EST195768	Fructosamine-3-kinase, deglycation	−2.8	AA891965	D	X	Fructosamine-3-kinase (FN3K gene)
	EST197246	Nuclear and cytoplasmic protein glycosylation	1.8	AA893443	I	X	UDP-N-acetylglu-cosamine-peptide N-acetylglucos-aminyltransferase 110 Kda subunit (OGT1)	100%, 1035 aa
	EST196352	N-glycosylation	2	AA892549	I	X	Mannosyl-oligosaccharide alpha-1,2-mannosidase
	EST196578	Hydrolysis and transglycosylation	2.9	AA892775	I	I	Rat lysozyme c, type 1 precursor	100%, 146 aa
Lipid metabolism	EST	Glycolipids catabolism	−2.5	AA859911	D	X	Cmp-n-acetyl-neuraminate-beta-galactosamide-alpha-2, 3-sialyltransferase (alpha 2,3-st) (gal-nac6s) (st3gala.2) (siat4-b)	100%, 349 aa
	Fatty acid transport protein	Membrane transporter for long-chain fatty acids	−2.6	U89529	D	X
	Low molecular weight fatty acid binding protein	Lipid transporter, tumor suppressor	−2.2	J02773	D	X
Lipid metabolism	C-FABP = cutaneous fatty acid-binding protein	Lipid binding, differentiation	2.4	S69874	I	I
	EST218294	Cholesterol biosynthesis	1.9	AI172293	I	X	RANP-1	100%, 292 aa
	EST	Oxidoreductase, cholesterol biosynthesis	3.1	E12625	I	X
	Isopentenyl diphosphate-dimethylallyl diphosphate isomerase	Isoprenoid biosynthesis, steroids synthesis	3.5	AF003835	I	X	/C-4 methyl sterol oxidase
	Peripheral-type benzodiazepine receptor (PKBS)	Benzodiazepine receptor, involving in the flow of cholesterol into mitochondria	11.3	J05122	I	I
	EST	LDL receptor, proteolysis and peptidolysis	∼5.6	AI071531	I	I	JE0111 lectin-like oxidized LDL receptor-mouse	78%, 360 aa
	Aminopeptidase-B	Exopeptidase, eukotriene A4 hydrolase	−1.8	D87515	D	X
Nucleotide metabolism	Adenosine kinase	Conversion of adenosine to AMP, ribonucleoside monophosphate biosynthesis	−2	U57042	D	X
	Proliferating cell nuclear antigen (PCNA/cyclin)	Delta-DNA polymerase cofactor, DNA replication and repair, cell cycle control	2.4	M24604	I	I
	EST	Production of the pyrimidine nucleoside triphosphates	∼11.8	AA859827	I	X	URK1 mouse uridine kinase	78%, 184 aa
	Phosphodiesterase I	Nucleotide pyrophosphatase, broad specificity	−2.7	D28560	D	X
	Phosphodiesterase I	Phosphodiesterase I. Nucleotide pyrophosphatase	∼6.2	D30649	I	X
	TBFII mRNA for polypyrimidine tract binding protein	RNA binding, mRNA splicing, mRNA processing	∼11.8	X74565	I	X
	EST189008	pre-mRNAs binding, pre-mRNA processing, and other aspects of mRNA metabolism and transport	2	AA799511	I	X	ROA2_human heterogeneous nuclear ribonucleoproteins A2/B1	97%, 41 aa
	EST	SnoRNP assembly, pre-18s rRNA processing	3	AF069782	I	X	/nucleolar protein 5 (NOP5)
	Gas-5 growth arrest homolog non-translated mRNA sequence	Processing of ribosomal RNA	3	U77829	I	X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 3.

Stress response genes

Category	Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
Oxidative stress	EST211851	Heavy metal binding, detoxifing metals, antioxidation	8.4	AI102562	I	I	MT1 rat metallothionein-1	100%, 60 aa
	Metallothionein-2 metallothionein-1	Heavy metal binding, detoxifing metals, antioxidation	8	M11794	I	I
	EST220041	Storage of trace-metals, cell stress	2.9	AI176456	I	I	Rat metallothionein-II	100%, 60 aa
	Glutathione S-transferase Yc1 subunit	Conjugation of reduced glutathione to a wide number of hydrophobic electrophiles	−2.2	S72505	D	D
	Glutathione S-transferase Yc subunit	Glutathione transferase	−2.3	K01932	D	D
	EST195844	Peroxidase and antioxidant activity	−2	AA892041	D	X	Antioxidant protein 2	100%, 223 aa
	EST 190084	Selenium-dependent glutathione peroxidase, reduction of hydrogen peroxide, organic hydroperoxide, and lipid peroxides	∼9.6	AA800587	I	I	A45207 glutathione peroxidase	94%, 166 aa
Heat shock protein	Heat shock protein 70	Heat shock protein	∼103.7	L16764, Z27118	I	I
	EST, EST191323	Heat shock protein	∼91.6	AA818604, AA848563	I	I	Rat heat shock 70 Kd protein 1/2	100%, 640 aa
	EST	Heat shock protein	2	AA875620	I	I	Heat shock 70 Kd protein 3 (HSP70.3)	100%, 640 aa
	Hsp70.2	Heat shock protein	52.6	Z75029	I	I
	Heat shock protein (Hsp27)	Heat shock protein	∼358.7	M86389	I	I
	EST	Heat shock protein	53	AA998683	I	I	Heat shock 27 protein—rat	100%, 204 aa
	EST220250	Heat shock protein	11.2	AI176658	I	I	Heat shock 27 protein—rat	100%, 204 AA
	Collagen-binding protein (gp46)	Heat shock protein	3.2	M69246	I	I
	EST216547	Heat shock protein	2	AI170613	I	I	10 Kd heat shock protein, mitochondrial	99%, 101 aa
	EST0020	Heat shock protein	2.8	AA108277	I	I	/Hsp105
	Alpha-crystallin B chain	Molecular chaperone	3.2	M55534, X60351	I	I/–
	EST223333	Heat shock protein	15.1	AI179610	I	I	A Chain A, Crystal structure of rat heme oxygenase-1 in complex with heme	100%, 288 aa
	Heme oxygenase gene	Heme oxygenase	∼88.5	J02722	I	I
DNA damage response	Progression elevated gene 3 protein (Gadd34)	DNA damage response, initiation of protein translation, proapoptotic function	3.2	AF020618	I	I
	EST	DNA damage response, inducing p38/JNK activation and	7.8	AI070295	I	I	Growth arrest and dna-damage-inducible protein GADD45	100%, 164 aa
	GADD45	DNA damage response, inducing p38/JNK activation and	17.4	L32591	I	I

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 4.

Neurotransmitter and hormone-related genes

Category	Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
Glutamate related	Glutamate receptor (GluR-B)	Subunit 2 of AMPA subtype of ionotropic glutamate receptor for synaptic transmission	−2	M38061	D	D
	Metabotropic glutamate receptor 3	Metabotropic glutamate receptor	−2.7	M92076	D	D
	Narp = neuronal activity regulated pentraxin (NPTX2)	Transportor, extracellular aggregating factor for AMPA receptors, synapses plasticity and uptake of extracellular material	∼101.5	S82649	I	I
	AMPA receptor interacting protein GRIP	Adapter protein, lustering of AMPA receptors	∼3.1	U88572	I	X
	VesI	Modulation of metabotropic glutamate and IP3 receptors	3	AB003726	I	X
	Activity and neurotransmitter-induced early gene 3 (ania-3)	Metabotropic glutamate receptor signaling pathway, neuronal immediate early gene	∼6.0	AF030088	I	X
GABA related	EST225364	GABA transporter	−2.1	AI228669	D	X	Sodium- and chloride dependent GABA transporter 1	100%, 598 aa
	GABA-A receptor gamma-2 subunit	Benzodiazepine receptor, gamma-aminobutyric acid-inhibited chloride channel	−2.2	L08497	D	D/I
Angiotensinogen	Angiotensinogen	Angiotensinogen, hormone	−5	M12112	D	X
Endothelin related	Endothelin-converting enzyme	Endothelin-converting enzyme, metalloendopeptidase	1.8	D29683	I	X
	EST	Endothelin receptor, G protein-coupled receptor	3.3	AA818970	I	I	Rat endothelin B receptor precursor	100%, 441 aa
Neuropeptide	Neuropeptide	Neuropeptide Y, neuropeptide hormone and inhibitory neuromodulator	2.3	M15880	I	I
	Alpha-type calcitonin gene-related peptide	Ligands for receptors found in nervous system- and peripheral tissues, inducing vasodilatation	∼9.5	M11597	I	X
	Beta-type calcitonin gene-related peptide	Neuropeptide hormone, calcium ion homeostasis, vasodilatation	∼10.3	M11596	I	I
	Adrenomedullin precurosr	A hypotensive peptide and vasodilatator agent	∼6.4	D15069	I	I
	Galanin (a neuropeptide)	Neuropeptide	5.5	J03624	I	I
Prostaglandin related	Cyclooxygenase isoform COX-2	Prostaglandin-endoperoxide synthase, the first rate-limiting step in the conversion of arachidonic acid to prostaglandins	7.6	S67722, L25925	I	I
Thyrotropin releasing hormone	Thyrotropin releasing hormone (TRH)	A regulator of the biosynthesis of TSH and as a neurotransmitter/neuromodulator in the central and peripheral nervous systems	∼11	M23643, M36317	I	I
Retinoic acid related	Cellular retinoic acid-binding protein II (CRABP II)	Retinoic acid-binding protein	2.2	U23407	I	X
	Cytosolic retinol-binding protein (CRBP)	Retinoid binding and transport	3.9	M19257	I	I
	Aldehyde dehydrogenase (ALDH)	Aldehyde dehydrogenase	−2.5	AF001898	D	X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 5.

Growth factor-related genes

Category	Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
IGF-related	Insulin-like growth factor II	Insulin-like growth factor receptor ligand	−2.5	X17012	D	I
	EST	Negative regulation of insulin signaling	2.2	AI113289	I	X	Protein-tyrosine phosphatase, non-receptor type 1 (PTP-1b) (PTN1)	100%, 431 aa
	EST203856	Insulin-like growth factor receptor binding protein	∼13.4	AIl009405	I	I	IBP3 rat insulin-like growth factor binding protein 3	100%, 291 aa
	Insulin-like growth factor-binding protein (IGF-BP3)	Insulin-like growth factor receptor binding protein	∼11.4	M31837	I	I
BDNF	EST	Growth factor	5.1	AI030286	I	I	Brain-derived neurotrophic factor precursor	100% 248 aa
	BDNF = brain-derived neurotrophic factor	Growth factor, neurogenesis, neuronal development	12, 7.3	S76758, D10938	I	I
TGF related	Transforming growth factor beta 1 (TGFB1)	Ligand of TGF receptor, cell proliferation, differentiation, and apoptosis. Fibrosis.	∼29.1	X52498	I	I
	EST	Binds and inactivates members of TGF-beta superfamily signaling proteins	−4.1	AA859752	D	X	Noggin	100%, 143 aa
	Furin	Processes hormone precursors: PTH, TGFb	2.2	X55660	I	I
CSF related	DOC-2 p59 isoform	Csf-1 signal transduction pathway, tumor suppressor	2.7	U95178	I	X
EGF related	Heparin binding EGF-like growth factor	Epidermal growth factor receptor ligand, positive control of cell proliferation	17.7	L05489	I	I
	EST216715	Linking EGFR and PDGFRB to Ras and Rac signaling pathways	1.9	AI170776	I	X	Growth factor receptor-bound protein 2 (GRB2)	100%, 202 aa
VEGF related	EST193502	Vascular endothelial growth factor I	2.3	AA850734	I	I	Mouse vascular endothelial growth factor precursor	98%, 213 aa
	Vascular endothelial growth factor form 3	Vascular endothelial growth factor receptor ligand, induces endothelial cell proliferation and vascular permeability	∼11.1	L20913	I	I
FGF receptor	Fibroblast growth factor receptor 1 beta-isoform	Fibroblast growth factor receptor	4	S54008	I	I

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 6.

Genes related to cytokines and chemokines

Category	Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
TNF related	Tumor necrosis factor receptor (TNF receptor)	Tumor necrosis factor receptor, apoptosis and nf-kappa b signaling	4.2	M63122	I	I
	Homocysteine respondent protein HCYP2	Serine-threonine kinase, interacting with TNFR1 and TRAF2 and cativting caspase	∼6.4	AF036537	I	I
	Homocysteine respondent protein HCYP2	Serine-threonine kinase, interacting with TNFR1 and TRAF2 and cativting caspase	∼6.4	AF036537	I	I
	EST190110	Zinc finger protein, single-stranded RNA biding, inhibiting, macrophage TNF-alpha production	5.2	AA800613	I	I	Rat tristetraproline	100%, 319 aa
	PRG1(PACAP-responsive gene 1)	Protection of cells from Fas or tumor necrosis factor alpha-induced apoptosis	5.5	X96437	I	X
	Osteoprotegerin (OPG)	Decoy receptor for osteoclast differentiation factor	5	U94330	I	X
IL related	Interleukin 1-beta	Interleukin-1 beta, apoptosis	3	M98820	I	I
	Interleukin 1 receptor antagonist	Interleukin-1 receptor antogonist, inhibiting the binding of IL-1-alpha and IL 1-beta	∼4.4	M63101	I	I
	Interleukin 6 (IL6)	IL6R ligand growth factor	∼18	M26744, M26745	I	I
	Interleukin 18 (IL-18)	Cytokine, immune response	2.9	AJ222813	I	I
Chemokine	CC chemokine ST38 precursor	C-C chemokine, chemotaxis	22.3	AF053312	I	I
	EST201236	CXC chemokine receptor, mediating intracellular calcium flux	∼3.2	AA945737	I	X	Chemokine receptor LCR1	100%, 331 aa
	Macrophage inflammatory protein-1alpha	Chemokine cc, inflammatory, and chemokinetic properties	∼20.4	U22414	I	I
	Macrophage inflammatory protein-1 beta (MIP-1 beta)	Chemokine, inflammatory, and chemokinetic properties	∼6.5	U06434	I	I
	Immediate-early serum-responsive JE gene	Chemotactic factor for monocyes, binding to ccr2 and ccr4	61.5	X17053	I	I
	Gro	Chemokine, inflammatory processes	∼9.2	D11445	I	I
Interferon related	Interferon induced mRNA	Cell surface receptor linked signal transduction, the transduction of antiproliferative and homotypic adhesion signals	5.6	X61381	I	I
	EST207718	Regulating gene activity in the proliferative and/or differentiative pathways induced by ngf, may be an autocrine factor	5	AI014163	I	X	Interferon-related protein PC4	100%, 448 aa
Others	Platelet-activating factor acetylhydrolase alpha 1 subunit (PAF-AH alpha 1)	Inactivates PAF	6.6	AF016047	I	I
	EST	A member of the S100 family of proteins, inhibition of casein kinase and as a cytokine	∼3.4	AA957003	I	X	S108 RAT calgranulin A	100%, 88 aa

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 7.

Genes related to signal transduction 1

Category	Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
Calcium related	Plasma membrane CA2+ ATPase-isoform 2	Calcium-transporting ATPase, transport of calcium out of the cell	−2.3	J03754	D	D
	EST199283	Calcium-transporting ATPase, transport of calcium out of the cell	−2.1	AA943784	D	D	Calcium-transporting atpase plasma membrane, brain isoform 2	100%, 1197 aa
	Calmodulin-sensitive plasma membrane Ca2+-transporting ATPase (PMCA3)	Calcium-transporting ATPase, transport of calcium out of the cell	−2.1	J05087	D	D
	EST	Calcium-binding, actin-cytoskeleton associated	−2.6	AI639532	D	X	Human troponin C, fast skeletal muscle	97%, 133 aa
	Activity and neurotransmitter-induced early gene 4 (ania-4)	Calcium/calmodulin-dependent protein kinase-related	∼4	AF030089	I	X
Kinase A related	cAMP phosphodiesterase	cAMP-specific phosphodiesterase	4.1	J04563	I	I
	cAMP phosphodiesterase (PDE4)	cAMP-specific phosphodiesterase	2.5	M25350	I	I/D
	Ssecks 322, PKC binding protein, and substrate mRNA	Protein kinase A anchoring protein	∼6.6	U75404, U41453, U23146	I	X
NO-kinase G related	EST191798	Guanylate cyclase, NO mediated signal transduction	−6.7	AA849036	D	X	Guanylate cyclase soluble, alpha-1 chain	100%, 689 aa
	Cyclic nucleotide phosphodiesterase (CaM-PDE)	CaM-dependent cyclic-nucleotide phosphodiesterase, hydrolase	−2.6	M94537	D	X
	EST	NO mediated signal transduction	2.1	AI058941	I	X	N(G), N(G)-dimethylarginine dimethylaminohydrolase1	100%, 284 aa
	GTP cyclohydrolase I	Synthesis of tetrahydrobiopterin, cofactor in aromatic amino acid hydroxylation, and nitric oxide synthesis	7.2	M58364	I	I
Membrane receptor tyrosine kinase	EST	Transmembrane receptor protein tyrosine kinase, neurogenesis	−1.7	M59814	D	X	Ephrin type-b receptor 1 precursor (elk)	100%, 983 aa
	Sky	Transmembrane receptor signaling protein tyrosine kinase, cell adhesion, particularly in CNS	−2.2	D37880	D	X
	EST	Receptor tyrosine kinase, neural crest development	5.2	AI639318	I	I	Proto-oncogene tyrosine-protein kinase reeptor RET precursor	92%, 1115 aa
Inositol phosphate metabolism/PKC	Phospholipase C-1	Inositol phosphate metabolism, producing IP3 and DAG. Translocation of tubby	−2.7	M20636	D	I/D/–	/cornichon-like protein
	Phospholipase C-beta1b	Inositol phosphate metabolism	−2.1	L14323	D	I/D/–
	Inositol 1,4,5-triphosphate 3-kinase	Inositol phosphate metabolism, converting IP3 to Ins(1, 3, 4, 5)P4	−2.2	X56917	D	D/–
	Protein kinase C delta subspecies	Diacylglycerol-activated/phosholipid dependent protein kinase C	∼15.5	M18330	I	I
MAP kinase related	Oxidative stress-inducible protein tyrosine phosphatase	Inactivation of MAP kinase (erk1/2), oxidative stress response	1.8	S81478	I	I
	Protein tyrosine phosphatase	Inactivation of MAP kinase(erk1/2), oxidative stress response	3.5	S74351 U02553	I	I
MAP kinase related	Dual-specificity protein tyrosine phosphatase (rVH6)	Inactivation of ERK2	3.5	U42627	I	I
	Dual specificity phosphatase, MKP-3	Inactivation of MAPK	6.3	X94185	I	I
	MAP-kinase phosphatase (cpg21)	Inactivates ERK1	10.7	AF013144	I	X
JAK-STAT cascade	Protein-tyrosine kinase (JAK2)	Protein tyrosine kinase, JAK-STAT cascade	∼6.0	U13396, AJ000557	I	X
	Stat3 protein	JAK-STAT cascade, transcription factor	3.2	X91810	I	I
	Suppressor of cytokine signaling-3 (SOCS-3)	SH2-containing protein, negative regulator in the JAK-STAT pathway	∼25.7	AF075383	I	I
Other	EST	Signaling role during T-cell activation	∼2.2	AA866291	D	X	/cornichon-like protein

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 8.

G protein-related genes

Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
EST	G protein	−2.9	AA925506	D	D	Guanine nucleotide-binding protein G(i)/G(s)/G(o) gamma-7 subunit	100%, 68 aa
N-chimaerin	GTPase activator, cooperating with Rac1 and Cdc42Hs	−1.5	X67250	D	X
EST19812	GTPase activator	−2.2	AA894317	D	X	N-CHIMAERIN/chimerin (chimaerin) 2 (CHN2)	100%, 333 aa
EST196299	GTPase activator	−2.9	AA892496	D	X
SPA-1 like protein	GTPase activator, stimulating Rsr1 GTPase	−1.8	AF026504	D	X
Ras-related rab1B protein	Small GTP-binding protein	−1.7	X13905	D	X
Ras-related protein (rad)	Small monomeric GTPase, member of the ras family of GTP binding proteins	14.8	U12187	I	I
G33A Rat mRNA for gene 33 polypeptide	RHO GTPase binding and activator, stress response	∼37.2	X07266	I	X
EST215655	RHO GTPase binding and activator, stress response	3.8	AI169756	I	X	Gene 33 polypeptide-rat	97%, 136 aa
EST195743	RHO small monomeric GTPase, ras-related, reorganization of the actin cytoskeleton	2.7	AA891940	I	X	RhoA:1 human GTP-binding protein rhoC 6	96%, 32 aa
Isoprenylated 67 kDa protein	GTP-binding protein/GTPase, immune response	∼3.7	M80367	I	X
Putative G-protein coupled receptor RA1c	G-protein coupled receptor	−5	AF079864	D	X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 9.

Transcriptional Regulation 1

Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
C-fos	Transcription factor	15.9	X06769	I	I
FBR-murine osteosarcoma provirus genome	Transcription factor	14.3	X03347	I	I	/c-fos
Fos-related antigen (Fra-1)	Transcription factor, similar to Fos	∼21	M19651	I	I
EST	Transcription factor	3.5	AA875032	I	I	/fos-related antigen-2(fra-2 gene)
c-jun oncogene	Transcription factor	∼17.6	X17163	I	I
EST201366	Transcription factor	2.3	AA945867	I	I	/C-jun
EST219534	Transcription factor	2.8	AI175959	I	I	/C-jun
EST194844	Transcription factor, growth factor response	6.4	AA891041	I	I	Rat transcription factor jun-B	100%, 343 aa
pJunB	Transcription factor, growth factor response	7.1	X54686	I	I
c-myc	Transcription factor, activating and repressing expression of target genes	∼10.4	Y00396	I	I
EST191797	Transcriptional regulatory protein, cell growth, and/or maintenance	−2.1	AA849035	D	X	MYCB	100%, 177 aa
Nerve growth factor-induced (NGFI-A) gene (egr1)	Transcription factor, mitogenesis, and differentiation	2.1	M18416	I	I
Immediate early gene transcription factor NGFI-B	Transcriptional factor, orphan nuclear receptor, steroid receptor family	2.3	U17254	I	I
NGFI-C3E exon#1-2	Transcription factor, positive control of cell proliferation	2.5	M92433	I	I
krox20	Transcription factor, nervous system development	7.2	U78102	I	I
EST	Transcription factor, repression of transcription, negative control of cell proliferation	3.6	AI071299	I	X	Mouse transforming growth factor-beta-inducible early growth response protein 1 (MGIF)	93%, 479 aa
Hypoxia-inducible factor 1	Transcription factor, stress response	2.7	Y09507	I	I
RL/IF-1	Transcription factor binding and cytoplasmic sequestering	∼16.4	X63594	I	I
NGF-inducible anti-proliferative putative secreted protein (PC3)	Transcription factor, DNA repair, negative control of cell proliferation	4.1	M60921	I	I
EST199655	Transcription factor, DNA repair, negative control of cell proliferation	2.6	AA944156	I	I	BTG2 rat btg2 protein precursor	100%, 157 aa

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 10.

Trancriptional Regulation 2

Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
FSH-regulated protein	Transcription factor, negative control of cell proliferation	∼5.5	L26292	I	X	/Kruppel-like factor 4 (KLF4)
RYB-a	Cell proliferation and DNA repair, repression of transcription	3	D28557	I	X
Sry-related HMG-box protein Sox11	Transcription activating factor, neurogenesis	∼5.0	AJ004858	I	X
EST198080	Transcription factor, neural development	−3.1	AA894277	D	X	/Soggy precursor and TEAD-2
BHF-1	Differentiation factor during neurogenesis	∼–7.7	D82074	D	X
EST	Transcription factor, differentiation factor during neurogenesis	∼–5.8	AI639109	D	X	/Neurogenic differentiation (NEUROD1, BHF-1, NIDDM)
LIM homeodomain protein (LH-2)	Transcription factor, cell differentiation	−2	L06804	D	X
Retinoic acid receptor alpha1	Nuclear hormone receptor, differentiation	∼9.8	AJ002940	I	X
Inhibitor of DNA-binding, splice variant Id1.25	Transcription factor, inhibiting dna binding, negative regulation of cell differentiation	1.7	L23148	I	X
High Mobility Group Protein I (Y)	AT DNA binding, transcription regulation	2.3	X62875	I	X
Chromosomal protein HMG2	DNA-binding protein, affecting transcription and cell differentiation, DNA repairing	5.2	D84418	I	X
Leucine zipper protein	Transcription factor, transcription co-repressor, cAMP responsive element binding	2.6	M63282	I	X
Transcriptional repressor CREM	Regulator of the transcription of cAMP-inducible genes	∼17.1	S66024	I	I
Interferon regulatory factor 1 (IRF-1)	Transcription activating factor, regulating apoptosis and tumor-suppression	2.6	M34253	I	I
EST, EST234097	Transcription factor	2.2	U53184, AI237535	I	X	Human LPS-induced TNF-alpha factor	80%, 134 aa
rNFIL-6 = C/EBP-related transcription factor	Transcription factor, immune and inflammatory responses, antiapoptosis	∼14.2	S77528	I	X
Sfb mRNA for silencer factor B	Transcription factor	6.2	X60769	I	I
CELF	Transcription factors, immune and inflammatory responses	14	M65149	I	I
EST	Transcription activating factor, immune response	∼43.8	AI045030	I	X	Rat CCAAT/enhancer binding protein delta	100%, 267 aa
Zinc finger protein	Transcriptional activator, b-cell growth and development, fibrogenesis	∼15.4	AF001417	I	X
EST	Transcription factor, activator	6.5	AA900476	I	X	MRG1 mouse MSG-related protein 1	98%, 268 aa

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 11.

Extracellular matrix-related genes

Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
Fibronectin gene 3 end	Extracellular matrix, cell adhesion and migration, signal transduction	1.7	X05834	I	I
Brevican core protein	Chondroitin sulfate proteoglycan, extracellular matrix, differentiation, gliotic response	−2.3	U37142	D	X
Dermatan sulfate proteoglycan-II (decorin)	Small proteoglycan, binding collagen, fibronectin, and tgf-beta. Suppressing the growth of various tumor cell lines	∼10.6	Z12298	I	X
EST206481	Calcium-binding of the extracellular matrix; inhibitor of calcification	5	AI012030	I	I	MGP rat matrix gla-protein precursor	100%, 102 aa
Osteopontin	Ligand for integrin alpha-v/beta-3 and CD44	60.9	M14656	I	I
Tenascin	Extracellular matrix glycoprotein, a ligand for integrins	∼7.7	U09401, U15550	I	X
Syndecan	Cell surface proteoglycan and receptor for the extracellular matrix, cell adhesion, action of growth factors	5.2	X60651, S61865	I	I
Ryudocan = heparan sulfate proteoglycan core protein	Cell surface proteoglycan, neutrophil migration, receptors or coreceptors	2.2	S61868	I	I
Neuroglycan C precursor	Chondroitin sulfate proteoglycan, CNS-specific functions	−2.3	U33553	D	X
UDP-glucose dehydrogenase	Excellular matrix biosynthesis	1.7	AB013732	I	X
Protein disulphide isomerase (PDI)	Procollagen-proline hydroxylation, extracellular matrix maintenance	2.3	X02918	I	I
Iodothyronine 5-monodeiodinase (5-MD)	Procollagen-proline hydroxylation, extracellular matrix maintenance	3.5	M21476	I	I
EST230748	Protein modification of extracellular matrix, tumor suppressor	1.8	AI234060	I	X	Protein-lysine 6-oxidase precursor	100%, 410 aa
Lysyl oxidase	Protein modification of extracellular matrix, tumor suppression	∼12.6	S77494, S66184	I	X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 12.

Genes related to cytoskeletal proteins

Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
Lamina associated polypeptide 2 (LAP2)	Binding lamin B1 and chromosomes, regulation of nuclear architecture	1.7	U18314	I	X
EST	Lamin B receptor, lamin/chromatin binding		AI145490	I	X	Rat NBP60	100%, 619 aa
EST189270	Crosslinking actin to membrane glycoproteins, transducing ligand-receptor binding to actin reorganization, neuronal migration	∼13.9	AA799773	I	X	ABP2_human endothelial actin-binding protein	60%, 124 aa
EST	Cross-linkers between plasma membranes and actin-based cytoskeletons, cell-cell recognition and signaling and cell movement	∼15.3	AF004811	I	I	Mouse moesin	99%, 576 aa
EST190459	Cytoskeletal protein, linking cytoskeleton to extracellular matrix receptors	∼7.1	AA800962	I	X	TALI_human talin	96%, 113 aa
Growth factor (Arc)	Actin binding, endoderm development	2.9	U19866	I	I
Myosin regulatory light chain (RLC)	Cytoskeleton organization and biogenesis	2.1	X05566	I	X
RLC-A gene for myosin regulatory light chain, exon 4	Cytoskeleton organization and biogenesis	2.5	X54617	I	X
SM22	Actin-binding proteins, muscle development	∼21	M83107	I	X
Tropmyosin (TM-4)	Binds actin filament	3	J02780	I	X
EST	Tropomyosin, cytoskeletal type, binds actin filament	5	AA859305	I	X	Tropomyosin isoform 6-rat	100%, 247 aa
CLP36 (clp36)	Cytoskeletal protein, adapter, actin binding	2.6	U23769	I	X
Striated muscle alpha-tropomyosin	Actin-binding and troponin-binding proteins	1.9	X02412	I	X
EST199921	Actin-binding proteins, inhibiting actomyosin Mg(2+)-ATPase	2.4	AA944422	I	I	Calponin, acidic isoform	100%, 329 aa
Nestin	Intermediate filament, neurogenesis	1.9	M34384	I	I
Vimentin	Intermediate filament, cytoskeletal protein	3.2	X62952	I	I
Glial fibrillary acidic protein alpha (GFAP) and glial fibrillary acidic protein delta (GFAP)	Intermediate filament, cell-specific marker	∼22.6	AF028784
Small proline-rich protein (spr)	Intermediate filament, cell shape and cell size control	5.6	L46593	I	I
EST195714	Member of the cornified envelope precursor protein family, structural protein, epidermal differentiation	13.5	AA891911	I	X	CORA rat cornifin alpha	100%, 151 aa
EST196136	Microtubule, cytoskeletal structural protein	16.8	AA892333	I	D	Tubulin alpha-1 chain	100%, 450 aa

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 13.

Ion, vesicular transport, and synaptic transmission

Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
Brain digoxin carrier protein	Na(+)-independent transporter of organic anions	∼−14.7	U88036	D	I
EST	Glycine-inhibited chloride channel	−2	AI145044	D	D	Glycine receptor alpha-2 chain variant A precursor	100%, 451 aa
Potassium channel, alpha subunit	Potassium channel regulator	∼−11	Y17606	D	X
Serum and glucocorticoid-regulated kinase (sgk)	Protein serine/threonine kinase, sodium transport	2.2	L01624	I	I
Sodium myo-inositol transporter (SMIT)	Small molecule transport, regulation of membrane potential and tissue osmolyte	2.9	AJ001290	I	I
Na-K-2Cl cotransporter (Nkcc1)	Sodium:chloride/potassium:chloride symporter	5	AF086758	I	X
Transferrin receptor	Iron transport	2.9	M58040	I	X
Ceruloplasmin	Ferroxidase, iron efflux	5	L33869	I	I
EST	Ferroxidase, iron efflux	∼5.1	AA817854	I	X	CERU rat ceruloplasmin precursor	100%, 1058 aa
Complexin II	Docking protein, exocytosis, neurotransmitter release	−1.8	U35099	D	X
Alpha-soluble NSF attachment protein	Membrane fusion, exocytosis, and intra-Golgi transport	−1.8	X89968	D	X
B/K protein	Calcium-dependent phospholipid binding	−3.6	U30831	D	X
EST	Membrane fusion, binds phospholipid vesicles	−3.4	AI639118	D	X	Calcium-dependent actin-binding protein	80%, 41 aa
Synaptotagmin IV homolog	Ca2+-dependent vesicular trafficking and exocytosis	1.9	U14398	I	I
S-100 related protein	Calcium binding, annexin II ligand, exocytosis and endocytosis	5	J03627	I	I
Clathrin assembly protein short form (CALM)	Retrieving synaptic vesicles	2.3	AF041373	I	X
EST	Synaptic transmission, synaptic uptake of extracellular material	−2	AI072943	D	D	Neuronal pentraxin I precursor (np-I) (np1) (47 kd taipoxin-binding protein)	100%, 431 aa
Secretogranin II	Packaging or sorting of peptide hormones and neuropeptides into secretory vesicles	2.2	M93669	I	I
Agrin	Synaptogenesis, extracellular matrix	−2	M64780	D	X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

TABLE 14.

Genes related to cell adhesion, motility, and immune responses

Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
CD14	Lps receptor, binding lipopolysaccharide binding protein, lbp/lps complex, and apoptotic cells, triggering phagocytosis	10.3	AF087943, AF087944	I	I
Lipopolysaccharide binding protein	A carrier for LPS and control LPS-dependent monocyte responses, the lbp/lps complex seems to interact with the cd14 receptor	∼15.3	L32132	I	X
EST188970	Cell adhesion	−2.1	AA799473	D	X	Lactadherin precursor (mfg-e8)	79%, 101 aa
Intercellular adhesion molecule-1	Binding integrin alpha-I/beta-2 and leukocyte adhesion IFA-1 protein, cell adhesion	3	D00913	I	I
P-Meta-1 mRNA for CD44 surface protein	Cell surface receptor for hyaluronate, matrix adhesion, lymphocyte activation and apoptosis	∼4	A30543	I	I
Glycoprotein CD44 (CD44)	Cell surface receptor for hyaluronate, matrix adhesion, lymphocyte activation and apoptosis	∼35.9	M61875	I	I
P41-Arc	Assembly of the actin cytoskeleton, cell motility and cell elongation	6.2	AF083269	I	I
Protein p9Ka homologous to calcium-binding protein	Calcium binding, motility, invasion, and tubulin polymerization	3.8	X06916	I	I
EST197807	Barbed-end actin capping, macrophage motile	2	AA894004	I	X	Mouse macrophagecapping protein	92%, 226 aa
MHC class I RT1.C-type protein	Human leukocyte antigen, presentation of foreign antigens	2.4	L40362	I	I
Proteasome subunit RC1	26S proteasome, non-lysosomal proteolytic pathway, class I-restricted antigen presentation	∼6.9	D10729	I	I
MHC class I antigen (RT1.EC2)	Human leukocyte antigen, presentation of foreign antigens	∼7.0	AF074608, M24324, M64795	I	I
MHC class II antigen RT1.B beta chain	Human leukocyte antigen, presentation of foreign antigens	∼2.3	U65217	I	I
MHC class II-like beta chain (RT1.DMb)	Human leukocyte antigen, presentation of foreign antigens	5.4	U31599	I	I
MHC-associated invariant chain gamma	Gamma chain antigen associated with MHC class II antigen	∼8.1	X13044	I	I
Tumor-associated glycoprotein E4 (Tage4)	A member of the immunoglobulin supergene family	∼7.9	L12025	I	X
Fc-gamma receptor	Fc fragment of IgG, low affinity IIIb, receptor	3.7	M32062	I	X
Fc gamma receptor	Receptor for the fc region of immunoglobulins gamma, low affinity receptor, downmodulation of cell activation state	∼11.2	X73371	I	I
IgE binding protein	Galactose-specific lectin, binding IgE, cell growth regulation, chemoattractant activity	21.7	J02962	I	I
Complement protein C1q beta chain	Complement component	2.9	X71127	I	I
Decay accelerating factor GPI-form precursor (DAF)	Preventing the formation of c4b2a and c3bbb; inhibiting the amplification of the complement cascade	∼23.9	AF039583	I	X

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

Real-time quantitative RT-PCR

Twenty genes were selected for RT-PCR based on whether they had important functions in cell death, were previously unreported, and had relatively high-fold changes on the microarrays. Real-time quantitative RT-PCR was performed on these genes (n = 3) using the ABI Prism 5700 Sequence Detection system (Applied Biosystems, Foster City, CA, U.S.A.) (Tang et al., 2001). Primer and probe sequences were selected from coding regions of each of the genes with the aid of Primer Express 2.0 (Applied Biosystems). All primers and probes were synthesized using PE Oligofactory (Applied Biosystems). Each probe was labeled at the 5′-end with the reporter dye VIC and at the 3′-end with quencher dye TAMRA (6-carboxytetramethyl-rhodamine) and was phosphate blocked at the 3′-end to prevent extension by AmpliTaq Gold DNA polymerase. One-step RT-PCR was performed according to the Taqman One-Step RT-PCR Master Mix Reagent kit protocol (Applied Biosystems). Fifty to 100-ng total RNA, 900-nmol/L primer and 250-nmol/L probe were added for the selected genes. Thermal cycling was carried out as follows. Reverse transcription: 48°C for 30 minutes; activation of hot started AmpliTaq Gold DNA polymerase: 95°C for 10 minutes; thermal cycling: 95°C for 15 seconds, and 60°C for 1 minute for 40 cycles. The amplified transcripts were quantified with the relative standard curve method and using GAPDH as a loading control.

RESULTS

Total number of regulated genes

A significant number of genes were regulated at 24 h after permanent focal ischemia (Figs. 1 and 2; Tables 1–15). Of the 328 transcripts (from 8,740 on the microarrays) that differed from the sham-operation group using the criteria above (all present and fold change >1.7 for all comparisons), 264 genes and ESTs were upregulated, and 64 genes and ESTs were downregulated. Figure 1 shows a scatter plot of increased expression (brown and red) and decreased expression (green) of transcripts in the 24-h ischemic samples compared with sham-operation controls. It is notable that many genes showed greater than 10-fold increases of expression. Figure 2 shows that the 328 regulated genes cluster into two groups: those that increase in animals with stroke and those that decrease in animals with stroke (Fig. 2). This unsupervised cluster also shows the relative consistency of expression of upregulated (red, threefold increase) and downregulated genes (blue, threefold decrease) in the three stroke animals compared to the three sham-operated animals (Fig. 2).

TABLE 15.

Others

Gene name	Function summary	Fold change	Gene bank #	RNA change in chips	RNA change in paper	Unigene/blast	Percent identity, aligned region
MG87	Iron-sulfur protein	−1.8	AF095741	D	X	Probable iron-sulfur protein b0947	32%, 279 aa
EST196401	Nucleotide binding	2.4	AA892598	I	X	/Putative nucleotide binding protein, estradiol-induced
EST221135	Compaction of DNA	2.1	AI177503	I	X	Human histone H3.3	100%, 135 aa
EST	Probably binding Ca2+ and an unknown ligand	2.6	AI639058	I	X	Human protein c18ORF1	73%, 141 aa
CD9	CD9 antigen, platelet activation and aggregation	2.1	X76489	I	X
ad1-antigen	Antigens, mast cell marker, platelet activation marker	2.9	X61654	I	X
Urinary plasminogen activator receptor 1 (uPAR-1)	Receptor for urokinase plasminogen activator, localizing and promoting plasmin formation, proteolysis-independent signal transduction	∼16.3	X71898	I	X
EST189300	Egf-like module	2.1	AA799803	I	X	Structure of the Egf-like module of human C1r, Nmr, 19 structures	76%, 134 aa
Major vault protein	Nucleocytoplasmic transport	3.1	U09870	I	X
Serine threonine kinase (pim-3)	Protein serine-threonine kinase, developmental processes	5.3	AF086624	I	X
EST105188	Similar to protein kinases	∼4.1	H31287	I	X	GS3955	61%, 85aa
Intracellular calcium-binding protein (MRP14)	Inhibition of casein kinase	∼16.2	L18948	I	X
EST229907	The domain of insulin growth factor-binding protein homologues	∼5.2	AI233219	I	X	MEGF6 protein-rat	37%, 78 aa
Epithelial cell transmembrane protein antigen precursor (RTI40)	An apical integral plasma membrane protein expressed in type I but not type II alveolar epithelial cells	10.6	U92081	I	I
EST197083	Globule surface membrane material, indication of adipocyte differentiation	12.3	AA893280	I	X	Mouse adipose differentiation-related protein	86%, 180 aa
Epithelial membrane protein-1	Membrane glycoproteins, cell differentiation	∼8.9	Z54212	I	X
ASM15	Encoding an untranslated RNA, lies at the end of a cluster of imprinted genes; tumor suppressor activity	∼40.3	X59864	I	I
Ri1	A LIM domain protein, LIM/double zinc finger domains	∼31.2	X76454	I	X
Activity and neurotransmitter-induced early gene 6 (ania-6)	Unclear; low similarity to cyclins	∼6.8	AF030091	I	X
EST196323	Unclear	3.5	AA892520	I	X	/Membrane protein of cholinergic synaptic vesicles (VATI), human BRCA1, Rho7 and vatI genes, complete cds, and ipf35 gene, partial cds

RNA change in paper: RNA change reported in previous ischemic stroke paper. I, Increase; D, Decrease; –, Unchange; X, not reported.

FIG. 1.

Scatter plot showing the average hybridization signal intensity of the genes in the ischemic (n = 3) compared with the sham surgery (n = 3) group. The diagonal lines indicate twofold change in the ischemic group compared with the sham group (twofold increase, upper line; twofold decrease, lower line). Significantly upregulated transcripts in the ischemic compared with sham groups are shown in red–brown; significantly downregulated transcripts in the ischemic compared to the sham groups are shown in green.

FIG. 2.

Hierarchical clustering of regulated genes in the ischemic group compared with sham group. Red represents threefold increases of expression compared with the means, and purple–blue indicates threefold decreases of expression compared with the means. The expression of the three sham animals is shown in the top three rows, and the expression of the three ischemic animals is shown in the bottom three rows. The expression of individual genes is shown in thin vertical columns. Note that a great many genes are induced in the three ischemic animals compared with the shams (left three fourths of the cluster), and a smaller set of genes are decreased in the three ischemic animals compared with the shams (right one fourth of the cluster).

By searching PubMed and performing Blast analyses, it was estimated that 165 (50%) of the 328 regulated genes had been reported in previous stroke studies. Of note, 147 known genes (45%) had not been reported to be regulated after stroke or other type of ischemia, and 16 (5%) were unknown ESTs. Thus, half (49.7%) of the transcripts reported in this study have not been reported in previous stroke or ischemia studies (Fig. 3).

FIG. 3.

Pie chart showing the percentages of genes identified as regulated by ischemia in the periinfarction cerebral cortex in this study. The proportion of genes reported in previous ischemic stroke studies (blue), not reported in previous ischemic stroke studies (lavender), and the percentages of expressed sequence tags (ESTs) (yellow) are shown.

Functional categories of differential expressed genes

By searching LocusLink, OMIM, GeneCards, and PubMed and reference gene ontology, we divided the regulated genes into 14 different functional categories (Fig. 4, Tables 1–15). Although some of the categories are somewhat artificial because some genes fall into several categories, these categories help in assessing the large amount of data (Figs. 4A–C).

FIG. 4.

(A) Percentages of the 328 genes regulated by ischemia in this study that were in each functional category. (B) Percentages of the genes in each functional category that were regulated more than fivefold. (C) Percentages of genes in each functional category that had not been reported in previous stroke studies. EST, expressed sequence tag.

Transcription factors and metabolism- and signal transduction–related categories had the most numbers of regulated transcripts (Fig. 4A). The genes showing the highest fold changes (more than fivefold) included the cytokines and chemokines, cell adhesion, motility and immune response–related genes, stress proteins, and transcriptional factors (Fig. 4B). Stress proteins, growth factors, cytokines and chemokines, cell adhesion, motility and immune response–related genes, and enzymes and enzyme inhibitors had been the most reported, whereas many of the G-protein–related genes and metabolism-related genes shown to be induced by stroke in this study had not been previously reported (Fig. 4C).

Enzymes and inhibitors. Several enzymes and proteases well known in cerebral ischemia were detected using the microarrays including the ICE-like cysteine protease, calpain and cathepsin L (cysteine protease), gelatinase B (collagenase), and TIM1 (metalloproteinase inhibitor). Additional upregulated genes included cathepsin K and C (cysteine protease) and PS-PLA1 (phospholipase); enzyme inhibitors, such as cystatin B (cysteine protease inhibitor); contrapsin-like protease inhibitor–related protein (serine protease inhibitor); and a ribonuclease inhibitor (Table 1).

Metabolism-related genes. Several genes in Table 2 known to be induced by stroke included GLUT1 (glucose transporter), ornithine decarboxylase (rate-limiting enzyme of polyamine biosynthesis), and PCNA/cyclin (DNA replication and repair). Additional regulated genes included glycerol 3-phosphate dehydrogenase (GPDH) that was induced more than 60-fold, PHAS-I (translation negative regulation), PKBS (benzodiazepine receptor, flow of cholesterol into mitochondria), and TBFII (RNA binding, splicing, and processing) (Table 2).

Stress response proteins. The transcripts of almost all heat shock proteins (Hsp) present on the microarrays were induced. Hsp27, Hsp70, and heme oxygenase were highly expressed as previously reported (Sharp et al., 2000). The DNA damage response gene GADD45 and glutathione peroxidase were also upregulated. The previously unreported antioxidant protein 2 transcript was decreased (Table 3).

Neurotransmitter and hormone–related genes. Unreported stroke-inducible genes, such as Ania-3 (metabotropic glutamate receptor signal pathway) and α-type calcitonin gene–related peptide (vasodilator) were highly expressed. Retinoic acid–related genes were significantly regulated, and the angiotensinogen transcript decreased. Reported stroke-inducible genes, such as Narp (extracellular aggregating for α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [AMPA] receptor), β-type calcitonin gene-related peptide (vasodilator), cyclooxygenase-2 (rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins), and thyrotropin-releasing hormone were also highly expressed (Table 4).

Growth factor–related genes. Several growth factors induced by ischemia were detected including brain-derived neurotrophic factor, transforming growth factor-β-1, heparin binding epidermal growth factor-like growth factor and vascular endothelial growth factor (VEGF) (Lipton, 1999). Other factors, not previously described in ischemic stroke, were also induced by ischemia: PT N1 (negative regulation of insulin signaling), DOC-2 p59 isoform (CSF1 signal transduction pathway), and GRB2 (linking EGFR and PDGFRB to Ras and Rac pathway). Of note, the Noggin transcript (binding and inactivating members of TGF-β superfamily signal protein) decreased (Table 5).

Cytokine and chemokine–related gene. Ischemia is known to induce many of these genes including tumor necrosis factor (TNF) receptor, interleukin (IL)-1β, IL-1 receptor antagonist, IL-6, CC chemokine ST38 precursor, MIP-1, and interferon-induced messenger RNA. Other inducible genes in this class included HCYP2 (serine/threonine kinase, interacting with TNFR1 and TRAF2), PRG1 (protection of cells from Fas or TNFα-induced apoptosis), gro, calgranulin, and IL-18 (Table 6).

Signal transduction–related genes. GTP cyclohydrolase I (NO-synthesis related), protein kinase C-δ, oxidative stress-inducible protein tyrosine phosphatase, MKP-3 (inactivation of MAPK), and SOCS-3 (negative regulator in the JAK-STAT pathway) are induced by ischemia as previously reported. Unreported genes included Ssecks 322 (PKA anchoring protein), Cpg21 (inactivating ERK1), JAK2 (JAK-STAT cascade), and several genes related to NO metabolism (Table 7).

G-protein–related genes. This was the most surprising group of genes because ischemia was not generally known to regulate them. Highly expressed transcripts included Rad (small monomeric GTPase) and G33A (Rho GTPase binding and activator) (Table 8). G-protein–coupled receptors, GTPase binding molecules, and GTP signaling are remarkably regulated in the periinfarction cortex after ischemia (Table 8).

Transcriptional regulatory proteins. Many transcription factors are known to be induced by ischemia and were also regulated on the microarrays including c-fos, Fra-1, c-jun, jun-B, c-myc, NGFI-A, krox20, and HIF1. Other highly regulated transcriptional factors detected on the microarrays included RL/IF-1 (IκB), retinoic acid receptor α1, chromosomal protein HMG2, transcriptional repressor CREM, C/EBP-related transcription factor, and CELF (Tables 9 and 10).

Extracellular matrix. Osteopontin, a ligand of integrin and CD44, was highly expressed along with decorin (binding fibronectin and TGF-β), tenascin (ligand of integrins), syndecan (receptor of extracellular matrix), and lysyl oxidase (protein modification of extracellular matrix) (Table 11). Other genes related to fibronectin and procollagen were also regulated as previously reported.

Cytoskeletal protein–related genes. Glial fibrillary acidic protein α and δ (intermediate filament) and tubulin α-1 chain (microtubule protein) were regulated by ischemia as expected. Other stroke-regulated genes included moesin (cross-linking actin to membranes), talin (linking cytoskeleton to extracellular matrix receptor), and spr (intermediate filament) (Table 12).

Genes-related ion, vesicular transport, and synaptic transmission. Brain digoxin carrier protein (transporter of organic anions), potassium channel α-subunit, neuronal pentraxin I, and agrin expression decreased. Transferrin receptor and ceruloplasmin (ferroxidase, iron efflux) increased (Table 13).

Cell adhesion, motility, and immune response–related genes. CD14 (binding apoptotic cell, triggering phagocytosis), CD44 (cell surface receptor, matrix adhesion, and apoptosis), and immunoglobulin E binding protein (galactose-specific lectin) increased as previously reported in other cerebral ischemia studies. Other regulated transcripts included lipopolysaccharide binding protein (carrier for lipopolysaccharide, interacting with CD14), proteasome subunit RC1 (26S proteasome, class I-restricted antigen presentation), and DAF (inhibiting amplification of complement cascade) (Table 14).

Others. Urinary plasminogen activator receptor 1, serine threonine kinase (pim-3), intracellular calcium–binding protein, the major vault protein—which is involved in nucleo-cytoplasmic transport—and neurotransmitter-induced early gene 6 were all regulated by ischemia (Table 15).

Real-time RT-PCR

Real-time RT-PCR was performed on 20 selected genes at 2, 4, and 24 h after ischemia (Fig. 5). The early times were chosen becausethese would be times when acute stroke treatment might be initiated. The 24-h time was chosen because this would be the time when infarct size was nearly stable but tissue repair and plasticity responses would likely have been initiated. The genes were chosen based on their high fold induction or their functions or functional classes.

FIG. 5.

Real-time quantitative reverse transcription polymerase chain reaction showing the expression of selected genes (y-axis) at 2, 4, and 24 h after cerebral ischemia in the periinfarction cerebral cortex. (A and B) Early upregulated genes that show a twofold or greater increase of expression at 2 or 4 h after cerebral ischemia compared with the sham-controls. (C and D) Late upregulated genes that showed a twofold or greater increase of expression at 24 h after cerebral ischemia compared with the sham-controls. (E) Downregulated genes. LPS, lipopolysaccharide; TBF, track-binding protein; DAF, decay accelerating factor.

The PCR results showed good agreement with the corresponding microarray data in terms of increase and decrease of expression. Early upregulated genes included glutamate receptor–related gene (Narp), G-protein–related genes (Rad and G33A), kinases and phosphatases (HYCP2, Pim-3, Cpg21, JAK2), and the transcriptional factor CELF (Figs. 5A and 5B). Two other genes showed modest regulation at early times and greater increases at 24h including the extracellular matrix gene Tenascin and the complement inhibitor, DAF (Fig. 5A). Early upregulated genes might be involved in acute neuronal injury caused by stroke.

Late upregulated transcripts included enzymes and inhibitors (Cathepsin C, Cip-26, Systatin B), metabolism-relatedgenes (PHAS-I, TBFII), cytoskeletal proteins (Spr), and the lipopolysaccharide binding protein (Figs. 5C and 5D). These genes may be related to tissue immune and repair responses or possibly to some aspect of plasticity after the stroke. Several transcripts were also downregulated on the microarrays (Tables 1–15). The expression of two of these transcripts, brain digoxin carrier protein and a potassium channel, was confirmed by RT-PCR (Fig. 5E).

DISCUSSION

Various members of virtually every class of genes throughout the entire genome appear to be regulated in response to cerebral ischemia. The challenge will be to develop strategies for identifying the best targets to treat acute stroke or to promote recovery and repair. Our previous studies identified groups of molecules that were regulated in common by several types of injuries that might provide such targets (Tang et al., 2002). The present study focuses on genes with high expression levels or on groups of genes that appear to signify activation of specific pathways. The multiplicity of genes induced after focal ischemia would suggest that pharmacologic approaches that targeted multiple pathways simultaneously might be more successful than single gene or even single gene pathway approaches. High-throughput drug screening might be designed to detect single compounds that modulated several different but critical injury or recovery pathways.

RNAs for calpain, ICE-like cysteine protease, cathepsin L, cathepsin K, cathepsin C, cystatin B, and contrapsin-like protease inhibitor–related protein (Cpi-26) were all induced after stroke. The ICE-like cysteine protease is homologous to caspase-3 (Juan et al., 1996). Caspase-3, a central executioner in mitochondrial apoptotic pathways (Hengartner, 2000), is induced along with caspase-9 in the penumbra of some (Benchoua et al., 2001; Namura et al., 1998) but not all focal ischemia studies of adult brain (Gill et al., 2002; Loetscher et al., 2001). The present results support the induction of several caspase-like RNAs by focal ischemia (Dirnagl et al., 1999). However, it is likely that regulation of most caspases occurs by cleavage-induced activation of their proteolytic domains rather than being transcriptionally regulated (Phan et al., 2002; Dirnagl et al., 1999).

Calpain is a calcium-activated nonlysosomal, intracellular cysteine protease that is induced and activated during cerebral ischemia (Neumar et al., 2001; Wang, 2000). Calpains can compromise lysosomal membrane integrity and lead to cathepsin B and L activation. During cerebral ischemia and neuronal apoptosis, cathepsin B and L were activated (Islekel et al., 1999; Yoshida et al., 2002). Cathepsin B activates caspase-3 via caspase-11 during liver cell apoptosis, and cathepsin L activates a caspase-3–like protease in liver cells. Calpain can also directly activate caspase-12 and cause endoplasmic reticulum stress. Calpain and caspase-3 cleave several common cytoskeletal, cytosolic, and nuclear protein substrates (Huang and Wang, 2001; Yamashima, 2000; Wang, 2000). It is not known whether caspase-11 or 12 are activated after cerebral ischemia, but it is likely that calpain and some caspase-related proteases are effectors of injury and tissue repair.

Cystatin B is an inhibitor of cathepsins B, H, and L (Cimerman et al., 2001). Cystatin B–deficient mice have increased expression of apoptosis/glial activation genes (Lieuallen et al., 2001), suggesting that Cystatin B may protect. Cathepsin C, a lysosomal cysteine protease, processes granzyme A and B. Granzymes are expressed in the granules of activated cytotoxic lymphocytes, the granules entering target cells via perforin after cytotoxic lymphocytes synapse with the target cells. Granzyme B can act upstream and downstream of caspases. Granzyme A causes apoptosis via a caspase-independent pathway (Podack, 1999). Since lymphocytes infiltrate ischemic brain (Jander et al., 1995), these cells may mediate apoptosis of target ischemic brain cells via these genes. It is not known whether cystatins, cathepsins, and granzymes play a role in neurons or glia after ischemia, although keratinocytes can express both perforin and granzymes (Berthou et al., 1997).

Contrapsin-like protease inhibitor related protein (Cpi-26), a member of the ‘serpins’ subfamily, is homologous to antichymotrypsin (Ohkubo et al., 1991). Antichymotrypsin can inhibit caspase activity and is antiapoptotic (Ikari et al., 2001). If Cpi-26 functions like antichymotrypsin, it may protect against focal cerebral ischemia.

Glycerol 3-phosphate dehydrogenase expression increased more than 60-fold at 1 d after focal ischemia. Glycerol 3-phosphate dehydrogenase is localized to mitochondria and is involved in mitochondrial hydrogen shuttles necessary for the reoxidation of glycolysis-derived NADH. Activated GPDH increases intracellular levels of NADH and increases cellular resistance against H₂O₂ injury (Hwang et al., 1999). It is possible that GPDH and PARP interact to modulate NADH/NADPH levels and cell survival and death.

Translation-related transcripts, including PHAS-I and polypyrimidine track-binding protein (TBFII), were also induced after focal ischemia. PHAS-I binds eIF4E and negatively regulates translation initiation. It dissociates from eIF4E when phosphorylated. Insulin treatment of adipose cells increased the phosphorylation of 4EBP1 and reduced the interaction of 4EBP1 with eIF4E (Pause et al., 1994). PHAS-I undergoes caspase-dependent cleavage in cells undergoing apoptosis. The N-terminal truncated polypeptide binds to eIF4E and fails to become sufficiently phosphorylated on insulin stimulation to bring about its release from eIF4E (Tee and Proud, 2002). Binding initiation factor eIF4E can rapidly induce apoptosis (Herbert et al., 2000). During global cerebral ischemia, eIF4E and 4E-BP1 were significantly dephosphorylated (Martin de la Vega et al., 2001). PHAS-I levels and phosphorylation state could modulate ischemic brain damage. Overexpression of polypyrimidine track-binding protein stimulates Apaf-1 internal ribosome entry site function (Mitchell et al., 2001). Polypyrimidine track-binding protein also modulates alternative splicing of caspase-2 (Cote et al., 2001), although the role for this gene in ischemic brain injury is unclear.

Narp expression, increased by 2- to 5-fold at 2 to 4 h of ischemia, increased more than 25-fold at 24 h after ischemia. Narp is a secreted immediate-early gene induced by a wide variety of stimuli. Since it is an extracellular aggregating factor for AMPA receptors (O'Brien et al., 1999), it might exacerbate early ischemic excitotoxicity. Agrin, another aggregating factor, was decreased after cerebral ischemia. Agrin is an extracellular heparan sulfate proteoglycan that signals AChR clustering, particularly during the formation of the neuromuscular junction. Agrin is also widely expressed in the central nervous system (Fong and Craig, 1999), although any effects of decreased agrin expression on central acetylcholine neurotransmission after ischemia are uncertain. The chronic changes of Narp and agrin and effects on glutamate and acetylcholine neurotransmission might also play important roles in synaptic plasticity and recovery after stroke.

Brain-derived neurotrophic factor RNA upregulation on the microarrays confirms many previous cerebral ischemia studies. Brain-derived neurotrophic factor may protect against cerebral ischemia in part by phosphorylation of ERK1/2 (Han and Holtzman, 2000; Encinas et al., 1999). Ischemia also induced the MAP-kinase phosphatase (cpg21) that inactivates ERK1 (Ishibashi et al., 1994). This might decrease the protective effects of brain-derived neurotrophic factor. However, ischemia-induced glutamate accumulation activates the P44/42 MAP-kinase pathway. Since inhibition of the MAP-kinase pathways protected neurons against glutamate excitotoxicity (Grant et al., 2001), induction of the cpg21 MAP-kinase phosphatase may also protect against brain ischemia. It is notable that cpg21 is induced more than 20-fold after 2 h of ischemia and more than 120-fold by 24 h of ischemia, suggesting an important but unstudied role for this gene.

Interleukin-6, Jak2, SOCS-3, and Pim-3 RNAs were also regulated by cerebral ischemia. Interleukin-6 induces gp130-homodimerization and activates Jak1, Jak2, Tyk2, STAT1, and STAT3. STAT1 and STAT3 translocate to the nucleus to activate transcription (Heinrich et al., 1998). Interleukin-6 protects the brain against focal and global ischemia (Loddick et al., 1998; Matsuda et al., 1996). SOCS-3 binds all four JAK kinases and inhibits their activity (Chen et al., 2000), possibly suggesting that SOCS-3 might counter the protective effects of IL-6. Co-expression of Pim kinases with Socs-1 results in phosphorylation and stabilization of Socs-1 protein (Chen et al., 2002). Lymphocytes from SOCS-1^−/− mice undergo accelerated apoptosis associated with increased Bax, and murine embryonic fibroblasts lacking SOCS-1 are more sensitive to TNF-induced cell death (Chen et al., 2000). Some Pim kinases prolong cell survival and inhibit apoptosis activity (Lilly et al., 1999). Thus, although the role of SOCS-3 is uncertain, IL-6, Pim kinases, and SOCS-1 may protect against cerebral ischemia.

rNFIL-6 (C/EBP β) and CELF (C/EBP δ) were highly induced. C/EBP plays a role in regulating other genes, since Egr-1 (NGFI-A) and MKP-1 mRNA were significantly reduced in liver of C/EBP knockout mice (Greenbaum et al., 1998). Our data show that, after stroke, both Egr-1 and oxidative stress-inducible protein tyrosine phosphatase (MKP-1) RNAs increased. The target genes of Egr-1, including PAI-1, VEGF, ICAM-1, IL1-β, and immediate-early serum-responsive JE gene (MIP-1) were also induced. Recent lung studies show that Egr-1 regulates many downstream genes and could have a central role in the pathogenesis of ischemic lung tissue damage (Yan et al., 2000). The present data support the possibility that C/EBP regulation of Egr-1 (NGFI-A) and its downstream genes including PAI-1, VEGF, ICAM-1, IL-1β, and MIP-1 could be an important signaling pathway in cerebral ischemia as well.

Transcripts for CD14, IgE binding protein (lectin), lectin-like oxidized LDL receptor (LOX-1), complement protein C1q, PS-PLA1 (serine-phospholipid-selective phospholipase A), and lipopolysaccharide binding protein were all increased after cerebral ischemia. CD14, lectin, and LOX-1 are phagocyte transmembrane molecules. CD14 and LOX-1 can bind phosphatidylserine (PtdSer) and other surface molecules to initiate apoptosis. C1q is an adhesive “bridging” molecule between apoptotic cells and phagocytes. These molecules can tether apoptotic cells and shuttle them to the phagocytic machinery (Savill and Fadok, 2000; Oka et al., 1998). PS-PLA1 hydrolyzes a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine (Sato et al., 1997). Lipopolysaccharide binding protein can bind CD14 (Gutsmann et al., 2001). The increase of the latter molecules can decrease phagocytic recognition of apoptotic cells, which can enhance cell survival when cells are subjected to weak proapoptotic signals (Hoeppner et al., 2001). During cerebral ischemia, microglia–macrophages are markedly activated (Stevens et al., 2002). Although inhibition of microglial activation is reported to protect against global brain ischemia (Yrjanheikki et al., 1998), macrophage and microglial engulfment of apoptotic cells can suppress the secretion of proinflammatory mediators such as tumor necrosis factor-α and suppress the inflammatory response to ischemia (Savill and Fadok, 2000). There seems to be a balance of the detrimental and neuroprotective effects of activated microglia and macrophages that may vary over time and with the severity of injury (Stoll et al., 1998). Exactly how these pathways influence acute injury or recovery after stroke is still unclear.

The focus of this study was to examine the periinfarction cortex at 24 h after a permanent MCA occlusion, at a time well beyond that when any treatment for acute stroke would likely be useful. The changes in many of the 328 transcripts may be related to the onset of tissue repair and the beginning of recovery. As many as two to three times as many molecules may be identified once the entire rat and human genomes are studied as a function of time and area of brain injured after stroke. Future approaches at defining those pathways crucial for recovery might include looking for genes shared with tolerance models, models where recovery is improved or is poor, and other approaches that will help refine the search for clinically important pathways. We have previously reported genes that are induced in common by several types of injury in brain and in blood (Tang et al., 2001, 2002). These common genes may relate to common mechanisms of injury and could represent common targets for a common therapy.