RESUMEN
Protein changes induced by traumatic or ischemic brain injury can serve as diagnostic markers as well as therapeutic targets for neuroprotection. The focus of this chapter is to provide a representative overview of preclinical brain injury and proteomics analysis protocols for evaluation and discovery of novel biomarkers. Detailed surgical procedures have been provided for inducing MCAo and implantation of chronic indwelling cannulas for drug delivery. Sample collection and tissue processing techniques for collection of blood, CSF, and brain are also described including standard biochemical methodology for the proteomic analysis of these tissues.The dynamics of proteomic analysis is a multistep process comprising sample preparation, separation, quantification, and identification of proteins. Our approach is to separate proteins first by two-dimensional gel electrophoresis according to charge and molecular mass. Proteins are then fragmented and analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Identification of proteins can be achieved by comparing the mass-to-charge data to protein sequences in respective databases.
Asunto(s)
Isquemia Encefálica/fisiopatología , Proteínas del Tejido Nervioso/análisis , Proteómica/métodos , Animales , Encéfalo/patología , Encéfalo/fisiología , Humanos , Infarto de la Arteria Cerebral Media , Proteoma/análisis , RatasRESUMEN
In an earlier study, we demonstrated that PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, provides protection against glutamate, staurosporine, veratridine, or hypoxia/hypoglycemia toxicities in primary cortical neuronal cultures by upregulating Bcl-2 expression [R.-W. Chen, C. Yao, X.C. Lu, Z.-G. Jiang, R. Whipple, Z. Liao, H.A. Ghanbari, B. Almassian, F.C. Tortella, J.R. Dave. PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, elicits its function in primary neuronal cultures by upregulating Bcl-2 expression. Neuroscience 135 (2005) 191-201]. Both JNK (c-Jun N-terminal kinase) and p38 MAP (mitogen-activated protein) kinase activation have a direct inhibitory action on Bcl-2 by phosphorylation. In the present study, we continued to explore the mechanism of PAN-811 neuroprotection. Our results indicate that treatment of cultured cortical neurons with glutamate (100 microM) induces phosphorylation of both JNK and p38 MAPK. Specifically, pretreatment of neurons with 10 microM PAN-811 (an optimal neuroprotective concentration) for 1h, 4h, or 24h significantly suppresses glutamate-mediated activation of both JNK and p38 MAPK. Furthermore, the p38 MAPK-specific inhibitor SB203580 and the JNK-specific inhibitor SP600125 prevented glutamate-induced neuronal death in these primary cultures. Our results demonstrate that glutamate-induced phosphorylation of JNK and p38 MAPK is suppressed by PAN-811, which might contribute to Bcl-2 upregulation and PAN-811 neuroprotection.
Asunto(s)
Antagonistas de Aminoácidos Excitadores , Ácido Glutámico/toxicidad , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Piridinas/farmacología , Tiosemicarbazonas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Antracenos/farmacología , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Femenino , Genes bcl-2/genética , Embarazo , Ratas , Ratas Sprague-Dawley , Sales de Tetrazolio , TiazolesRESUMEN
In focal cerebral ischemia, peri-infarct depolarizations (PIDs) cause an expansion of core-infarcted tissue into adjacent penumbral regions of reversible injury and have been shown to occur through 6 hr after injury. However, infarct maturation proceeds through 24 hr. Therefore, we studied PID occurrence through 72 hr after both transient and permanent middle cerebral artery occlusion (MCAo) via continuous DC recordings in nonanesthetized rats. PIDs occurred an average 13 times before reperfusion at 2 hr and then ceased for an average approximately 8 hr. After this quiescent period, PID activity re-emerged in a secondary phase, which reached peak incidence at 13 hr and consisted of a mean 52 PIDs over 2-24 hr. This phase corresponded to the period of infarct maturation; rates of infarct growth through 24 hr coincided with changes in PID frequency and peaked at 13 hr. In permanent MCAo, PIDs also occurred in a biphasic pattern with a mean of 78 events over 2-24 hr. Parameters of secondary phase PID incidence correlated with infarct volumes in transient and permanent ischemia models. The role of secondary phase PIDs in infarct development was further investigated in transient MCAo by treating rats with a high-affinity NMDA receptor antagonist at 8 hr after injury, which reduced post-treatment PID incidence by 57% and provided 37% neuroprotection. Topographic mapping with multielectrode recordings revealed multiple sources of PID initiation and patterns of propagation. These results suggest that PIDs contribute to the recruitment of penumbral tissue into the infarct core even after the restoration of blood flow and throughout the period of infarct maturation.
Asunto(s)
Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Animales , Muerte Celular , Conotoxinas/farmacología , Depresión de Propagación Cortical , Conductividad Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Cinética , Masculino , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
Penetrating brain injury (PBI) is a leading cause of mortality and morbidity in modern warfare and accounts for a significant number of traumatic brain injuries worldwide. Here we characterize the pathophysiology of a new rat model of PBI that simulates the large temporary cavity caused by energy dissipation from a penetrating bullet round. Male Sprague-Dawley rats (250-300 g) were subjected to a simulated ballistic wound to the right frontal hemisphere implemented by an inflatable penetrating probe. Three levels of injury severity were compared to control animals. Neurological and physiological outcome was assessed over a 3-day recovery period and brain tissue collected at 72 h for histopathological evaluation. Brain-injured regions included the ipsilateral frontal cortex and striatum with volumetric increases in intracranial hemorrhage (5-18 mm3) and lesion size (9-86 mm3) related to severity. Similarly, hemispheric swelling increased (3-14%) following PBI, associated with a significant rise in intracranial pressure. Astrogliosis was present in regions adjacent to the core-injury along with microglial and leukocyte infiltration. Injury remote to the lesion was observed in the cerebral peduncle that may have accounted, in part, for observed neurological deficits. Neurological and balance beam testing revealed sensorimotor deficits that persisted through 72 h. Severe electroencephalographic disturbances included the occurrence of cortical spreading depression, slow-waves, and brain seizure activity. In conclusion, this rat PBI model replicates diverse, salient features of clinical PBI pathology, generates reproducible and quantifiable measures of outcome, and is scalable by injury severity, rendering it an attractive vehicle for experimental brain trauma research.
Asunto(s)
Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Modelos Animales de Enfermedad , Heridas por Arma de Fuego/patología , Heridas por Arma de Fuego/fisiopatología , Animales , Lesiones Encefálicas/complicaciones , Hemorragia Cerebral/etiología , Hemorragia Cerebral/patología , Hemorragia Cerebral/fisiopatología , Electroencefalografía , Presión Intracraneal , Masculino , Actividad Motora/fisiología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Índices de Gravedad del Trauma , Heridas por Arma de Fuego/complicacionesRESUMEN
The potent NMDA receptor antagonist, Conantokin-G (CGX-1007), a snail peptide, has an 8-h therapeutic window in rat focal cerebral ischemia. We hypothesized that the mechanism of neuroprotection is the inhibition of 'secondary phase' peri-infarct depolarizations (PIDs), recently shown to recur 6--24 h post-reperfusion. Rats were implanted with intrathecal (i.t.) catheters for drug delivery and DC-compatible electrodes for continuous PID monitoring and subjected to transient (2 h) middle cerebral artery occlusion. Four groups were studied. In two groups (C(40)C and C(20)C), continuous infusion of CGX--1007 was administered over 8--24 h post-occlusion at 0.1 microg/h (0.04 nmol/h) following either a 40- or 20-nmol bolus dose at 8 h. Another group (C(40)S) received the 40-nmol bolus followed by saline infusion, and a control group received saline. Intrathecal drug treatment reduced infarct volumes relative to controls by 61%, 31%, and 10% in C(40)C, C(40)S, and C(20)C groups, respectively, but also induced dose-dependent paralysis and elevated mortality. All rats had PID rates similar to the control group prior to treatment, but following treatment secondary phase PIDs were reduced by 47--57% in each drug group compared to controls. Because several animals exhibited PID inhibition but no neuroprotection, there was no significant correlation between these endpoints across groups. However, drug-treated animals that did not exhibit secondary phase PIDs prior to treatment had significantly smaller infarcts and reduced subsequent PID activity than corresponding control rats. Results suggest that post-reperfusion PIDs play a substantial, though still undefined pathogenic role in delayed maturation of cerebral infarction and NMDA receptor-targeted neuroprotection.
Asunto(s)
Infarto Encefálico/prevención & control , Conotoxinas/administración & dosificación , Depresión de Propagación Cortical/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Isquemia/tratamiento farmacológico , Análisis de Varianza , Animales , Temperatura Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Infarto Encefálico/etiología , Infarto Encefálico/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Electrodos , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Isquemia/patología , Masculino , Ratas , Ratas Sprague-Dawley , Reperfusión/métodos , Factores de TiempoRESUMEN
Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid alphaII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature alphaII-spectrin breakdown products (alphaII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved alphaII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length alphaII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas alphaII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific alphaII-SBDPs were significantly increased in CSF after injury. However, caspase-3 alphaII-SBDPS were observed in CSF of some injured animals. These results indicate that alphaII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.
Asunto(s)
Química Encefálica/fisiología , Calpaína/líquido cefalorraquídeo , Caspasas/líquido cefalorraquídeo , Arteria Cerebral Media/fisiología , Fragmentos de Péptidos/líquido cefalorraquídeo , Espectrina/líquido cefalorraquídeo , Animales , Biomarcadores , Caspasa 3 , Corteza Cerebral/química , Corteza Cerebral/metabolismo , Densitometría , Immunoblotting , Infarto de la Arteria Cerebral Media/líquido cefalorraquídeo , Ratas , Daño por Reperfusión/líquido cefalorraquídeo , Accidente Cerebrovascular/líquido cefalorraquídeoRESUMEN
Brain edema formation associated with trauma-induced intracerebral hemorrhage (ICH) is a clinical complication with high mortality. Studies have shown that heme oxygenase-1 (HO-1) plays an important role in ICH-induced brain edema. In order to understand the role of HO-1 in the protective effect of selective brain cooling (SBC), we investigated the time course of HO-1 changes following penetrating ballistic-like brain injury (PBBI) in rats. Samples were collected from injured and control animals at 6, 24, 48, and 72 h, and 7 days post-injury to evaluate HO-1 expression, heme concentration, brain water content, and immunohistochemistry (IHC). Following a 10% frontal PBBI, HO-1 mRNA and protein was increased at all time points studied, reaching maximum expression levels at 24-48 h post-injury. An increase in the heme concentration and the development of brain edema coincided with the upregulation of HO-1 mRNA and protein during the 7-day post-injury period. SBC significantly decreased PBBI-induced heme concentration, attenuated HO-1 upregulation, and concomitantly reduced brain water content. These results suggest that the neuroprotective effects of SBC may be partially mediated by reducing the heme accumulation, which reduced injury-mediated upregulation of HO-1, and in turn ameliorated edema formation. Collectively, these results suggest a potential value of HO-1 as a diagnostic and/or therapeutic biomarker in hemorrhagic brain injury.
Asunto(s)
Edema Encefálico/enzimología , Edema Encefálico/terapia , Hemorragia Cerebral/enzimología , Hemorragia Cerebral/terapia , Traumatismos Penetrantes de la Cabeza/enzimología , Traumatismos Penetrantes de la Cabeza/terapia , Hemo Oxigenasa (Desciclizante)/fisiología , Hipotermia Inducida/métodos , Animales , Agua Corporal/metabolismo , Edema Encefálico/fisiopatología , Hemorragia Cerebral/fisiopatología , Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Traumatismos Penetrantes de la Cabeza/complicaciones , Hemo/antagonistas & inhibidores , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo Oxigenasa (Desciclizante)/genética , Masculino , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/fisiologíaRESUMEN
One of the histopathological consequences of a penetrating ballistic brain injury is the formation of a permanent cavity. In a previous study using the penetrating ballistic-like brain injury (PBBI) model, engrafted human amnion-derived multipotent progenitor (AMP) cells failed to survive when injected directly in the injury tract, suggesting that the cell survival requires a supportive matrix. In this study, we seated AMP cells in a collagen-based scaffold, injected into the injury core, and investigated cell survival and neuroprotection following PBBI. AMP cells suspended in AMP cell conditioned medium (ACCS) or in a liquefied collagen matrix were injected immediately after a PBBI along the penetrating injury tract. Injured control rats received only liquefied collagen matrix. All animals were allowed to survive two weeks. Consistent with our previous results, AMP cells suspended in ACCS failed to survive; likewise, no collagen was identified at the injury site when injected alone. In contrast, both AMP cells and the collagen were preserved in the injury cavity when injected together. In addition, AMP cells/collagen treatment preserved some apparent brain tissue in the injury cavity, and there was measurable infiltration of endogenous neural progenitor cells and astrocytes into the preserved brain tissue. AMP cells were also found to have migrated into the subventricular zone and the corpus callosum. Moreover, the AMP cell/collagen treatment significantly attenuated the PBBI-induced axonal degeneration in the corpus callosum and ipsilateral thalamus and improved motor impairment on rotarod performance. Overall, collagen-based scaffold provided a supportive matrix for AMP cell survival, migration, and neuroprotection.
Asunto(s)
Colágeno , Matriz Extracelular/trasplante , Traumatismos Penetrantes de la Cabeza/cirugía , Células Madre Multipotentes/trasplante , Degeneración Nerviosa/patología , Recuperación de la Función , Amnios , Animales , Movimiento Celular , Supervivencia Celular , Cuerpo Calloso/patología , Modelos Animales de Enfermedad , Traumatismos Penetrantes de la Cabeza/patología , Traumatismos Penetrantes de la Cabeza/fisiopatología , Humanos , Masculino , Microinyecciones , Actividad Motora , Degeneración Nerviosa/cirugía , Ratas , Ratas Sprague-Dawley , Prueba de Desempeño de Rotación con Aceleración Constante , Trasplante de Células Madre , Tálamo/patología , Andamios del Tejido , Resultado del TratamientoRESUMEN
To identify a viable cell source with potential neuroprotective effects, we studied amnion-derived multipotent progenitor (AMP) cells in a rat model of penetrating ballistic-like brain injury (PBBI). AMP cells were labeled with fluorescent dye PKH26 and injected in rats immediately following right hemispheric PBBI or sham PBBI surgery by ipsilateral i.c.v. administration. At 2 weeks post-injury, severe necrosis developed along the PBBI tract and axonal degeneration was prominent along the corpus callosum (cc) and in the ipsilateral thalamus. Injected AMP cells first entered the subventricular zone (SVZ) in both sham and PBBI rats. Further AMP cell migration along the cc only occurred in PBBI animals. No significant difference in injury volume was observed across all treatment groups. In contrast, treatment with AMP cells significantly attenuated axonal degeneration in both the thalamus and the cc. Interestingly, PKH26-labeled AMP cells were detected only in the SVZ and the cc (in parallel with the axonal degeneration), but not in the thalamus. None of the labeled AMP cells appeared to express neural differentiation, as evidenced by the lack of double labeling with nestin, S-100, GFAP, and MAP-2 immunostaining. In conclusion, AMP cell migration was specifically induced by PBBI and requires SVZ homing, yet the neuroprotective effect of intracerebral ventrical treatment using AMP cells was not limited to the area where the cells were present. This suggests that the attenuation of the secondary brain injury following PBBI was likely to be mediated by mechanisms other than cell replacement, possibly through delivery or sustained secretion of neurotrophic factors.
Asunto(s)
Lesiones Encefálicas/patología , Lesiones Encefálicas/cirugía , Células Madre Multipotentes/trasplante , Degeneración Nerviosa/cirugía , Amnios/citología , Animales , Axones/patología , Diferenciación Celular , Movimiento Celular , Humanos , Inmunohistoquímica , Masculino , Células Madre Multipotentes/citología , Degeneración Nerviosa/patología , Ratas , Ratas Sprague-DawleyRESUMEN
To gain additional insights into the pathogenic cellular and molecular mechanisms underlying different types of brain injury (e.g., trauma versus ischemia), recently attention has focused on the discovery and study of protein biomarkers. In previous studies, using a high-throughput immunoblotting (HTPI) technique, we reported changes in 29 out of 998 proteins following acute injuries to the rat brain (penetrating traumatic versus focal ischemic). Importantly, we discovered that one protein, endothelial monocyte-activating polypeptide II precursor (p43/pro-EMAPII), was differentially expressed between these two types of brain injury. Among other functions, p43/pro-EMAPII is a known pro-inflammatory cytokine involved in the progression of apoptotic cell death. Our current objective was to verify the changes in p43/pro-EMAPII expression, and to evaluate the potentially important implications that the differential regulation of this protein has on injury development. At multiple time points following either a penetrating ballistic-like brain injury (PBBI), or a transient middle cerebral artery occlusion (MCAo) brain injury, tissue samples (6-72 h), CSF samples (24 h), and blood samples (24 h) were collected from rats for analysis. Changes in protein expression were assessed by Western blot analysis and immunohistochemistry. Our results indicated that p43/pro-EMAPII was significantly increased in brain tissues, CSF, and plasma following PBBI, but decreased after MCAo injury compared to their respective sham control samples. This differential expression of p43/pro-EMAPII may be a useful injury-specific biomarker associated with the underlying pathologies of traumatic versus ischemic brain injury, and provide valuable information for directing injury-specific therapeutics.
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Lesiones Encefálicas/diagnóstico , Isquemia Encefálica/diagnóstico , Citocinas/metabolismo , Proteínas de Neoplasias/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Biomarcadores/metabolismo , Lesiones Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Recuento de Células , Immunoblotting , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Cellular and molecular pathways underlying ischemic neurotoxicity are multifaceted and complex. Although many potentially neuroprotective agents have been investigated, the simplicity of their protective mechanisms has often resulted in insufficient clinical utility. We describe a previously uncharacterized class of potent neuroprotective compounds, represented by PAN-811, that effectively block both ischemic and hypoxic neurotoxicity. PAN-811 disrupts neurotoxic pathways by at least two modes of action. It causes a reduction of intracellular-free calcium as well as free radical scavenging resulting in a significant decrease in necrotic and apoptotic cell death. In a rat model of ischemic stroke, administration of PAN-811 i.c.v. 1 h after middle cerebral artery occlusion resulted in a 59% reduction in the volume of infarction. Human trials of PAN-811 for an unrelated indication have established a favorable safety and pharmacodynamic profile within the dose range required for neuroprotection warranting its clinical trial as a neuroprotective drug.
Asunto(s)
Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Isquemia , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Piridinas/farmacología , Tiosemicarbazonas/farmacología , Animales , Apoptosis , Western Blotting , Calcio/metabolismo , Corteza Cerebral/metabolismo , Quelantes/farmacología , Técnicas de Cocultivo , Cuerpo Estriado/metabolismo , Fragmentación del ADN , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fluoresceínas/farmacología , Depuradores de Radicales Libres/metabolismo , Humanos , Hipoxia , L-Lactato Deshidrogenasa/metabolismo , Masculino , Necrosis , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Factores de TiempoRESUMEN
Excessive glutamate release is associated with neuronal damage. A new strategy for the treatment of neuronal injury involves inhibition of the neuropeptidase glutamate carboxypeptidase II (GCP II), also known as N-acetylated alpha-linked acidic dipeptidase. GCP II is believed to mediate the hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to glutamate and N-acetyl-aspartate, and inhibition of NAAG peptidase activity (by GCP II and other peptidases) is neuroprotective. Mice were generated in which the Folh1 gene encoding GCP II was disrupted (Folh1-/- mice). No overt behavioral differences were apparent between Folh1-/- mice and wild-type littermates, with respect to their overall performance in locomotion, coordination, pain threshold, cognition and psychiatric behavioral paradigms. Morphological analysis of peripheral nerves, however, showed significantly smaller axons (reduced myelin sheaths and axon diameters) in sciatic nerves from Folh1-/- mice. Following sciatic nerve crush, Folh1-/- mice suffered less injury and recovered faster than wild-type littermates. In a model of ischemic injury, the Folh1-/- mice exhibited a significant reduction (p < 0.05) in infarct volume compared with their wild-type littermates when subjected to middle cerebral artery occlusion, a model of stroke. These findings support the hypothesis that GCP II inhibitors may represent a novel treatment for peripheral neuropathies as well as stroke.
Asunto(s)
Isquemia Encefálica/enzimología , Glutamato Carboxipeptidasa II/deficiencia , Glutamato Carboxipeptidasa II/genética , Enfermedades del Sistema Nervioso Periférico/enzimología , Animales , Conducta Animal/fisiología , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Electrofisiología , Infarto de la Arteria Cerebral Media/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/patología , Compresión Nerviosa , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/patología , Fenotipo , Desempeño Psicomotor/fisiología , Nervio Ciático/patologíaRESUMEN
Temporal changes in gene expression were measured using DNA microarrays after 30-min or 2-hr transient middle cerebral artery occlusion (MCAo) in rats. Total RNA was extracted from the injured hemisphere at 30 min, 4 hr, 8 hr, 24 hr, 3 days, and 7 days after MCAo for GeneChip analysis using Affymetrix U34 Rat Neurobiology arrays (1,322 functional genes). In total, 267 genes were expressed differentially: 166 genes were upregulated, 94 genes were downregulated, and 7 genes were biphasically up- and downregulated. Among all differentially expressed genes, 88 were newly identified as associated with ischemic brain injury. Most affected genes were distributed among 12 functional categories. Immediate early genes, transcription factors, and heat shock proteins were upregulated as early as 30 min after MCAo, followed by the upregulation of inflammation, apoptosis, cytoskeletal, and metabolism genes, which peaked within 4-24 hr of injury. Neurotrophic growth factors exhibited a sustained upregulation beginning 24 hr after MCAo and persisting through 7 days post-injury. Three classes of genes were downregulated with distinct temporal patterns: ion channel genes and neurotransmitter receptor genes were downregulated between 8-24 hr after injury, whereas synaptic proteins genes were downregulated between 3-7 days after MCAo. Downregulation of synaptic protein gene expression after ischemic injury is of particular interest because of its conspicuously delayed pattern as a functional group, which has not been reported previously and may play a role in post-injury recovery.