In vivo carotid strain imaging using principal strains in longitudinal view.

Carotid plaque rupture can result in stroke or transient ischemic attack that can be devastating for patients. Ultrasound strain imaging provides a noninvasive method to identify unstable plaque likely to rupture. Axial, lateral and shear strains in carotid plaque have been shown to be linked to carotid plaque instability. Recently, there has been interest in using principal strains, which do not depend on angle of insonification of the carotid artery for quantifying instability in plaque along the longitudinal view. In this work relationships between angle dependent axial, lateral and shear strain along with axis independent principal strains are compared. Three strain indices were defined, 1) Average Mean Strain (AMS), 2) Maximum Mean Strain (MMS) and 3) Mean Standard Deviation (MSD) to identify relationships between these five strain image types in a group of 76 in vivo patients. The maximum principal strain demonstrated the highest strain values when compared to axial strain for all patients with a linear regression slope of 1.6 and a y intercept of 2.4 percent strain for AMS. The maximum shear strain when compared to shear strain had a slope of 1.15 and a y intercept of 0.21 percent for AMS. Next, the effect of insonification angle, which is the angle subtended by the artery at the location of plaque was studied. Patients were divided into three sub groups, i.e. less than 5 degrees (n = 31), between 5 and 10 degrees (n = 24) and above 10 degrees (n = 21). The angle of insonification did not make a significant difference between the three angle groups when comparing the relationship between the angle dependent and independent strain values.

A Cross-Sectional Investigation of Cognition and Ultrasound-Based Vascular Strain Indices.

OBJECTIVE: We examine the relationship between variability in the plaque strain distribution estimated using ultrasound with multiple cognitive domains including executive, language, visuospatial reasoning, and memory function. METHOD: Asymptomatic (n = 42) and symptomatic (n = 34) patients with significant (>60%) carotid artery stenosis were studied for plaque instability using ultrasound strain imaging and multiple cognitive domains including executive, language, visuospatial reasoning, and memory function. Correlation and ROC analyses were performed between ultrasound strain indices and cognitive function. Strain indices and cognition scores were also compared between symptomatic and asymptomatic patients to determine whether there are significant group differences. RESULTS: Association of high-strain distributions with dysexecutive function was observed in both asymptomatic and symptomatic patients. For memory, visuospatial, and language functions, the correlations between strain and cognition were weaker for the asymptomatic compared to symptomatic group. CONCLUSIONS: Both asymptomatic and symptomatic patients demonstrate a relationship between vessel strain indices and executive function indicating that silent strokes and micro-emboli could initially contribute to a decline in executive function, whereas strokes and transient ischemic attacks may cause the further decline in other cognitive functions.

Quantification of carotid artery plaque stability with multiple region of interest based ultrasound strain indices and relationship with cognition.

Vulnerability and instability in carotid artery plaque has been assessed based on strain variations using noninvasive ultrasound imaging. We previously demonstrated that carotid plaques with higher strain indices in a region of interest (ROI) correlated to patients with lower cognition, probably due to cerebrovascular emboli arising from these unstable plaques. This work attempts to characterize the strain distribution throughout the entire plaque region instead of being restricted to a single localized ROI. Multiple ROIs are selected within the entire plaque region, based on thresholds determined by the maximum and average strains in the entire plaque, enabling generation of additional relevant strain indices. Ultrasound strain imaging of carotid plaques, was performed on 60 human patients using an 18L6 transducer coupled to a Siemens Acuson S2000 system to acquire radiofrequency data over several cardiac cycles. Patients also underwent a battery of neuropsychological tests under a protocol based on National Institute of Neurological Disorders and Stroke and Canadian Stroke Network guidelines. Correlation of strain indices with composite cognitive index of executive function revealed a negative association relating high strain to poor cognition. Patients grouped into high and low cognition groups were then classified using these additional strain indices. One of our newer indices, namely the average L - 1 norm with plaque (AL1NWP) presented with significantly improved correlation with executive function when compared to our previously reported maximum accumulated strain indices. An optimal combination of three of the new indices generated classifiers of patient cognition with an area under the curve (AUC) of 0.880, 0.921 and 0.905 for all (n = 60), symptomatic (n = 33) and asymptomatic patients (n = 27) whereas classifiers using maximum accumulated strain indices alone provided AUC values of 0.817, 0.815 and 0.813 respectively.

Improved Correlation of Strain Indices with Cognitive Dysfunction with Inclusion of Adventitial Layer with Carotid Plaque.

Plaque instability may lead to chronic embolization, which in turn may contribute to progressive cognitive decline. Accumulated strain tensor indices over a cardiac cycle within a pulsating carotid plaque may be viable biomarkers for the diagnosis of plaque instability. Using plaque-only carotid artery segmentations, we recently demonstrated that impaired cognitive function correlated significantly with maximum axial and lateral strain indices within a localized region of interest in plaque. Inclusion of the adventitial layer focuses our strain or instability measures on the vessel wall-plaque interface hypothesized to be a region with increased shearing forces and measureable instability. A hierarchical block-matching motion tracking algorithm developed in our laboratory was used to estimate accumulated axial, lateral, and shear strain distribution in plaques identified with the plaque-with-adventitia segmentation. Correlations of strain indices to the Repeatable Battery for the Assessment of Neuropsychological Status Total score were performed and compared with previous results. Overall, correlation coefficients (r) and significance (p) values improved for axial, lateral, and shear strain indices. Shear strain indices, however, demonstrated the largest improvement. The Pearson correlation coefficients for maximum shear strain and cognition improved from the previous plaque-only analyses of -0.432 and -0.345 to -0.795 and -0.717 with the plaque-with-adventitia segmentation for the symptomatic group and for all patients combined, respectively. Our results demonstrate the advantage of including adventitia for ultrasound carotid strain imaging providing improved association to parameters assessing cognitive impairment in patients. This supports theories of the importance of the vessel wall plaque interface in the pathophysiology of embolic disease.

Estimation of ultrasound strain indices in carotid plaque and correlation to cognitive dysfunction.

Carotid plaque prone to release emboli may be predicted by increased strain variations within plaque due to arterial pulsation over a cardiac cycle. Non-invasive ultrasound strain imaging may therefore be a viable surrogate to determine the risk of embolic stroke and possible cognitive impairment. Ultrasound strain imaging was performed on 24 human subjects with significant plaque, who also underwent standardized cognitive assessment (Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)) prior to a carotid endarterectomy (CEA) procedure. Radiofrequency signals were acquired using a Siemens Antares with a VFX 13-5 linear array transducer. Plaque regions were segmented by a radiologist at end-diastole using the Medical Imaging Interaction Toolkit. A hierarchical block-matching motion tracking algorithm was utilized to estimate the cumulated axial, lateral, and shear strains within the imaging plane. The maximum strain indices of the plaque, defined as mean accumulated strain over a small region of interest in the plaque with large deformations, were obtained. All the strain indices were then correlated with RBANS Total score. Overall cognitive performance was negatively associated with maximum axial and lateral strains respectively. The results demonstrate a direct relationship between the maximum axial and lateral strain indices in carotid plaque and cognitive impairment.

Ethanol promotes cytotoxic effects of tumor necrosis factor-related apoptosis-inducing ligand through induction of reactive oxygen species in prostate cancer cells.

BACKGROUND: Effective treatment of prostate cancer (PCa) remains a major challenge due to chemoresistance to drugs including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Ethanol and ethanol extracts are known apoptosis inducers. However, cytotoxic effects of ethanol on PCa cells are unclear. METHODS: In this study we utilized PC3 and LNCaP cell culture models. We used immunohistochemical analysis, western blot analysis, reactive oxygen species (ROS) measurement, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) Cell Proliferation Assay, Annexin-V staining and flow cytometry for quantification of apoptosis. In vitro soft agar colony formation and Boyden chamber invasion assays were used. Tumorigenicity was measured in a xenotransplantation mouse model. RESULTS: Here, we demonstrate that ethanol enhances the apoptosis-inducing potential of TRAIL in androgen-resistant PC3 cells and sensitizes TRAIL-resistant, androgen sensitive LNCaP cells to apoptosis through caspase activation, and a complete cleavage of poly (ADP)-ribose polymerase, which was in association with increased production of ROS. The cytotoxicity of ethanol was suppressed by an antioxidant N-acetyl cystein pretreatment. Furthermore, ethanol in combination with TRAIL increased the expression of cyclin-dependent kinase inhibitor p21 and decreased the levels of Bcl-2 and phosphorylated-AKT. These molecular changes were accompanied by decreased proliferation, anchorage-independent growth and invasive potential of PC3 and LNCaP cells. In vivo studies using a xenotransplantation mouse model with PC3 cells demonstrated significantly increased apoptosis in tumors treated with ethanol and TRAIL in combination. CONCLUSIONS: Taken together, use of ethanol in combination with TRAIL may be an effective strategy to augment sensitivity to TRAIL-induced apoptosis in PCa cells.

Methods for robust in vivo strain estimation in the carotid artery.

A hierarchical block-matching motion tracking algorithm for strain imaging is presented. Displacements are estimated with improved robustness and precision by utilizing a Bayesian regularization algorithm and an unbiased subsample interpolation technique. A modified least-squares strain estimator is proposed to estimate strain images from a noisy displacement input while addressing the motion discontinuity at the wall-lumen boundary. Methods to track deformation over the cardiac cycle incorporate a dynamic frame skip criterion to process data frames with sufficient deformation to produce high signal-to-noise displacement and strain images. Algorithms to accumulate displacement and/or strain on particles in a region of interest over the cardiac cycle are described. New methods to visualize and characterize the deformation measured with the full 2D strain tensor are presented. Initial results from patients imaged prior to carotid endarterectomy suggest that strain imaging detects conditions that are traditionally considered high risk including soft plaque composition, unstable morphology, abnormal hemodynamics and shear of plaque against tethering tissue can be exacerbated by neoangiogenesis. For example, a maximum absolute principal strain exceeding 0.2 is observed near calcified regions adjacent to turbulent flow, protrusion of the plaque into the arterial lumen and regions of low echogenicity associated with soft plaques. Non-invasive carotid strain imaging is therefore a potentially useful tool for detecting unstable carotid plaque.

Ultrasonic attenuation estimation in small plaque samples using a power difference method.

Many studies have shown that atherosclerosis changes the ultrasonic attenuation properties of the vessel wall and plaque. Accurate estimation of the attenuation coefficient slope could therefore provide an early indication of atherosclerosis and the differentiation between low, mild and highly-attenuating plaque within the vessel. However, the traditional reference phantom method that fits the power spectrum in a region of interest fails to accurately estimate the attenuation coefficient for small irregular shaped ex-vivo plaque specimens. This discrepancy was primarily due to partial volume effects and the unknown backscatter coefficient of the plaque sample. We have developed a method based on the reference-phantom method that utilizes the difference in the acoustic power above and below the sample to accurately compute values of the attenuation coefficient ex vivo. Our results demonstrate that this approach overcomes the two drawbacks mentioned earlier and provides accurate estimates of the attenuation coefficient slope for small excised tissue samples.

Lipids and lipidomics in brain injury and diseases.

Lipidomics is systems-level analysis and characterization of lipids and their interacting moieties. The amount of information in the genomic and proteomic fields is greater than that in the lipidomics field, because of the complex nature of lipids and the limitations of tools for analysis. The main innovation during recent years that has spurred advances in lipid analysis has been the development of new mass spectroscopic techniques, particularly the "soft ionization" techniques electrospray ionization and matrix-assisted laser desorption/ionization. Lipid metabolism may be of particular importance for the central nervous system, as it has a high concentration of lipids. The crucial role of lipids in cell signaling and tissue physiology is demonstrated by the many neurological disorders, including bipolar disorders and schizophrenia, and neurodegenerative diseases such as Alzheimer's, Parkinson's, and Niemann-Pick diseases, that involve deregulated lipid metabolism. Altered lipid metabolism is also believed to contribute to cerebral ischemic (stroke) injury. Lipidomics will provide a molecular signature to a certain pathway or a disease condition. Lipidomic analyses (characterizing complex mixtures of lipids and identifying previously unknown changes in lipid metabolism) together with RNA silencing, using small interfering RNA (siRNA), may provide powerful tools to elucidate the specific roles of lipid intermediates in cell signaling and open new opportunities for drug development.

Carotid atherosclerotic plaques from symptomatic stroke patients share the molecular fingerprints to develop in a neoplastic fashion: a microarray analysis study.

Identification of genetic mechanisms that promote the onset of stroke and transient cerebral ischemic attack symptoms in carotid atherosclerotic patients would further our understanding of the pathophysiology of this disease and could lead to new pharmacological and molecular therapies. Using Affymetrix Human Genome 230 GeneChip set, the present study evaluated the gene expression differences in geometrically similar carotid artery plaque samples extricated from six symptomatic stroke patients and four asymptomatic patients. There was no significant difference in the degree of stenosis between the two groups. Of the 44,860 transcripts analyzed, 289 (approximately 0.6% of the total transcripts) were differentially expressed between the plaques from the symptomatic and asymptomatic groups (236 were expressed more abundantly and 53 were expressed less abundantly in the symptomatic group). Of the 236 transcripts expressed more abundantly in the symptomatic plaques, 71% (167 transcripts) indicate an active cell proliferation and neoplastic process. These include oncogenes, growth factors, tumor promoters, tumor markers, angiogenesis promoters, transcription factors, RNA splicing factors, RNA processing proteins, signal transduction mediators and those that control the metabolism. Real-time polymerase chain reaction confirmed the increased expression of 63 transcripts in the symptomatic plaques. The other groups of transcripts expressed more abundantly in the symptomatic plaques are those that control ionic homeostasis, those that participate in the progression of degenerative neurological diseases (Alzheimer's disease, amyotrophic lateral sclerosis and Huntington's disease) and epilepsy. This indicates that symptomatic plaques are molecularly and biochemically more active than the asymptomatic plaques, or active plaque growth precipitates stroke symptoms.

Osteopontin infusion into normal adult rat brain fails to increase cell proliferation in dentate gyrus and subventricular zone.

In the first week after focal ischemia in adult brain, the basal level of neurogenesis increases dramatically in two distinct areas: The dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. It is possible that this remotely induced neurogenesis is the result of a proliferation inducing factor, or factors, diffusing from the infarction to the neurogenic regions. The secreted protein osteopontin (OPN) is a possible factor. In this study, OPN mRNA levels were measured in the cerebral infarction of adult rats that underwent I hour of middle cerebral artery occlusion (MCAO). OPN mRNA levels increased 36.0, 55.0 and 46.7 fold at 6, 24 and 72 hours reperfusion respectively. We also determined whether OPN alone could be responsible for this ischemia-induced neurogenesis. OPN (2.4 microg/day) was infused into the lateral ventricles of the brain in non-ischemic adult male rats, continuously over three days. Bromodeoxyuridine (BrdU) immunohistochemistry was performed and the total BrdU positive (BrdU+) cells were counted. OPN, compared to aCSF infusion, decreased BrdU+ cells in DG and had no significant effect on cell proliferation in the SVZ. This study indicates that osteopontin alone does not increase cell proliferation in the normal adult brain.

Citicoline: neuroprotective mechanisms in cerebral ischemia.

Cytidine-5'-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in a number of CNS injury models and pathological conditions of the brain. Citicoline improved the outcome in several phase-III clinical trials of stroke, but provided inconclusive results in recent clinical trials. The therapeutic action of citicoline is thought to be caused by stimulation of PtdCho synthesis in the injured brain, although the experimental evidence for this is limited. This review attempts to shed some light on the properties of citicoline that are responsible for its effectiveness. Our studies in transient cerebral ischemia suggest that citicoline might enhance reconstruction (synthesis) of PtdCho and sphingomyelin, but could act by inhibiting the destructive processes (activation of phospholipases). Citicoline neuroprotection may include: (i) preserving cardiolipin (an exclusive inner mitochondrial membrane component) and sphingomyelin; (ii) preserving the arachidonic acid content of PtdCho and phosphatidylethanolamine; (iii) partially restoring PtdCho levels; (iv) stimulating glutathione synthesis and glutathione reductase activity; (v) attenuating lipid peroxidation; and (vi) restoring Na(+)/K(+)-ATPase activity. These observed effects of citicoline could be explained by the attenuation of phospholipase A(2) activation. Based on these findings, a singular unifying mechanism has been hypothesized. Citicoline also provides choline for synthesis of neurotransmitter acetylcholine, stimulation of tyrosine hydroxylase activity and dopamine release.

GeneChip analysis after acute spinal cord injury in rat.

Spinal cord injury (SCI) leads to induction and/or suppression of several genes, the interplay of which governs the neuronal death and subsequent loss of motor function. Using GeneChip, the present study analyzed changes in the mRNA abundance at 3 and 24 h after SCI in adult rats. SCI was induced at T9 level by the New York University impactor by dropping a 10-g weight from a height of 25 mm. Several transcription factors, immediate early genes, heat-shock proteins, pro-inflammatory genes were up-regulated by 3 h, and persisted at 24 h, after SCI. On the other hand, some neurotransmitter receptors and transporters, ion channels, kinases and structural proteins were down-regulated by 3 h, and persisted at 24 h, after SCI. Several genes that play a role in growth/differentiation, survival and neuroprotection were up-regulated at 24 h after SCI. Using real-time quantitative PCR, the changes observed by GeneChip were confirmed for seven up-regulated (interleukin-6, heat-shock protein-70, heme oxygenase-1, suppressor of cytokine signaling 2, suppressor of cytokine signaling 3, interferon regulatory factor-1, neuropeptide Y), two down-regulated (vesicular GABA transporter and cholecystokinin precursor) and two unchanged (Cu/Zn-superoxide dismutase and phosphatidyl inositol-3-kinase) genes. The present study shows that inflammation, neurotransmitter dysfunction, increased transcription, ionic imbalance and cytoskeletal damage starts as early as 3 h after SCI. In addition to these effects, 24 h after SCI the repair and regeneration process begins in an attempt to stabilize the injured spinal cord.

Attitudes and approaches to acute ischemic stroke in Wisconsin hospitals.

Although acute stroke is a common presentation to an emergency room, the presentation of a patient with acute ischemic stroke, within a limited time window as an appropriate candidate for cerebral thrombolysis, is not common. In many of these patients, their candidacy can be improved through community education toward emergent transfer to an emergency room if they manifest symptoms of stroke. This would improve the "symptom-to-door" time. Another goal is to improve the recognition and approach of the hospital itself toward improving the "door-to-drug" time in appropriate patients. The obstacle to this second goal does not seem to be a nihilistic or evasive attitude on the basis of this study. Contrary to what was expected, enthusiasm for the use of cerebral thrombolysis was found in emergency physicians of all hospital categories, particularly of small remote hospitals. Instead, educational initiatives should focus on the facilitation of protocols for present and future ischemic stroke therapy, particularly in larger remote facilities that may be more self-dependent in their approach to acute stroke. An equally important focus should be toward more active participation by local neurologists who may be available for acute stroke care. Further, as this study demonstrates a correlation between the involvement of a local neurologist and the use of a stroke protocol, neurologists of non-tertiary facilities should be recruited to participate in these educational initiatives.

Protective effects of glial cell line-derived neurotrophic factor on hippocampal neurons after traumatic brain injury in rats.

OBJECT: The purpose of this study was to evaluate whether glial cell line-derived neurotrophic factor (GDNF) can protect against hippocampal neuronal death after traumatic brain injury (TBI). METHODS: Male Sprague-Dawley rats were subjected to moderate TBI with a controlled cortical impact device while in a state of halothane-induced anesthesia. Then, GDNF or artificial cerebrospinal fluid ([aCSF]; vehicle) was infused into the frontal horn of the left lateral ventricle. In eight brain-injured and eight sham-operated rats, GDNF was infused continuously for 7 days (200 ng/day intracerebroventricularly at a rate of 8.35 ng/0.5 microl/hour). An equal volume of vehicle was infused at the same rate into the remaining eight brain-injured and eight sham-operated rats. Seven days post-injury, all rats were killed. Their brains were sectioned and stained with cresyl violet, and the hippocampal neuronal loss was evaluated in the CA2 and CA3 regions with the aid of microscopy. A parallel set of sections from each brain was subjected to immunoreaction with antibodies against glial fibrillary acidic protein (GFAP; astroglia marker). In the aCSF-treated group, TBI resulted in a significant neuronal loss in the CA2 (60%, p < 0.05) and CA3 regions (68%, p < 0.05) compared with the sham-operated control animals. Compared with control rats infused with aCSF, GDNF infusion significantly decreased the TBI-induced neuronal loss in both the CA2 (58%, p < 0.05) and CA3 regions (51%, p < 0.05). There was no difference in the number of GFAP-positive astroglial cells in the GDNF-infused rats in the TBI and sham-operated groups compared with the respective vehicle-treated groups. CONCLUSIONS: The authors found that GDNF treatment following TBI is neuroprotective.

Effects of citicoline on phospholipid and glutathione levels in transient cerebral ischemia.

BACKGROUND AND PURPOSE: Cytidine-5'-diphosphocholine (citicoline or CDP-choline) is an essential intermediate in the biosynthesis of phosphatidylcholine, an important component of the neural cell membrane. Citicoline provided significant neuroprotection after transient forebrain ischemia in gerbils. This study was undertaken to examine changes and effects of citicoline on phospholipids and glutathione synthesis after transient cerebral ischemia and reperfusion. METHODS: Ten-minute transient forebrain ischemia was induced by bilateral carotid artery occlusion in male Mongolian gerbils with reperfusion up to 6 days. Citicoline (500 mg/kg IP in saline) was given to gerbils just after the end of ischemia, at 3-hour reperfusion, and daily thereafter until 1 day before euthanasia. Hippocampal lipids were extracted and analyzed by thin-layer and gas chromatography. Glutathione was measured by using an enzymatic recycling assay. Glutathione reductase activity was determined by measuring NADPH oxidation. RESULTS: Significant decreases in phospholipids occurred at 1-day reperfusion. Citicoline significantly restored the phosphatidylcholine, sphingomyelin, and cardiolipin levels but did not affect phosphatidylinositol and phosphatidylserine at 1 day. The phospholipids returned to sham levels over days 2 to 6 and were unaffected by citicoline. Ceramide levels significantly increased by 3 and 6 days of reperfusion and were unaltered by citicoline. Ischemia resulted in significant decreases in glutathione and glutathione reductase activity over 3 days of reperfusion. Citicoline significantly increased total glutathione and glutathione reductase activity and decreased the glutathione oxidation ratio, an indicator of glutathione redox status. CONCLUSIONS: Our data indicated that the effects of citicoline on phospholipids occurred primarily during the first day of reperfusion, with effects on glutathione being important over the 3-day reperfusion period.

Ornithine decarboxylase knockdown exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain.

Transient cerebral ischemia leads to increased expression of ornithine decarboxylase (ODC). Contradicting studies attributed neuroprotective and neurotoxic roles to ODC after ischemia. Using antisense oligonucleotides (ODNs), the current study evaluated the functional role of ODC in the process of neuronal damage after transient focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. Transient MCAO significantly increased the ODC immunoreactive protein levels and catalytic activity in the ipsilateral cortex, which were completely prevented by the infusion of antisense ODN specific for ODC. Transient MCAO in rats infused with ODC antisense ODN increased the infarct volume, motor deficits, and mortality compared with the sense or random ODN-infused controls. Results of the current study support a neuroprotective or recovery role, or both, for ODC after transient focal ischemia.

Na+-K+-Cl- cotransporter in rat focal cerebral ischemia.

In cultured neurons, the authors previously demonstrated that the Na+-K+-Cl- cotransporter is significantly stimulated by elevated extracellular potassium and glutamate, which are important factors in cerebral ischemic damage. These findings led the authors to hypothesize that stimulation of the cotransporter after ischemia might result in Na+, K+, and Cl- influx, and might contribute to neuron damage. In the current study, the authors investigated such a role of the Na+-K+-Cl- cotransporter in focal cerebral ischemia. Cerebral ischemia was induced by 2-hour occlusion of the left middle cerebral artery (MCA) and 24-hour reperfusion in male spontaneously hypertensive rats (SHRs). Immunocytochemical staining and immunoblotting revealed an up-regulation of expression of the cotransporter protein in neurons in cortex at 24 hours of reperfusion. Artificial cerebral spinal fluid (aCSF) or 100 micromol/L bumetanide (a cotransporter inhibitor) in aCSF were continuously microdialyzed through a microdialysis probe into left cortices throughout 2-hour MCA occlusion and 24-hour reperfusion. Compared with the aCSF-treated group, infarction volume was significantly reduced in the bumetanide-treated group (25%, P < 0.05). In addition, brain water content in the bumetanide-treated brains was decreased by 70% (P < 0.05). These results strongly suggest that the Na+-K+-Cl- cotransporter may play an important role in cerebral ischemic neuronal damage.

Expression of Na(+)-K(+)-Cl(-) cotransporter in rat brain during development and its localization in mature astrocytes.

Na(+)-K(+)-Cl(-) cotransporter has been proposed to play an important role in the regulation of intracellular Cl(-) concentration in neurons during development. In this study, the expression pattern of the cotransporter in different regions of rat brain was examined at birth (P0), postnatal days 7 (P7), P14, P21, and adult by Western blotting analysis. In cortex, thalamus, cerebellum and striatum, the cotransporter expression level was low at P0 and significantly increased at P14 (P<0.05). The expression peaked at P21 and was maintained at the same level in adulthood. However, in hippocampus, a peak level of the cotransporter expression was detected in adult brain. The immunocytochemistry study of adult rat brain revealed that an intense staining of the Na(+)-K(+)-Cl(-) cotransporter protein was observed in dendritic processes of CA1-CA3 hippocampal pyramidal neurons. In contrast, abundant immuno-reactive signals of the cotransporter were found in somata of thalamic nucleus. Immunofluorescence double staining demonstrates that the Na(+)-K(+)-Cl(-) cotransporter was expressed in astrocytes within cortex, corpus callosum, hippocampus and cerebellum. In addition, co-localization of the cotransporter and glial fibrillary acidic protein (GFAP), or with aquaporin 4, was found in perivascular astrocytes of cortical cortex and white matter. The results indicate that a time-dependent expression of the Na(+)-K(+)-Cl(-) cotransporter protein occurs not only in cortex but also in hippocampus, striatum, thalamus and cerebellum. In addition, the cotransporter is expressed in astrocytes and perivascular astrocytes of adult rat brain.

Neuroprotection by memantine, a non-competitive NMDA receptor antagonist after traumatic brain injury in rats.

This study investigated whether memantine, a non-competitive NMDA receptor antagonist is neuroprotective after traumatic brain injury (TBI) induced in adult rats with a controlled cortical impact device. TBI led to significant neuronal death in the hippocampal CA2 and CA3 regions (by 50 and 59%, respectively), by 7 days after the injury. Treatment of rats with memantine (10 and 20 mg/Kg, i.p.) immediately after the injury significantly prevented the neuronal loss in both CA2 and CA3 regions. This is the first study showing the neuroprotective potential of memantine to prevent the TBI-induced neuronal damage.