Delayed and Prolonged Local Brain Hypothermia Combined with Decompressive Craniectomy: A Novel Therapeutic Strategy That Modulates Glial Dynamics

Hypothermia is considered a useful intervention for limiting pathophysiological changes after brain injury. Local hypothermia is a relatively safe and convenient intervention that circumvents many of the complications associated with systemic hypothermia. However, successful hypothermia treatment requires careful consideration of several factors including its practicality, feasibility, and associated risks. Here, we review the protective effects-and the cellular mechanisms that underlie them-of delayed and prolonged local hypothermia in rodent and canine brain injury models. The data show that the protective effects of therapeutic hypothermia, which mainly result from the modulation of inflammatory glial dynamics, are limited. We argue that decompressive craniectomy can be used to overcome the limitations of local brain hypothermia without causing histological abnormalities or other detrimental effects to the cooled area. Therefore, delayed and prolonged local brain hypothermia at the site of craniectomy is a promising intervention that may prove effective in the clinical setting.

Blood-Brain Barrier Experiments with Clinical Magnetic Resonance Imaging and an Immunohistochemical Study

OBJECTIVE: The purpose of study was to evaluate the feasibility of brain magnetic resonance (MR) images of the rat obtained using a 1.5T MR machine in several blood-brain barrier (BBB) experiments. METHODS: Male Sprague-Dawley rats were used. MR images were obtained using a clinical 1.5T MR machine. A microcatheter was introduced via the femoral artery to the carotid artery. Normal saline (group 1, n = 4), clotted autologous blood (group 2, n = 4), triolein emulsion (group 3, n = 4), and oleic acid emulsion (group 4, n = 4) were infused into the carotid artery through a microcatheter. Conventional and diffusion-weighted images, the apparent coefficient map, perfusion-weighted images, and contrast-enhanced MR images were obtained. Brain tissue was obtained and triphenyltetrazolium chloride (TTC) staining was performed in group 2. Fluorescein isothiocyanate (FITC)-labeled dextran images and endothelial barrier antigen (EBA) studies were performed in group 4. RESULTS: The MR images in group 1 were of good quality. The MR images in group 2 revealed typical findings of acute cerebral infarction. Perfusion defects were noted on the perfusion-weighted images. The MR images in group 3 showed vasogenic edema and contrast enhancement, representing vascular damage. The rats in group 4 had vasogenic edema on the MR images and leakage of dextran on the FITC-labeled dextran image, representing increased vascular permeability. The immune reaction was decreased on the EBA study. CONCLUSION: Clinical 1.5T MR images using a rat depicted many informative results in the present study. These results can be used in further researches of the BBB using combined clinical MR machines and immunohistochemical examinations.

Accumulated Mannitol and Aggravated Cerebral Edema in a Rat Model of Middle Cerebral Artery Infarction

OBJECTIVE: Repeated administration of mannitol in the setting of large hemispheric infarction is a controversial and poorly defined therapeutic intervention. This study was performed to examine the effects of multiple-dose mannitol on a brain edema after large hemispheric infarction. METHODS: A middle cerebral artery was occluded with the rat suture model for 6 hours and reperfused in 22 rats. The rats were randomly assigned to either control (n=10) or the mannitol-treated group (n=12) in which intravenous mannitol infusions (0.8 g/kg) were performed six times every four hours. After staining a brain slice with 2,3,5-triphenyltetrazolium chloride, the weight of hemispheres, infarcted (IH) and contralateral (CH), and the IH/CH weight ratio were examined, and then hemispheric accumulation of mannitol was photometrically evaluated based on formation of NADH catalyzed by mannitol dehydrogenase. RESULTS: Mannitol administration produced changes in body weight of -7.6+/-1.1%, increased plasma osmolality to 312+/-8 mOsm/L. It remarkably increased weight of IH (0.77+/-0.06 gm versus 0.68+/-0.03 gm : p<0.01) and the IH/CH weight ratio (1.23+/-0.07 versus 1.12+/-0.05 : p<0.01). The photometric absorption at 340 nm of the cerebral tissue in the mannitol-treated group was increased to 0.375+/-0.071 and 0.239+/-0.051 in the IH and CH, respectively from 0.167+/-0.082 and 0.162+/-0.091 in the IH and CH of the control group (p<0.01). CONCLUSION: Multiple-dose mannitol is likely to aggravate cerebral edema due to parenchymal accumulation of mannitol in the infarcted brain tissue.

Gene Expression Profile in Microglia following Ischemia-Reperfusion Injury

Microglial activation is thought to play a role in the pathogenesis of many brain disorders. Therefore, understanding the response of microglia to noxious stimuli may provide insights into their role in disorders such as stroke and neurodegeneration. Many genes involved in this response have been identified individually, but not systematically. In this regards, the microarray system permitted to screen a large number of genes in biological or pathological processes. Therefore, we used microarray technology to evaluate the effect of oxygen glucose deprivation (OGD) and reperfusion on gene expression in microglia under ischemia-like and activating conditions. Primary microglial cultures were prepared from postnatal mice brain. The cells were exposed to 4 hrs of OGD and 1 h of reperfusion at 37 degrees C. Isolated mRNA were run on GeneChips. After OGD and reperfusion, > 2-fold increases of 90 genes and > 2-fold decrease of 41 genes were found. Among the genes differentially increased by OGD and reperfusion in microglia were inflammatory and immune related genes such as prostaglandin E synthase, IL-1beta, and TNF-alpha. Microarray analysis of gene expression may be useful for elucidating novel molecular mediators of microglial reaction to reperfusion injury and provide insights into the molecular basis of brain disorders.

Microarray Analysis of Oxygen-Glucose-Deprivation Induced Gene Expression in Cultured Astrocytes

Since astrocytes were shown to play a central role in maintaining neuronal viability both under normal conditions and during stress such as ischemia, studies of the astrocytic response to stress are essential to understand many types of brain pathology. The microarray system permitted screening of large numbers of genes in biological or pathological processes. Therefore, the gene expression patterns in the in vitro model of astrocytes following exposure to oxygen-glucose deprivation (OGD) were evaluated by using the microarray analysis. Primary astrocytic cultures were prepared from postnatal Swiss Webster mice. The cells were exposed to OGD for 4 hrs at 37 degrees C prior to cell harvesting. From the cultured cells, we isolated mRNA, synthesized cDNA, converted to biotinylated cRNA and then reacted with GeneChips. The data were normalized and analyzed using dChip and GenMAPP tools. After 4 hrs exposure to OGD, 4 genes were increased more than 2 folds and 51 genes were decreased more than 2 folds compared with the control condition. The data suggest that the OGD has general suppressive effect on the gene expression with the exception of some genes which are related with ischemic cell death directly or indirectly. These genes are mainly involved in apoptotic and protein translation pathways and gap junction component. These results suggest that microarray analysis of gene expression may be useful for screening novel molecular mediators of astrocyte response to ischemic injury and making profound understanding of the cellular mechanisms as a whole. Such a screening technique should provide insights into the molecular basis of brain disorders and help to identify potential targets for therapy.

Gene Expression Analysis of Murine Primary Microglia Stimulated with LPS using Microarray

BACKGROUND: Since heightened microglial activation was shown to play a role in the pathogenesis of many brain disorders, understanding the molecular mechanisms of microglial activation may lead to new treatment strategies. The microarray system permitted screening of large numbers of genes in biological or pathological processes. Therefore, we evaluated the gene expression pattern during microglial activation using microarray analysis. METHODS: Primary microglial cultures were prepared from postnatal Swiss Webster mice. The cells were activated by lipopolysaccharide (LPS, 10 microgram/ml) for 5 hours prior to cell harvesting. From the cultured cells, we isolated mRNA, synthesized cDNA, converted to biotinylated cRNA and then reacted with GeneChips (Affymetrix MU74A-v2). The data were normalized and analyzed. RESULTS: After microglial activation with LPS, we found >4 fold increases in the expression of 139 genes and >4 fold decreases of 16 genes expression compared with control. Most of the induced or suppressed genes were known to regulate inflammation, immune reactions, injury responses, cell death or survival related mechanisms. CONCLUSIONS: These results suggest that microarray analysis of gene expression may be useful for screening novel molecular mediators of microglial activation and making profound understanding of the cellular mechanisms as a whole. Such screening techniques should provide insights into the molecular basis of brain disorders and help to identify potential targets for therapy.

Effect of Minocycline on Activation of Glia and Nuclear Factor kappa B in an Animal Nerve Injury Model

Glial cells are activated in neuropathy and play a key role in hyperalgesia and allodynia. This study was performed to determine whether minocycline could attenuate heat hyperalgesia and mechanical allodynia, and how glial cell activation and nuclear factor kappa B (NF-kappaB) were regulated by minocycline in a model of chronic constriction of sciatic nerve (CCI). When minocycline (50 mg/kg, oral) was daily administered from 1 day before to 9 days after ligation, heat hyperalgesia and mechanical allodynia were attenuated. Furthermore, when minocycline treatment was initiated 1 or 3 days after ligation, attenuation of the hypersensitive behavior was still robust. However, the effect of attenuation was less when minocycline was started from day 5. In order to elucidate the mechanism of pain attenuation by minocycline, we examined the changes of glia and NF-kappaB, and found that attenuated hyperalgesia and allodynia by minocycline was accompanied by reduced microglial activation. Furthermore, the number of NF-kappaB immunoreactive cells increased after CCI treatment and this increase was attenuated by minocycline. We also observed translocation of NF-kappaB into the nuclei of activated glial cells. These results suggest that minocycline inhibits activation of glial cells and NF-kappaB, thereby attenuating the development of behavioral hypersensitivity to stimuli.

Partial Outlet Obstruction Induced Alterations of Rat Urinary Bladder Muscle / 대한해부학회지

Partial outlet obstruction induced by benign prostatic hypertrophy or urethral stricture are common urologic conditions showing voiding dysfunction. This study was performed to compare the effects of short-term partial outlet obstruction on the contractile responses to different stimuli and to elucidate the mechanism of altered contractile response during the development of significant voiding dysfunction. Sprague-Dawley rats, 10 week old, were subjected to partial outlet obstruction for 7 days. Contractile responses of the urinary bladder detrusor muscle strips to electrical field stimulation (1, 2, 5, 10 and 20 Hz), KCl (50 and 100 mM) and carbachol (10(-9 to 5) M) were measured. Total protein and DNA contents of the bladder were measured. Connexin 43 mRNA expression was measured using RT-PCR and connexin 43 protein was observed in the fixed bladder tissue using immunohistochemistry. The electrical field stimulation-induced contractile response and KCl-induced contraction was not changed after partial outlet obstruction. Contraction induced by carbachol was enhanced by the partial outlet obstruction. Total protein and DNA contents were increased in the partial outlet obstruction group. Connexin 43 mRNA and protein expression were detectable in the normal bladders and increased after 7 days of obstruction. These results suggest that the altered contractile responses during the early stage of the partial outlet obstruction are the result of the changes of the contraction mechanisms or structure of bladder muscle. Connexin 43 may play an important role in those alterations.

Effect of Minocycline on Nociceptive Change and Microglial Activation in a Rat Model of Sciatic Neuropathy / 대한해부학회지

Nerve injury leads to chronic neuropathic pain syndromes. Activation of microglia has been studied to investigate the role in pain development. Minocycline is known as a potent inhibitor of microglial activation in many types of the brain injury models. But it is not known whether minocycline interferes with pain and microglial activation after the peripheral nerve injury. In this study, we investigated the time course of pain and microglial activation after sciatic nerve injury and also tested the effect of minocycline using sciatic nerve ligation model. All experiments were performed using 150~180 g male Sprague-Dawley rats. The chronic constriction injury (CCI) of the sciatic nerve with four 4.0 chromic gut suture was used to induce neuropathic pain in the left sciatic nerve. The behavioral response of rats to the stimuli (heat, cold & pressure) was assessed by measuring the lifting of the foot and the avoidance of touching the floor at pre-surgical day 1, post-surgical day 1, 4, 7, and 10. The L4 ~6 spine was fixed and used to detect microglia. Oral minocycline (50 mg/kg) was administered daily to the last day of the experiment. Minocycline was administered to one group of rats from pre-surgical day 1 and minocycline treatment was initiated from post-surgical day 1, 3, and 5 in other groups. Neuropathic pain was evident from day 4 and the peak response was observed at 10 days after CCI. Minocycline significantly attenuated neuropathic pain even when treatment was delayed by 3 days. But, it had no effet when treatment initiated 5 days after injury. Minocycline also attenuated microglial activation. In summary, a correlation was evident between the neuropathic pain and microglial activation in our model and minocycline reduced both development of pain and microglial activation. Thus, minocycline can be a good candidate for the treatment of neuropathic pain. However, the administration should be initiated prior to microglial activation.

Effect of Mild Hypothermia on the Mitogen Activated Protein Kinases in Experimental Stroke

Middle cerebral artery occlusion (MCAO) results in cell death by activation of complex signal pathways for cell death and survival. Hypothermia is a robust neuroprotectant, and its effect has often been attributed to various mechanisms, but it is not yet clear. Upstream from the cell death promoters and executioners are several enzymes that may activate several transcription factors involved in cell death and survival. In this study, we immunohistochemically examined the phosphorylation of mitogen- activated protein kinase, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase during early period of the ischemic injury, following 2 hours (h) of transient MCAO. Increased phosphorylation of ERK and p38 was observed in the vessels at 3 h, neuron-like cells at 6 and 12 h and glia-like cells at 12 h. Activation of JNK was not remarkable, and a few cells showed active JNK following ischemia. Phosphorylation of Elk-1, a transcription factor, was reduced by ischemic insult. Hypothermia attenuated the activation of ERK, p38 and JNK, and inhibited reduction of Elk-1. These data suggest that signals via different MAPK family members converge on the cell damage process and hypothermia protects the brain by interfering with these pathways.

Effect of Age on Proliferation of Rat Vascular Smooth Muscle Cells Stimulated with Fetal Bovine Serum, Angiotensin II and Phorbol 12-Myristate 13-Acetate / 대한해부학회지

The present study investigated effects of fetal bovine serum (FBS), angiotensin II (Ang II) and phorbol 12 -myristate 13 -acetate (PMA) on proliferation of vascular smooth muscle cells (VSMC) isolated from aorta of 5, 9, 12 and 17 week old Sprague -Dawley rats. With 10% FBS, proliferation rate of VSMCs was markedly greater in 17 week old group than in 5 week old group. Treatment of 0.1 ~10 micro M Ang II or 10 and 100 micro M PMA in 0.1% bovine serum albumin and 10% FBS media did not influence the cell proliferation. However, in 5% FBS media, Ang II and PMA enhanced the proliferation of the VSMC from all age groups except 5 week. To test the effects of Ang II in the FBS, captopril, an Ang converting enzyme inhibitor, and ibersartan, an Ang II receptor antagonist, were pretreated. Neither 10 micro M captopril nor ibersartan in 5% FBS influenced the VSMC proliferation. These results indicate that the VSMCs from older rats proliferate faster than those from 5 week -old rats, and 5% FBS -induced proliferation was enhanced by Ang II and PMA.

Characterization of cytoplasmic Form of Human CTLA - 4 Molecule / 대한면역학회지

CTLA-4 (=CD152), a T cell activation antigen, has been known to be homologous to CD28 in its molecular and genomic structure. Both of these two molecules are sharing their counterreceptors, B7 (CDSO) and B7-2 (CD86) and are known to play a crucial role in T cell activation. In previous our study it was reported that there are 2 forms of CTLA-4 antigen in activated human T cells, 30 kD membrane-bound form and 34 kD cytosolic-sequestered form and the former was less than 5 % of total of this antigen induced. Aims of this study are to confirm previous finding by using flow cytometry and to characterize the cytoplasmic form of human CTLA-4 by using ultrafiltration and immunoprecipitation techniques. In PHA stimulated peripheral blood lymphocyte surface expression of CTLA-4 was less than 2.1% of any of CD4+, CD8+ and CD56+ subsets. And the 34 kD form of CTLA-4 was detected in CDS+ subset only. This discrepancy confirms that 34 kD antigen is the cytoplasmic form of human CTLA-4. In ultrafiltration and subsequent Western blot analysis study this 34 kD antigen was detected in >100 kD fraction only. And in non-reducing condition this antigen formed high molecular weght complex (MW > 350 kD). In immunoprecipitation study using anti-peptide A antibody it was found that this high molecular weight complex consists of the 34 kD cytoplasmic form of CTLA-4 and previously unknown 54 kD antigen and 46 kD antigen at 1:1:8-10 ratio. And none of these 3 molecules were identified in membrane fraction of activated human T cell. The result of this study implies that CTLA-4 molecule induced upon T cell activation mainly sequestered in cytoplasrn and another signal is necessary to target this antigen on the activated T cell surface.