Characterization of metal element distributions in the rat brain following ischemic stroke by synchrotron radiation microfluorescence analysis

Ischemic stroke is one of the leading causes of death worldwide,and effective treatment strategies in the chronic phase of this disease remain insufficient.Homeostasis of metals in the brain plays an important role in maintaining normal brain function.However,the dynamic spatial distributions of iron,zinc,calcium,potassium,and copper in a rat brain following ischemic stroke and the association between structural distribution and function remain to be elucidated.In this study,we used a synchrotron radiation-based micro-X-ray fluorescence technique to image element mapping changes in special rat brain regions after ischemic stroke,showing the distribution characteristics of iron,zinc,calcium,potassium,and copper.We demonstrated,for the first time,the consistent dynamic spatial distributions of metal elements at a series of time points(3 h,4.5 h,6 h,12 h,1 d,3 d,5 d,7 d,10 d,14 d,28 d)after brain ischemia,which revealed that the homeostasis of iron,zinc,calcium,potassium,and copper in the brain was disturbed with distinctive change trends,providing clear insights in understanding the underlying pathogenesis of stroke from a novel perspective,thus laying the foundation of further developing new drug targets for stroke treatment.

Thermoacoustic tomography of in vivo rat brain

We present for the¯rst time in vivo imaging of rat brain using microwave-induced thermoacoustic tomography(TAT).The in vivo imaging of rat brain was realized through an unconventional delivery of microwave energy from the front of rat brain(while the transducer was scanned along coronal plane of the animal brain),which maximized the microwave penetration into the brain.In addition,we found that the imaging contrast was highly dependent on the direction of the electric¯eld polarization(EFP)and that more tissue structures/compositions could be revealed when both X-and Y-EFPs were used for TAT.The in vivo TAT images of rat brain obtained were compared with the 3.0 T MRI images and histological photographs,and numerous important brain anatomical structures were identi¯ed.An example of our TAT approach for imaging a foreign object embedded in a rat brain was also demonstrated.This study suggests that TAT has a great potential to be used in neuroscience studies and in noninvasive imaging of brain disorders.

Inhibition of the Specific ~3H-DL-Glu Binding in the Hippocampus of Rat Brain by Lead

The effect of Pb2+ on 3H-DL-Glutamate (3H-DL-Glu) binding in the membrane preparations from the hippocampus of rat brain was investigated with a view to explaining the cognitive and learning deficits produced by the heavy metal. The results indicated that Pb2+ (3. 1-25. 0 μmol.L -1 ) inhibited 3 H-DL-Glu binding in a concentration-dependent manner . Scatchard analysis further revealed that at a concentration of 6. 3 μmol.L-1 Pb2 + interfered with binding mainly through significantly decreasing the density of binding sites. This finding provided an important insight into Pb2+ -induced impairments in learning and memory previously documented in children and in experimental animals chronically exposed to Pb^(2+)

Distribution of phosphorylated Elk-1 in rat brain after Y-maze active avoidance training in a temporal manner

BACKGROUND： Elk-1 mRNA distributes extensively in the neurons of mice, rat and human brains, and the Elk-1 expression may be correlated with the synaptic plasticity, learning and memory. OBJECTIVE： To observe the distribution of phosphorylated Elk-1 （pEIk-1） in whole brain of rats received Y-maze active avoidance training and the changes of pEIk-1 expression at different time points after training. DESIGN ： A randomized controlled study SETTING ： Research Room of Neurobiology, the Second Affiliated Hospital of Southern Medical University MATERIALS ： Fifty-five male clean-degree SD rats of 3-4 months old, weighing 200-250 g, were provided by the Experimental Animal Center of Southem Medical University. The rabbit anti-monoclonal pEIk-1 antibody was purchased from Cell Signal Transduction Company, and ABC kit from Vector Company. METHODS ： The experiments were carried out in the Research Room of Neurobiology, Second Affiliated Hospital of Southern Medical University from September 2004 to February 2005. ① Grouping： The rats were randomly divided into training group （n = 25）, sham-training group （n = 25） and normal control group （n = 5）, and the training and sham-training groups were observed at 0, 1, 3, 6 and 24 hours after training, which represented the five phases in the process of leaming and memory. ② Y-maze training： The rats were preconditioned in the electrical Y-maze apparatus, 20 minutes a day for 3 days continuously, and training began from the 4^th day. In the training group, the rats were trained with the combination of light and electddty. Each rat repeated for 60 times in each training, and the correct times were recorded, those correct for less than 25 times were taken as unqualified, and excluded from the training group, and supplemented by other rats in time. In the sham-training group, there was no fixed correlation between the application of light and electricity. The rats in the normal contrel group were given not any training. ③Detection of pEIk-1 expression： The rats were anesthetized after Y-maze training, brain tissue was removed to prepare coronal freezing sections, and the pEIk-1 expression was detected with routine ABC method. MATN OUTCOME MEASURES： ① Distribution of pEIk-1 immuno-positive neurons in whole brain of rats in the normal control group. ②Comparison of the expression of pEIk-1 immuno-positive neurons in whole brain at different time points after training between the training group and sham-training group. RESULTS ： All the 55 rats were involved in result analysis. ③ Distribution of pEIk-1 immuno-positive neurons in the whole brain of rats in the normal control group： Strong expressions of pEIk-1 immuno-positive neurons were observed in prefrontal lobe, granular layer of olfactory bulbs, Purkinje cell layer and granular layer of cerebellum, whole stdate cortex, temporal cortex, pre-pyriform cortex, hypothalamic supraoptic nucleus, hypothalamic paraventricular nucleus and periventricular nucleus, thalamic paraventricular nucleus, pronucleus and postnucleus of amygdala cortex, central nucleus of amygdala, medial amygdaloid nucleus, entorhinal cortex, hippocampal dentate gyros, CA1-4 regions, caudate-putamen, material division, brain stem spinal nucleus of trigeminal nerve, and superior olivary nucleus, and those in hippocampal dentate gyrus and CA1 region were the strongest.② Distribution of pEIk-1 immuno-positive neurons in the whole brain of rats at different time points after training in the training group and sham-training group： In the training group, the expressions were obviously enhanced in caudate-putamen of striatum, material division, most cortexes, hippocampal dentate gyrus, hippocampal CA regions, nucleus amygdalae, thalamic paraventricular nucleus, Purkinje cell layer of cerebellum, entorhinal cortex, hypothalamic supraoptic nucleus, hypothalamic paraventricular nucleus, and periventricular nucleus at 0 hour after training, and the enhancement lasted for 6 hours at least, and those at 24 hours were decreased to normal. In the sham-training group, obvious enhanced expressions of pEIk-1 immuno-positive neurons could be observed in most cortexes, nucleus amygdalae, entorhinal cortex, hypothalamic supraoptic nucleus, hypothalamic paraventdoular nucleus and periventricular nucleus, brain stem spinal nucleus of trigeminal nerve, Purkinje cell layer and granular layer of cerebellum at O, 1, 3 and 6 hours, and decreased to normal after 24 hours. The expressions in material division, caudate-putamen of striatum, hippocampus were not obviously enhanced as compared with those in the normal control group, but significantly different from those in the training group （0, 1, 3, 6 hours after training, material division： F= 0.576, 0.023, 0.116, 8.873, P〈 0.01; caudate-putamen： F= 0.157, 0.427, 0.030, 0.001, P〈 0.01; hippocampus： F= 6.716, 2.405, 14.137, 1.416, P 〈 0.05-0.01 ）. CONCLUSION： The expression of activated pEIk-1 can be detected in the learning related brain areas under normal status, and the perk-1 expression in the brain areas dynamically changed in a time-dependent manner after Y-maze training, and it is indicated that pEIk-1 is involved in the learning and memory process in Y-maze related brain areas.

In vitro release of 1,3-bis(2-chloroethyl)-1-nitrosourea sustained-release microspheres and the distribution in rat brain tissues

BACKGROUND: The implantation of released chemotherapeutic drugs, which takes biodegradable polymer as vector, into the tumor site can get high concentration and release the drug for a long time, it can directly act on the tumor cells, and reduce the general toxicity. OBJECTIVE: To explore the in vitro and in vivo course of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sustained-release from BCNU-loaded polylactide (PLA) microspheres (MS) and location in rat brain tissue. DESIGN: A repetitive measurement. SETTING:Central Pharmacy, General Hospital of Chinese People’s Armed Police Forces. MATERIALS: Thirty male SD rats were used. PLA (Mr5000, batch number: KSL8377) was produced by Wako Pure Chemical Inc.,Ltd. (Japan); BCNU (batch number: 021121) by Tianjin Jinyao Amino Acid Co., Ltd.; BCNU-PLA-MS was prepared by the method of solvent evaporation and pressed into tablets (10 mg/tablet). High-performance liquid chromatography (HPLC) Agilent 1100 (USA); LS9800 liquid-scintillation radiometric apparatus (Beckman). Chromatographic conditions: Elite Hypersil ODS2 C18 chromatographic column (5 μm, 4.6 mm×150 mm); Mobile phase: methanol: water (50:50), flow rate was 1.0 mL per minute, wave length of ultraviolet detection was 237 nm, and the inlet amount of samples was 10 μL. METHODS: The experiments were carried out in the experimental animal center of the General Hospital of Chinese Armed Police from May 2004 to July 2005. ① In vitro BCNU-PLA-MS release test: BCNU-PLA-MS was prepared by the method of solvent evaporation, then placed in 0.1 mol/L phosphate buffered solution (PBS, pH 7.4, 37 ℃), part of MS were taken out at 1, 2, 3, 7, 10 and 15 days respectively, and the rest amount of BCNU in MS was determined by HPLC, then the curve of BCNU-PLA-MS release was drawn. ②In vivo BCNU-PLA-MS release and distribution test: The rats were anesthetized, then BCNU-PLA-MS were implanted to the site 1 mm inferior to the cortex of frontal lobe. Five rats were killed postoperatively at 4 hours, 1, 2, 3, 7 and 15 days, the residual MS was removed from the brain tissue. The rest amount of BCNU was determined with HLPC, and the curve of BCNU-PLA-MS release was drawn as compared with the amount of BCNU in the implanted tablets. Besides, brain tissues (1 g) at the implanted side and the contralateral one were obtained respectively, blood sample (0.5 mL) was also collected, 3H-BCNU was counted radioactively in radioactive liquid flash solution. The distributions of BCNU-PLA-MS in normal rat brain tissue and serum were detected. The analysis of variance was applied to compare the intergroup differences of the measurement data. MAIN OUTCOME MEASURES: ① Characteristics of BCNU-PLA-MS release in phosphate buffered solution (PBS) and rat brain tissue; ② Distributions of BCNU-PLA-MS in normal rat brain tissue and serum. RESULTS: ① Release of BCNU-PLA-MS in PBS and rat brain tissue: The BCNU released from BCNU-PLA-MS could be sustained for over 2 weeks both in PBS and brain tissue. In PBS, the released rate of BCNU was over 15% at 24 hours, nearly 50% at 72 hours and over 90% at 15 days. In brain tissue, the released rate was 8% at 4 hours, 16% at 24 hours, 60% at 72 hours, respectively, and BCNU could be sustained released for over 15 days. ② Distributions of BCNU-PLA-MS in normal rat brain tissue and serum: The concentrations of BCNU in the ipsilateral brain tissue were 6 to 70 times higher than those in the contralateral one. The concentrations of BCNU in the ipsilateral brain tissue were obviously higher than those in serum and contralateral brain tissue (F =103.47, P < 0.01). CONCLUSION: BCNU-PLA-MS can increase the drug concentration in targeted brain tissue, decrease that in the non-targeted brain tissue, reduce general toxic and side effects, and have good releasing function.

EXTRACTION AND ISOLATION OF PROTEOGLYCANS FROM RAT BRAIN

This paper reports a comparative study of the extraction rate of rat brain proteoglycans (PGs) by three different methods, with chromatography, papain digestion and electrophoretic technique. The results showed: ① The extraction rate of brain PGs by 4mol/L guanidine HCl (GuHCl)was higher than that by phosphate-buffered saline (PBS) In any method, however the protein/PGs ratio in the GuHCl-extract was lower than that in the PBS-extract. ② PBS mainly extracted the soluble chondroitin sulfate proteoglycan (CSPG), whereas the 4mol/L GuHCl could extracted both soluble CSPG and insoluble heparan sulfate proteoglycan (HSPG). ③ After delipidation of brain by organic reagents, the extraction rate of delipidized brain PGs either by the PBS or by the 4mol/L GuHCl decreased obviously. ④ By direct extraction with PBS, GuHCl seguentially, few amount of PGs in the residue from brain was found.

A ggregation-induced emission nanoparticles for in vivo three-photon fuorescence microscopic rat brain angiography

Rodents are popular biological models for physiological and behavioral research in neuroscience and rats are better models than mice due to their higher genome similarity to human and more accessible surgical procedures.However,rat brain is larger than mice brain and it needs powerful imaging tools to implement better penetration against the scattering of the thicker brain tissue.Three-photon fluorescence microscopy(3PFM)combined with near-infrared(NIR)excitation has great potentials for brain circuits imaging beause of its abilities of anti scattering,deep-tissue imaging,and high signal-to-noise ratio(SNR).In this work,a type of AIE lumninogen with red fuorescence was synthesized and encapsulated with Pluronic F-127 to make up form nano-particles(NPs).Bright DCDPP-2TPA NPs were employed for in trino three-photon fuorescent laser scanning microscopy of blood vessels in rats brain under 1550 nm femtosecond laser exci-tation.A fine three-dimensional(3D)reconstruction up to the deepness of 600 pm was achieved and the blood flow velocity of a selected vessel was measured in vrito as well.Our 3PFM deep brain imaging method simultaneously recorded the morphology and function of the brain blood vessels in vivo in the rat model.Using this angiography combined with the arsenal of rodent's brain disease,models can accelerate the neuroscience research and clinical diagnosis of brain disease in the future.

Effects of Lead on pH and Temperature-Dependent Substrate-Activation Kinetics of ATPase System and its Protection by Thiol Compounds in Rat Brain

Lead (Pb) inhibited the activities of Na+ -K+ ATPase (IC50= 2.0×10^(-6) M), K + -Para-Nitrophenyl phosphatase (PNPPase) (IC50= 3.5×10^(-6) M) and [3H]-ouabain binding (IC50 = 4.0×10^(-5) M) in rat brain P2 fraction. A variable temperature or pH significantly elevated the inhibition of Na+-K+ ATPase by Pb in buffered acidic, neutral and alkaline pH ranges. Noncompetitive inhibition with respect to activation of Na+ -K+ ATPase by ATP was indicated by a variation in Vmax values with no significant changes in Km values at any temperature studied. In the presence of Pb, for Na+ -K+ ATPase at pH 6.5 and 8.5, Vmax was decreased with an increase in Km values suggesting a mixed type of inhibition. Sulfhydryl agents such as dithiothreitol (DTT) and cvsteine (Cyst), but not glutathione (GSH) offered varied levels of protection against Pb-inhibition of Na + -K+ ATPase at pH 7.5 and 8.5. The present data suggest that inhibition of Na+ -K+ ATPase by Pb is both temperature and pH-dependent. These results also indicate that Pb inhibited Na + -K + ATPase by interfering with phosphorylation of enzyme molecule and dephosphorylation of the enzyme-phosphoryl complex and exerted an effect similar to that of SH-blocking agents.

Validation of a HPLC Method for Quantification of Thiamine and Its Phosphate Esters in Rat Brain Tissue

The present data show a fast and efficient biological sample processing method for the extraction of thiamine (vitamin B1) and its mono-(TMP) and di-(TDP) phosphate esters from hippocampus, thalamus and prefrontal cortex (PFC) and blood sample of the rodents. In addition, using the hippocampus and standards of these three compounds we validated an isocratic fluorescence HPLC procedure for a simultaneous detection of them in a single chromatogram within a total run time of about 12 min. Reproducibility for TDP, TMP and B1 was 2.66%, 4.50% and 7.43% (intraday) and 37.54%, 25.39% and 25.87% (interday), respectively. Recovery assays were between 96.0% and 101.7%. The calibration curves were linear and the concentrations of the three compounds, all in nanomolar range, were determined in the brain areas and in the blood samples. When compared to the current methods in the literature, this new method provides information on essential variables, such as linearity range and limit of detection, reproducibility and stability of thiamine, TMP and TDP in rat brain samples. The present data on sample processing and B1 and its phosphate ester level determinations are the first to be validated using hippocampus samples of rats.

Effects of Calcium on the GABA_A-Coupled CI^-/HCO₃^--ATPase from Plasma Membrane of Rat Brain

The work is a study of the influence of Ca2+ (0.01 - 1 mM) on neuronal CI-, HCO3-, -ATPase complex: an enzyme that is a CI--pump which is functionally and structurally coupled to GABAA-receptors. It is found that influence of Ca2+ on the multifunctional complex starts at concentration of 50·M and at concentration of 0.1 mM, it reduces the “basal” one and increases the CI-, HCO3-, -stimulated Mg2+-ATPase activities. GABA (0.1 - 100μM) activates the “basal” Mg2+-ATPase activity in the ab-sence of calcium. The effect of GABA on the enzyme in the presence of 0.01 ·M Ca2+ does not change. At the same time, 1 mM Ca2+eliminates the GABA effect on the “basal” Mg2+-ATPase activity. Competitive blocker of GABAA-receptors bicuculline (5 - 20 μM) in the absence of Ca2+ ions elimi-nates the stimulation of the “basal” Mg2+-ATPase by anions. When 0.25 mM Ca2+ is added to the in-cubation medium the inhibitory bicuculline effect on the enzyme does not appear. We found that 0.1 mM o-vanadate (protein tyrosine phosphatase blocker) reduces the GABA-activated ATPase activity. At the same time, 0.1 mM genistein (a protein tyrosine kinase blocker) has no effect on enzyme activity. In the presence of Ca2+ (0.25 mM), the effect of o-vanadate on the “basal” and CI-, HCO3-, -ATPase activities does not appear. It is shown for the first time that high concentrations of Ca2+prevent the action of GABAA-ergic ligands on the study ATPase. It is assumed that there is the involvement of protein kinases and protein phosphatases in the modulation of the enzyme activity by calcium. The observed effect of calcium on the ATPase may play an important role in the study of the mechanisms of epileptogenesis and seizure activity.

Effect of cyproheptadine on changes of 5-HT and 5-HIAA levels in brain cortex and serum following rat brain injury

Changes of 5-hydroxytryptamine(5-HT)and 5-hydroxyindoleacetic acid(5-HIAA)levels in brain cortex and serum were studied using spectrofluorometer follow-ing brain injury in rats.Cyproheptadine,a receptor antagonist of 5-HT,was employedand its effect on alterations of 5-HT and 5-HIAA contents in brain cortex and serum af-ter brain injury was investigated.The results showed that after brain injury levels of5-HT and 5-HIAA in the brain cortex increased markedly,The increase of 5-HT in thebrain cortex reached its peak at 48h after brain injury,and its value was 4.29±0.44μg/g,which was 4.8 times of control value.The increase of 5-HIAA in the brain cortexreached its peak at 24h after brain injury,and its value was 5.48±0.41μg/g,which was5.8times of control value.The change of 5-HT in the serum was not significant,but thelevel of 5-HIAA in the serum was increased significantly.Cyproheptadine could reducelevels of 5-HT and 5-HIAA in this experiment.The value of 5-HT in the brain cortexdecreased to 1.32±0.09μg/g,and the value of 5-HIAA decreased to 1.36±0.10μg/g.Thelevel of 5-HIAA in the serum was also reduced significantly.It was 0.27±0.03μg/ml.

HIGH FREQUENCY ELECTROACUPUNCTURE INDUCED CHANGES OF IP_3 LEVEL IN RAT BRAIN AND SPINAL CORD

In the present study, the radioreceptor binding method was used to determine the changes of IP3 content in rat brain and dorsal spinal cord of high frequency (100 Hz) electroacupuncture (EA) analgesia and EA tolerance rat. The control levels of IP3 in rat brain (less cerebellum and cortex) and dorsal spinal cord were 6.3± 0.78 pmol / mg protein and 3.4± 0.60 pmol / mg protein, respectively. The results showed that IP, in brain increased gradually within 45 min after stimulation in EA analgesia rat. Meanwhile, the dorsal spinal cord IP, content decreased significantly 15 min, 30 min after EA stimulation and recovered to control level 45 min after EA stimulation. In EA tolerance rat, IP3 contents markedly increased in brain. And IP3 content in the spinal cord also increased dramatically within 30 min, but decreased rapidly to control level 45 min after EA stimulation. The IP3 level in EA tolerance rat brain and spinal cord was much higher than that in EA analgesia rat (P<0.01). The studies first reported that high frequency (100 Hz) EA may be linked to PI system in its signal transduction pathways.

AN IMMUNOHISTOCHEMICAL STUDY OF PERIVASCULAR MACROPHAGES IN BRAINS OF SPONTANEOUSLY HYPERTENSIVE RATS

In order to investigate the possible role of macrophages in pathological changes of cerebrovascular diseases, we observed and counted the numbers of perivascular macrophages in brains of spontaneously hypertensive rats(SHR) and normotensive Wistar-Kyoto(WKY)rats using monoclon al antibodies(ED2)against rat macrophages.The immunohistochemistry(ABC method)and perfu sion-fixed frozen brain sections(16μm)were used in the study.The results showed that the ED2-specific staining in brain sections was restricted to macrophages in a perivascular location.The number of perivascular macrophages was significantly greater in SHR than in WKY.The results suggest that the hypertension is asseciated with increased subendothelial accumulation of macrophages.

Activation of endogenous neural stem cells in experimental intracerebral hemorrhagic rat brains

Background Many researchers suggest that adult mammalian central nervous system (CNS) is incapable of completing self-repair or regeneration. And there are accumulating lines of evidence which suggest that endogenous neural stem cells (NSCs) are activated in many pathological conditions, including stroke in the past decades, which might partly account for rehabilitation afterwards. In this study, we investigated whether there was endogenous neural stem cell activation in intracerebral hemorrhagic (ICH) rat brains.Methods After ICH induction by stereotactical injection of collagenase type Ⅶ into globus pallidus, 5-Bromo-2 Deoxyuridine (BrdU) was administered intraperitoneally to label newborn cells. Immunohistochemical method was used to detect Nestin, a marker for neural stem cells, and BrdU.Results Nestin-positive or BrdU-Labeled cells were predominantly located at 2 sites: basal ganglion around hemotoma, ependyma and nearby subventricular zone (SVZ). No positive cells for the 2 markers were found in the 2 sites of normal control group and sham group, as well as in non-leisoned parenchyma, both hippocampi and olfactory bulbs in the 4 groups. Nestin+ cells presented 4 types of morphology, and BrdU+ nucleus were polymorphologic. Postive cell counting around hemotoma showed that at day 2, Nestin+ cells were seen around hemotoma in model group , the number of which increased at day 4, day 7(P<0.01), peaked at day 14(P<0.05), and reduced significantly by day 28(P<0.01).Conclusion Endogenous neural stem cells were activated in experimental intracerebral hemorrhagic rat brains.

Lipid Peroxidation and Ultrastructural Modifications in Brain after Perinatal Exposure to Lead and/or Cadmium in Rat Pups

Objective To assess lipid peroxidation and ultrastructural modifications in rat brains following perinatal exposure to lead （Pb） and/or cadmium （Cd）. Methods Female rats were divided into four groups： control group, Pb （300 mg/L） group, Cd group （10 mg/L） and Pb＋Cd （300 mg/L, 10 mg/L） group. The compounds were delivered in the drinking water throughout pregnancy and lactation. Results The levels of compounds in blood and brain of the Pb＋Cd group were similar to those of other groups, but the effects of Pb＋Cd on pups＇ body and brain weights were higher than on other compounds. Electron microscopy revealed that Pb and Cd had effects on mitochondrial swelling, disruption and cristae loss, Nissl body dissolution, degenerated organelles and vacuoles, cytomembrane disappearance, and nuclear chromoplasm concentration. The activity of glutathione peroxidase （GSH-Px）, superoxide dismutase （SOD）, catalase （CAT）, acetylcholinesterase （ACHE） was decreased, whereas the activity of maleic dialdehyde （MDA） was increased. Conclusion Perinatal exposure to low doses of Pb and Cd can produce alterations in lipid peroxidation and ultrastructural modifications in rat brains, and exposure to both metals can result in greater damages.

Developmental study on the immunocytochemical localization of NOV protein-containing neurons in the rat brain

The localization and development of nephroblastoma overexpressed gene (nov) protein-immunoreactive neurons in the brain of E8-P300 rats have been studied using immunocytochemistry and image analysis. Results are as follows: No NOV protein-immunoreactive cells were detected in the rat brain during prenatal development. A few of positive cells were detected at the early postnatal stage. However, the number and the immunoreactivity of these cells increased gradually at later stages. NOV-immunoreactive cells were widely distributed in the rat brain during P30-P60. The number of immunoreactive cells and their intensity also peaked within this stage. The number and staining intensity of NOV-positive cells decreased gradually with age. The positive cells were mainly located in cingulate cortex, striatum, hippocampus, hypothalamus, cerebellum and brain stem. The present results indicate that nov may play an important role in the development and differentiation of brain as well as maintaining the function of

Correlation Study on Expression of GST-π Protein in Brain Tissue and Peripheral Blood of Epilepsy Rats Induced by Pilocarpine

Previous studies have suggested that glutathione-S-transferase π （GST-π） over-expression in the brain tissue is associated with refractory epilepsy. However, whether the change in GST-π level in the peripheral blood is in line with that in brain tissue remains unknown. This study examined the correlation between GST-π in brain tissue and that in peripheral blood in rat models of pilocarpine-induced refractory epilepsy. The animals were divided into drug-resistant group and drug-responsive group according to the response to anti-epileptic drugs. GST-π expression in brain tissue was immunohistochemically determined, while the expression of GST-π in peripheral blood was analyzed by Western blotting. In the hippocampus and cortex, GST-π was mainly found in the cytoplasm and membrane of neurons, and the GST-π expression level was higher in drug-resistant group than in the drug-responsive group and saline control group （P〈0.05）. Moreover, there was no significant difference between responders and saline control animals （P〉0.05）. The change in expression of GST-π in peripheral blood showed the same pattern as that in brain tissues, suggesting GST-π might contribute to drug resistance in epilepsy. Importantly, the GST-π over-expression in peripheral blood could be used as a marker for resistance to anti-epileptic agents.

Alterations in enterocyte mitochondrial respiratory function and enzyme activities in gastrointestinal dysfunction following brain injury

AIM:To determine the alterations in rat enterocyte mitochondrial respiratory function and enzyme activities following traumatic brain injury(TBI).METHODS:Fifty-six male SD rats were randomly divided into seven groups(8 rats in each group):a control group(rats with sham operation)and traumatic brain injury groups at 6,12,24 h,days 2,3,and 7after operation.TBI models were induced by Feendy’s free-falling method.Mitochondrial respiratory function(respiratory control ratio and ADP/O ratio)was measured with a Clark oxygen electrode.The activities of respiratory chain complexⅠ-Ⅳand related enzymes were determined by spectrophotometry.RESULTS:Compared with the control group,the mitochondrial respiratory control ratio(RCR)declined at 6 h and remained at a low level until day 7 after TBI(control,5.42±0.46;6 h,5.20±0.18;12 h,4.55±0.35;24 h,3.75±0.22;2 d,4.12±0.53;3 d,3.45±0.41;7 d,5.23±0.24,P<0.01).The value of phosphate-to-oxygen(P/O)significantly decreased at12,24 h,day 2 and day 3,respectively(12 h,3.30±0.10;24 h,2.61±0.21;2 d,2.95±0.18;3 d,2.76±0.09,P<0.01)compared with the control group(3.46±0.12).Two troughs of mitochondrial respiratory function were seen at 24 h and day 3 after TBI.The activities of mitochondrial complex Ⅰ (6 h:110±10,12 h:115±12,24 h:85±9,day 2:80±15,day 3:65±16,P<0.01)and complexⅡ(6 h:105±8,12 h:110±92,24 h:80±10,day 2:76±8,day 3:68±12,P<0.01)were increased at 6 h and 12 h following TBI,and then significantly decreased at 24 h,day 2 and day3,respectively.However,there were no differences in complex Ⅰ andⅡactivities between the control and TBI groups.Furthermore,pyruvate dehydrogenase(PDH)activity was significantly decreased at 6 h and continued up to 7 d after TBI compared with the control group(6 h:90±8,12 h:85±10,24 h:65±12,day 2:60±9,day 3:55±6,day 7:88±11,P<0.01).The changes inα-ketoglutaric dehydrogenase(KGDH)activity were similar to PDH,except that the decrease in KGDH activity began at 12 h after TBI(12 h:90±12,24 h:80±9,day 2:76±15,day 3:68±7,day7:90±13,P<0.01).No significant change in malate dehydrogenase(MDH)activity was observed.CONCLUSION:Rat enterocyte mitochondrial respiratory function and enzyme activities are inhibited following TBI.Mitochondrial dysfunction may play an important role in TBI-induced gastrointestinal dysfunction.

Altered blood-brain barrier permeability in rats with prehepatic portal hypertension turns to normal when portal pressure is lowered

AIM: To study the blood-brain barrier integrity in prehe-patic portal hypertensive rats induced by partial portal vein ligation, at 14 and 40 d after ligation when portal pressure is spontaneously normalized. METHODS: Adult male Wistar rats were divided into four groups: GroupⅠ: Sham14d, sham operated; GroupⅡ: PH14d, portal vein stenosis; (both groups were used 14 days after surgery); GroupⅢ: Sham40d, Sham operated and GroupⅣ: PH40d Portal vein stenosis (GroupsⅡandⅣused 40 d after surgery). Plasma ammonia, plasma and cerebrospinal fluid protein and liver enzymes concentrations were determined. Trypan and Evans blue dyes, systemically injected, were investigated in hippocampus to study blood-brain barrier integrity. Portal pressure was periodically recorded. RESULTS: Forty days after stricture, portal pressure was normalized, plasma ammonia was moderately high, and both dyes were absent in central nervous system parenchyma. All other parameters were reestablished. When portal pressure was normalized and ammonia level was lowered, but not normal, the altered integrity of blood-brain barrier becomes reestablished. CONCLUSION: The impairment of blood-brain barrier and subsequent normalization could be a mechanism involved in hepatic encephalopathy reversibility, Hemo-dynamic changes and ammonia could trigger blood-brain barrier alterations and its reestablishment.

Orphanin FQ antagonizes the analgesic effect of 5-HT in rat brain

Orphanin FQ (OFQ) is a new modulatory peptide which was found in 1994. It mediates in morphine analgesia and electroacupuncture analgesia. The interaction between OFQ and 5-hydroxytryptamine (5-HT) on analgesia was observed using the method of intracerebroventricular microinjection. The results showed that cumulative i. c.v. administration of gradually increasing doses of 5-HT produced a dose-dependent analgesia, and administration of different doses of OFQ separateness had no significant effect on tail flick latency, but 1 or 10 μg OFQ could reverse the analgesia induced by 5-HT, suggesting that OFQ could antagonize the analgesia induced by 5-HT in rat brain.