TheAssociationoftheNeuronalProtectiveEffectofCerebralIschemicPreconditioningwithConcentrationsofExtracellularAminoAcidsinRats1.doc

精品論文大集合the association of the neuronal protective effect of cerebral ischemic preconditioning with concentrations of extracellular amino acids in rats1zhang min, li wenbin, mei heshan, wang yongli, li lihebei medical university, shijiazhuang (050017)e-mail: abstractthe present study was undertaken to observe changes in concentrations of glutamate, aspartate and-aminobutyric acid (gaba) in the ca1 hippocampus during the acquirement of brain ischemic tolerance induced by cerebral ischemic preconditioning (cip) using cerebral microdialysis and high performance liquid chromatography techniques in rats. an initial, moderate and synchronous increasein glutamate, aspartate and gaba was first evoked by the lethal ischemic insult for 8 min which wasverified to induce delayed neuronal death (dnd) in the ca1 hippocampus. gaba quickly recovered to and kept at control level after reperfusion, but both glutamate and aspartate levels increased secondly in more magnitudes within the early stage of reperfusion. when the animals were pretreated 2 days before the lethal ischemic insult with a cip for 3 min which protected the pyramidal neurons against dnd normally induced by the lethal brain ischemia, the second peaks of glutamate and aspartate was prevented completely. the results suggested first in vivo that the protective effect of the cip against dnd of pyramidal neurons in the ca1 hippocampus normally induced by lethal brain ischemia might be associated with suppressing the increase in extracellular concentrations of both glutamate and aspartate.keywords: cerebral ischemic preconditioning; microdialysis; glutamate; aspartate; gaba1. introductiontransient sublethal cerebral ischemia could protect hippocampal neurons against delayed neuronal death (dnd) induced normally by lethal ischemic insult. the transient cerebral ischemia is usually referred to as cerebral ischemic preconditioning (cip). the phenomenon was first found by kitagawa et al 1 and proved by many other studies 2, 3. the mechanisms underlying the protective effect of cip on neurons are yet not completely understood although a variety of evidence involved in the mechanisms have been provided such as immediate early genes and their proteins 4, adenosine receptors and atp-sensitive k+ channels 5, nitric oxide 6, heat shock proteins7, mitogenactivated protein kinase (mapk) family members 8, etc.many studies indicated that changes in brain extracellular concentrations of amino acids such as glutamate, aspartate and -aminobutyric acid (gaba) play an important role in some pathological processes including epilepsy, stroke, and other neurodegenerative disorders 9, 10. glutamate is a major excitatory amino acid (eaa) neurotransmitter in the mammalian brain. glutamate uptake is transiently reduced and the extracellular glutamate is increased after hypoxia-ischemia insults in the brain 11, 12. aspartate is another eaa, and released along with glutamate in certain excitatory pathways in the rat hippocampus. aspartate level was found to be significantly increased by cerebral ischemia 11, 13. the excessive glutamate and aspartate in cerebral ischemia triggered a cascade of cellular and molecular events such as calcium overload, excessive production and release of no and free radicals which then induce neuronal degeneration or death including necrosis and dnd 14, 15. gaba is a major inhibitory transmitter and considered as putatively protective substance. continuous increase in extracellular gaba level in the hippocampus after stroke was important for neuronal survival and thus attenuated ischemic damage16.considering the involvement of these amino acids in neuro-damages or neuro-protections, we1 本課題得到國(guó)家自然科學(xué)基金(30770738),教育部博士點(diǎn)基金(20050089001),河北省自然科學(xué)基金(c200500720),河北省自然科學(xué)基金(c2008001042)的資助。- 15 -presumed that changes in concentrations of these amino acids may be associated with the protection of cip on neurons. to the best of our knowledge, no direct evidence supporting the above presumption in vivo has been provided up to now. therefore, the aim of the present study was to observe changes in concentrations of extracellular glutamate, aspartate and gaba in the ca1 hippocampus during the induction of brain ischemic tolerance by cip in rats using cerebral microdialysis and high performance liquid chromatography techniques. these findings can improve the knowledge involved in the development of brain tolerance to ischemic insults.2. materials and methods2.1animal and groupingtwenty four male wistar rats (280-320 g in weight) were provided by the experimental animal center of hebei medical university. before experiment, the animals were fed in the experimental room for 2-3 days to habituate to the environment of the laboratory. animal care and use conformed to guidelines for care and use of laboratory animals. the rats were divided randomly into sham, cip, brain ischemic insult, and cip + brain ischemic insult groups (n=6 in each group).2.2establishment of global brain ischemic modelthe rat global brain ischemic model was established by four-vessel occlusion 17. first, the bilateral vertebral arteries were electrocauterized under chloral hydrate anesthesia (350 mg/kg) administered by abdominal injection as following. an incision of skin, 1.5 cm in length, directly overlying the first cervical vertebra was made on the dorsal side of the neck behind the occipital bone. the paraspinal muscles were separated from the middle, and the bilateral alar foramina of the first cervical vertebra were exposed. an electrocautery needle burned in advance was inserted into the alar foramen and the bilateral vertebral arteries were electrocauterized permanently. silver ball electrodes were mounted on the parietal bone to record the electroencephalogram (eeg). after a recovery for 2 days from the operations, the bilateral common carotid arteries (bccas) of the rats were occluded to produce global brain ischemia. briefly, the bccas of the rats were exposed under ether anesthesia and local anesthesia with 1% procaine solution. after the rats recovered from the ether anesthesia, the bccas were clamped using clips. an occlusion in a short period of 3 min was normally used as cip, or a relative long one for 8 min was used as ischemic insult, which is lethal for ca1 pyramidal neurons and usually results in dnd. when the cip was followed by the 8 min ischemic insult, the interval between them was 2 days. changes in pupils and eeg were observed to determine whether the brain ischemia was induced. global brain ischemia was considered to be successful only in cases of animals in which the pupils enlarged, the eeg showed decreases in frequency and amplitude or even approached isoelectric level, and the righting reflex disappeared during the four-vessel occlusion.the wounds were sutured after each operation. the body temperature of the animals was kept at approximately 37 c by irradiating the whole body of animals with a heating lamp during the above operations and treatments until the rats returned to normal.the rats in sham group were subjected to all procedures for the global brain ischemia except for occlusion of the bccas.2.3preparation of artificial cerebrospinal fluid (acsf)the acsf (ph 7.4) was prepared according to methods described by thmen 18, which contained 125 mm nacl, 4 mm kcl, 2 mm (cacl22h2o), 1.14 mm (mgso47h2o), 1.29 mm kh2po4, and 25 mm nahco3. the ascf was stored at 4c. before using, 5.3 mg glucose was added in every 5 ml ascf.vao and igsi2-day intervalcip for 3 min7-day interval2.4recovery experiments of the microdialysis probe in vitrobefore inserting the microdialysis probe into the brain, the recovery rate for each probe was determined. in brief, the probe connected via polyethylene tube to a syringe selector, and to an 1 ml syringe mounted on a bee syringe pump (mf-9090, bas company, usa) was immersed into the calibration solution containing 39.06 m glutamate, 19.53 m aspartate and 39.06 m gaba. then, the probe was perfused with the acsf at a constant rate of 2 l/min by a bee syringe pump. the outflow of acsf passed the probe was collected in a period of 20 min and stored at 80 c for later analyzing the recovery rate by high performance liquid chromatography (hplc) system. the recovery rate in vitro was used for normalizing the recovery rate of microdialysis probe in vivo.2.5brain microdialysisthe experiments were carried out on conscious, freely moving rats. bas beekeeper system for awake animals (md-1576, bas company, usa) and br-2 brain microdialysis probes were used for the microdialysis. first, an intracerebral guide and stylet (md-2250, bas company, usa) was implanted into the dorsal hippocampus of the rats. the head of the rats was secured on a stereotaxic frame (sas-4100, asi instrument company, usa) under anesthesia of chloral hydrate (350 mg/kg, i.p.). according to the atlas of paxinos and watson (1997), a hole was made on the parietal bone 4 mm caudal to the bregma and 1.7 mm lateral to the midline using a high-speed dental drill (xl-30w, osada company, usa). after the dura was carefully incised, the intracerebral guide and stylet was inserted vertically into the ca1 subfield of the dorsal hippocampus (1.5 mm ventral to the surface) and fixed to the skull with dental cement. after recovery from the anesthesia, animals were returned to individual cages and allowed access to food and water ad libitum. the rats then were used for furtherexperiments after a recovery for 2 days. the details of the protocols were indicated in figure 1.vao and igsi2-day intervalbcca exposure7-day intervalsham groupcip groupi i groupcip+i i groupvao and igsi2-day intervalii for 8 min7-day intervalvao and igsi2-day intervalcip for 3 min2-day intervalii for 8 min7-day intervalmicrodialysisneuropathologic evaluationstabilization period120 minsample collection250 minprobe insertion60 min 30 min160 mins1s3 s4s13s14s21figure 1 experimental protocol for each group. the lower inset shows the details for brain microdialysis and sample collection. briefly, after a stabilization period of 120 min from the insertion of the microdialysis probe intothe ca1 hippocampus, the dialysate were collected. dialysate were first collected every 20 min for 1 h to detect control levels of the amino acid (sample 1 to sample 3 in order). from the beginning (pointed out by the arrow head) of the bcca exposure in sham group or the last time of the bcca occlusion in other groups, the samples were collected consecutively for 190 minutes as follows. the outflow of dialysate during a period of 3 min was collected as a sample and total 10 samples (sample 4 to sample 13 ) were gained within the initial 30 min. afterward, the outflow of dialysate during a period of 20 min was collected as a sample and total 8 samples (sample 14 to sample 21) were gained within the following 160 min. abbreviations: vao, bilateral vertebral artery occluding; igsi, intracerebral guide and stylet inserting; bcca, bilateral common carotid arteries; cip, cerebral ischemic preconditioning; ii, lethal ischemic insult; s1s3, sample 1 to sample 3; s4s13, sample 4 to sample 13; s14s21, sample 14 to sample 21on the brain microdialysis time as shown in figure 1, the microdialysis probe was inserted into the rat ca1 region of the dorsal hippocampus through the chronically implanted intracerebral guide under ethylether anaesthesia to prevent animals from distress or pain 3 h before the bcca exposure in the sham group or the last time of brain ischemia in the other groups. before the insertion, the integrity of the probe was checked and recovery rate was tested in vitro as mentioned above. after the insertion, the probe was continuously perfused with the acsf at a constant rate of 2 l/min by a bee syringe pump. after a stabilization period of 120 min, the dialysate were collected to an eppendorf tube by a refrigerated fraction collector (mf-9096, bas company, usa). the detail protocols for the collection of dialysate were shown in figure 1. briefly, dialysate were collected every 20 min for 1 h, which was named as sample 1 to sample 3 in order, to detect control amino acid levels before the bccas exposing in the sham group or the last time of the bccas occlusion in the other groups. from the beginning of the bccas exposing or the last time of the bccas occlusion, the samples were collected consecutively for 190 minutes as follows. the outflow of dialysate during a period of 3 min was collected as a sample and total 10 samples (sample 4 to sample 13) were gained within the initial 30 min. afterward, the outflow of dialysate during a period of 20 min was collected as a sample and total8 samples (sample 14 to sample 21) were gained within the following 160 min. all dialysate samples were kept at 80 c until analysis.after microdialysis, the rats were returned to the cages and allowed access to food and water ad libitum. all animals were sacrificed 7 days after the exposure of the bcca or the last time of brain ischemia for neuropathologic evaluation (details showing in figure 1).2.6neuropathological evaluationto check the position of the microdialysis probe and neuropathological changes, neuropathological evaluation was performed. briefly, on the 7th day after the exposure of the bcca or the last time of brain ischemia, the animals were deeply anesthetized with 10% chloral hydrate and perfused through the ascending aorta with normal saline and followed by 4% paraformaldehyde. the brain was then removed and a 3 mm-thick brain slice including the bilateral hippocampus was excised coronally behind the optical chiasm. the brain slice was sectioned coronally on a freezing microtome in a thickness of 20 m after post fixation with the same fixative. the sections were stained with thionin for verification of the probe placement and neuropathological evaluation under light microscopy. only dialysate samples collected from rats in which the probe was verified to locate in the ca1 region of the dorsal hippocampus were used for analysis.the histological changes of the hippocampal ca1 subfield were divided into four grades (hg) according to the methods described by kitagawa et al 1 and kato et al 19. the standard of the division was as follows: grade 0, no neuron death; grade i, scattered single neuron death; grade ii, mass neuron death; grade iii, almost complete neuron death. the neuronal density (nd) of the hippocampal ca1 subfield was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus, and clear nucleolus within 1 mm linear length in the ca1 hippocampus. theaverage number of pyramidal neurons in six areas of the hippocampal ca1 subfield was calculated as the value of nd.2.7amino acid determinationthe concentrations of glutamate (glu), aspartate (asp) as well as gaba in the dialysates were analyzed by a high performance liquid chromatography (hplc) system.2.7.1 mobile phasethe mobile phase a contained 20 mm naac3h2o buffer (ph 7.2), including 0.018% triethylamine (tea) and 0.3% tetrahydrofuran (thf). the mobile phase b consisted of 20% 100 mm naac3h2o (ph 7.2), 40% methyl alcohol and 40% acetonitrile.2.7.2 preparation of o-phthaldialdehyde (opa)five mg opa (sigma company, usa) was dissolved in 150 l methanol, followed by adding 5 l mercaptoethanol and 1.1 ml boric acid buffer (0.4 m, ph 9.6). the opa solution was saved away from light at 4 c and added 2 l mercaptoethanol every day. the opa solution was used not more than one week.2.7.3 preparation of the reference solutionthe internal reference was norvaline (nor) solution (250 m). soliatary references of each amino acid solution were prepared in a concentration of 10 mm glutamate, 5 mm aspartate, and 10 mm gaba, respectively. the solvent of all the reference solutions was purified water that was filtered by0.45 m filter membrane. each solution was subpackaged and kept at -80 c for future use.2.7.4 detection methods liquid chromatography conditionsagilent 1100 hplc system with fluorescent detector was used for assaying concentrations of glu, asp and gaba in the dialysate samples. the system consisted of an quaternary pump (g1379a, agilent company, usa) with a degasser unit, an auto sample injector (g1313a, agilent company, usa), and a multi wavelength fluorescence detector (g1321a, agilent company, usa). the detector was operated at excitation wavelength of 340 nm and emission at 450 nm. separation was carried out by a 20-mm 3.9-mm sentry guard column (c18 bonded silica) (dikma technologies) connected to a5 m brave bds c18 column (250 mm 4.6 mm i.d., alltech associates, inc.). the concentrations of the 3 kinds of amino acids were detected synchronously with a gradient elution program (table 1). the flow rate of thetable 1 gradient elution program for high performance liquid chromatography systemstepstime (min)mobile phase a (%)mobile phase b (%)1082182206436325406043201005350100stop time: 35min; post time: 10minfigure 2 a representative chromatogram of high performance liquid chromatography for internal reference norvaline, and the mixed references of glutamate, aspartate and gaba. abbreviations: asp, aspartate; glu, glutamate; gaba, -aminobutyric acid; nor, norvaline.mobile phase was 1 ml/min. two l of the reference solution or microdialysate to be detected, 2 l norvaline solution, and 4 l opa was imb