| Epilepsy is one of the most common chronic neurologic diseases definedas spontaneously and repetitively synchronous abnormal discharge of cerebralcortical neuron resulting in cerebral dysfunction. As the characteristic ofepilepsy, transience, paroxysmal, repetition, stereotype can be observed. It hasbeen reported that the morbidity is24-53%per1hundred thousand people indeveloped countries. According as the current data, there are about ninemillion patients in China where the mobidity is about35per1hundredthousand people with ascending trend year by year. Epileptic seizurescontribute to the loss of neurons, gliosis and subsequently the dysfunction oflocal and remote cerebral areas leading to the declined quality of life and eventhe death of patients. Recently, the pathophysiological mechanisms underlyingthe brain injury attributed to epilepsy have been further clarified resulting inthe emergence of some novel antiepileptic compounds; however, theoptimized therapeutic effect has not been achieved. The focus problems ofcurrent antiepileptic treatments are that the toxic and side effects of drugs, theconsiderable complications, the remedy when refractory and the relativelylack of methods for status epilepticus (SE).The second injury of brain, which involves the oxdative stress, theinflammatory reaction, apoptosis, intracellular calcium overload andexcitatory amino acids toxicity, is attributed to epileptic seizures andcontributes to not only the deteriorated condition but also the enhancement ofepileptogenesis. All of the pathophysiological changes interact resulting inpathological cascade reaction and regulatory network. Especially theexcitatory amino acids toxicity, oxidative stress and apoptosis acting in theprimary and non-primary epileptic focus play an important role in neuronaldysfunction. Accordingly, inhibition of the excitatory amino acids toxicity,anti-oxidative stress and discontinuation of apoptosis process are important ways to protect the brain from the injury associated with epilepsy.Dexmedetomidine (DEX), a high selective alpha2-adrenoceptor agonist,has been widely used in sadation and general/local anaesthesia. Reports haveasserted that DEX can inhibit the excessive release of excitatory amino acids,attenuate the oxidative stress and inflammatory reaction and discontinue theprocess of apoptosis. Meanwhile, researchers have concluded theanti-epiepsia effect of clonidine, another alpha2-adrenoceptor agonist. Thepossible mechanisms involve the pre and postsynaptic inhibition associatedwith Ca2+influx and excessive release of excitatory amino acids; however,clonidine is still unacceptable because of the simultaneous selection ofalpha1-adrenoceptor. About8times selectivity for alpha2-adrenoceptorcompared to clonidine, DEX is an appropriate choice for treatment ofepilepsy.In the current experiment, male healthy Wistar rats were chosen for theacute/chronic basal lateral amygadala (BLA) electrical kindling epilepsiamodels. According as Racine behavioral classification and electro-encephalogram (EEG), the severity and duration of epileptic seizure wererecorded to examine if it can be induced by BLA electrical stimulation. Theexcitatory amino acid-glutamate (Glu)/inhibitory amino acid-γ-aminobutyricacid (GABA), the glutathione (GSH)/malondiadehyde (MDA), the Bax/Bcl-2and the neuclear transcription factor-κB (NF-κB) in hippocampus tissuewere tested to observe the unbalance between excitatory and inhibitory aminoacids, the injury associated with the oxidative stress and the process ofapoptosis, as well as their changes after intraperitoneal injection of DEX. Allthe data would be analysised to assess the anti-epilepsy and brain protectiveefficacy of DEX and to explore the possible mechanisms. There were threeparts of the current study; each one of them was stated as below. PartⅠEstablishment of the acute and chronic epilepsy in rat BLAelectrical kindling modelObjective: To test the inductive effect of epilepsy under the differentBLA electrical stimulus parameter. The success rate of kindling, theafterdischarge threshold, the Racine’s classification and the EEG wereobserved and recorded to explore the differentiation of the influences underthe diffent parameters and to assess the values of them.Methods: Sixty male healthy adult Wistar rats weight250-350gramswere randomly devided into three groups. There were15rats in group1and3respectively, while30rats in group2were devided randomly and averagelyinto two subgroups again. Following the intraperitoneally injected chloralhydrate, the twisted enamel-covered nickel-cadmium electrode was implantedinto the left BLA (Bregma: anterior:2.8mm; lateral:4.8mm; ventral:8.5mm from skull surface) of the rat via brain stereotactic instrument. After oneweek recovery, the afterdischarge threshold (ADT), which was the minimumcurrent intensity (≤400μA) as the emergence of spike wave (duration≥3s),of each rat was detected. Subsequently, the rats were electrically stimulatedwith different kindling parameters. Experiment (1): the estabilishment ofchonic BLA kindling model. After the detection of ADT, the fifteen rats ingroup1were subject to BLA kindling program with a constent current (500μA), waviness width1ms, monophasic square-wave pulses, and60Hz for1s,once every day, during which the behavioral and EEG changes were recorded.The severity of seizure was assessed according to the Racine’s classification,and the rats were thought kindled successfully after consecutive5times of5stage seizures. The durations of afterdischarge were measured on the basis ofEEG. Experiment (2): the estabilishment of acute BLA kindling model. Thirtyrats were devided randomly and averagely into two subgroups after thedetection of ADT, group A and group B. Rats were subject to the BLAkindling program with the different parameters: group A, rats were kindledwith the constent current, intensity500μA, frequency16Hz, waviness width1.0ms, train length10.0s, biphasic square-wave pulse stimulation. Threr was7min interval among each stimulation, and rats were stimulated no more than2days with30times every day untile the consecutive3Racine5stage seizures were observed, which was thought the mark of kindled successfully.Group B, rats were kindled with the constent current, intensity500μA,frequency60Hz, waviness width1.0ms, train length10.0s, biphasicsquare-wave pulse stimulation. There was30min interval among eachstimulation, and rats were stimulated2days with10times every day untile theconsecutive3Racine5stage seizures were observed, which was thought themark of kindled successfully. Experiment (3): the establishment of a rat modelof self-sustaining SE (SSSE) with prolonged amygdala stimulating: after thedetectiong of ADT,15rats were kindled with the following paradigm: theconstent current, intensity500μA, frequency16Hz, waviness width1.0ms,train length1.0s, monophasic square-wave pulse stimulation. The rats werethought kindled successfully until consecutive5Racine5stage seizures. Aftera2weeks recovery period, the induction process of SSSE was performed asdescribed below: stimulus duration of25min and stimulus program consistingof100ms trains of1.0ms square-wave pulses. The trains were given at afrequency of2/s and the intra-train pulse frequency was50/s. Peak pulseintensity was700μA. Development and duration of SSSE were monitored for12h by EEG-recording.Results:1Chonic BLA kindling models: of15rats,14conformed to the standardof ADT (≤400μA) and were subject to the subsequent test. The rest one wasexcluded from experiment considering of the unmeasurable ADT associatedwith the incorrect placement of electrode. During the kindling process,1of14rats was excluded from test because of the deciduous electrode. Thirteen ratsfinished the process; however,12of them appeared the consecutive5Racine5stage seizure and were thought kindled successfully, the rest one had evershown Racine5stage seizure followed by descending seizure grades and wasthought failing to kindling. The time taken by kindling process was10-19days,the total successful kindling rate (successful kindling number vs total kindlingnumber) was85.7%, the actual successful kindling rate (successful kindlingnumber vs finished kindling number) was92.3%, and all of the rats were survival of the kindling process. Anytime after kindled, the same stimulationcan induce the Racine4-5stage seizure.2Acute BLA kindling models:30rats were devided randomly andaveragely into two groups, group A and group B. In group A,13of the15ratsconformed to the standard of ADT (≤400μA) and were subject to thesubsequent test, while2rats were excluded. The electrodes of2within the13rats were come off; however the rest11rats finished the kindling process, inwhich8rats showed consecutive3Racine5stage seizures, while3ratsshowed nonconsecutive5stage seizres or5stage followed by descendingseizure grades. The time taken by kindling process were1-2days, the totalsuccessful kindling rate was61.5%, the actual successful kindling ratewas72.7%. In group B,14of the15rats conformed to the standard of ADT(≤400μA) and were subject to the subsequent test, while1rat was excluded. Theelectrodes of4rats within the14rats were come off; however the rest10ratsfinished the kindling process, in which8rats showed consecutive3Racine5stage seizures, while2rats showed nonconsecutive5stage seizres. The timetaken by kindling process was2days, the total successful kindling rate was57.1%, and the actual successful kindling rate was80.0%.3The BLA electrical stimulated self-sustaining SE (SSSE) models:15rats conformed to the standard of ADT (≤400μA) and were subject to thesubsequent test. The electrodes of1rat within the14rats were come off;however the rest13rats finished the kindling process, in which13rats weresuccessfully kindled and subject to the subsequent induction process, while1rat failed to be kindled. The time taken by kindling process was10-19days,the total successful kindling rate was86.7%, and the actual successful kindlingrate was92.9%. After the inductiong of SSSE with prolonged25min BLAelectrical stimulation, all of the13rats showed self-sustaining intermittentRacine5seizures defined as SSSE, duration time5-12h. There were2ratsdied during SSSE, and the death rate was15.4%.Conclusion:The rat BLA electrical kindling epilepsia models can well imitate the pathophysiological progress of humen-bing temporal lobe epilepsy. Thecurrent data has demonstrated that the implanted electrode was safe andreliable without the death of the operated rats. During kindling process, thetime required for successfully kindled, the total successful kindling rate andthe actual successful kindling rate are significantly different among threegroups. In the chornic BLA kindling models, the time is about10-19days;however, the total successful kindling rate and the actual successful kindlingrate are significantly higher than the other two groups. In group A of the acuteBLA kindling models, the time is shorter than that of chronic models; however,the the total successful kindling rate and the actual successful kindling rate arenot so optimized. The group B has shown the same short time as group A, butmore lower the the total successful kindling rate. The actual successfulkindling rate of group B is statistically higher than group A, but still lowerthan the chronic models. In the BLA electrical stimulated SSSE models, all ofthe rats undergone the prolonged inductiong progress can show SSSE after thesuccessfully chronic BLA-kindling process, and the death rate during SSSE issimilar to human bing.PartⅡ Anticonvulsant effect of dexmedetomidine in a rat model ofself-sustaining status epilepticus with prolonged amygdalastimulationObjective: To explore the effect of DEX on the number and accumulatedtime of seizure in the rat model of SSSE with prolonged amygdale stimulation,and detect the level of Glu/GABA and GSH/MDA in hippocampus of rat afterSSSE to assess its anticonvulsant and cerebral protective action and discussthe involved mechanisms.Methods: Sixty-five adult male healthy Wistar rats were devidedrandomly and averagely into5groups, Group L (SSSE plus low dosage DEX):intraperitoneally DEX (50μg/kg)-injected and electrically stimulated animals(n=15); Group H (SSSE plus high dosage DEX): intraperitoneally DEX(100μg/kg)-injected and electrically stimulated animals (n=15); GroupSE (SSSE): electrically stimulated and non-DEX-injected animals (n=15);Group S (sham): electrode-implanted but non-DEX-injected and non-electrically-stimulated animals (n=10); and Group C (control): without anytreatment (n=10). The kindling and induction programs were performd asdescribed in part I. The rats in group L/H were intraperitoneally injected DEX50/100μg/kg5min after the successful inductiong of SSSE. The number andaccumulative time were recorded according to the EEG; the levels ofGlu/GABA and GSH/MDA in hippocampus tissure of rat collected12h afterthe emergence of SSSE were detected by the liquid chromatography-tandemmass spectrometry (LC-MS) and the enzyme-linked immunosorbent assay(ELISA).Results:1Threr are13rats in group L,11rats in group H, and15rats in SE werekindled successfully, and showed5-12h induced SSSE. Eventually,13rats inL,11rats in H and13rats in SE were survived after SSSE,2rats died ofSSSE in SE were excluded from the experiment (death rate=13.33%).Compared with group SE, the number and accumulative time in group L werenot statistically decreased (p>0.05); however, the ones in group H weresignifically decreased (p<0.05). There were not epileptic seizures in the rats ofgroup S and C.2In LC-MS. In the hippocampus tissures,12h after the SSSE, thecontents of Glu were increased in group L, H, and SE compared with group C(p<0.05). There was no difference between group S and group C (p>0.05), orbetween group L and SE (p>0.05). But significant decrease was observed ingroup H compared to that in group SE (p<0.05). The levels of GABA wereobviously increased in group L, H, SE compared with group C (p<0.05).There was no different between group S and C (p>0.05), or group L and SE(p>0.05). However, the level of GABA in group H was significantly decreasedthan that in group L and SE (p<0.05). The present data suggested that the levelof GABA was not decreased or increased corresponding to the dosage of DEX but to the level of Glu.3In ELSIA. In the hippocampus tissures,12after the SSSE, the contentsof GSH were decreased in group L, H, SE compared with group C (p<0.05).There was no difference between group S and C (p>0.05), or between group Land SE (p>0.05). The level of GSH in group H was still lower than that ingroup S and C; however, it was higher than that in group L and SE (p<0.05).Simultaneously, the levels of MDA were significantly increased in group L, H,and SE compared to the group C (p<0.05). There was no difference betweengroup S and C (p>0.05), or between group L and SE (p>0.05). But the levelwas significantly decreased in group H than that in group L and SE (p<0.05).The current data suggested that high dosage DEX (100μg/kg) effectivelyattenuated the oxidative stress associated with SSSE.Conclusions：After kindling process, SSSE can be successfully inducedin all rats with a25min prolonged stimulus program. Twelve hours afterSSSE, the levels of Glu/GABA and GSH/MDA in hippocampus tissure canbe increased/compensatively increased and decreased/increased, suggestingthe close relation between SSSE and the excitatory amino acid toxicity or theoxidative stress. The high dosage DEX can show anticonvulsant andbrain-protective effect by decreasing the number and accumulative time ofthe seizures during SSSE, the level of Glu and MDA, and increasing the levelof GSH.PartⅢ Anti-epilepsia effect of dexmedetomidine and the underlyingmechanism in rat chronic BLA electric kindling modelObjective: to observe the action of DEX on the duration and Racineclassfication in the chronic BLA kindling epileptic rat models, detect the levelof Bax/Bcl-2and NF-κB in the hippocampus tissue, assess the effect of DEXon the inflammatory and apoptosis responses associated with epilepsy, andexplore the involved mechanisms underlying the brain-protective effect ofDEX. Methods: Sixty-five male adult healthy Wistar rats were devided into5groups randomly and averagely. Group L (chronic BLA electric kindlingprocess plus stimulation after kindling plus low dosage DEX): chronickindling process followed by intraperitoneally DEX (50μg/kg)-injected andelectrically stimulated animals (n=15); Group H (chronic BLA electrickindling process plus stimulation after kindling plus high dosage DEX):chronic kindling process followed by intraperitoneally DEX(100μg/kg)-injected and electrically stimulated animals (n=15); Group E (chronic BLAelectric kindling process only): chronic kindling process followed byelectrically stimulated and non-DEX-injected animals (n=15); Group S (sham):electrode-implanted but non-DEX-injected and non-electrically-stimulatedanimals (n=10); and Group C (control): without any treatment (n=10). The ratsin group L, H, E were subject to continuing electrical stimulation once a dayafter successful BLA electrical kindling and the rats in group L/H wereintraperitoneally injected DEX50/100μg/kg every5min before stimulation.The Racine classification and ADD were recorded when the last stimulationfor the assessment of severity. At the1week after kindling, after the laststimulation, all rats in5groups were executed and the hippocampus tissueswere reserved for detection of Bax/Bcl-2and NF-κB.Results:1There were13rats in group L,13rats in group H, and12rats in groupE were kindled successfully, and no rats were dead during the kindling process.After kindled, all rats in group L, H, E were subject to electrical stimulationagain, once a day. The rats in group E showed stably5stage seizure and theADD was not significantly varied. After1week, the seizure degree and ADDof the rat in group L were not statistically different from that in group E(p>0.05). However, the seizure degree and ADD in group H were significantlydifferent from that in group H (p<0.05). There were no epileptic seizures inrats of group S and C.2In Western blot. One week after the electrical stimulation once a day,the levels of Bcl-2in group L, H, E were significantly decreased compared with group C (p<0.05) in the hippocampus tissure. There was no differencebetween group L and group E (p>0.05), or between group S and group C(p>0.05); however, the level in group H was significantly increased comparedto that in group E (p<0.05). The level of Bax in group L, H, E weresignificantly increased compared to group C (p<0.05). there were nodifference in the level of Bax between group L and E (p>0.05), or betweengroup S and C (p>0.05); however, the level in group H was significantlydecreased compared to that in group E (p<0.05).3In Western blot, after1week electrical stimulation once a day, thelevels of NF-κB in rat hippocampus tissues in group L, H, E weresignificantly decreased compare to group C (p<0.05). There was nodifference between group L and E (p>0.05), or group S and C (p>0.05);however, the level in group H was higher than that in group E (p<0.05).Conclusions：DEX can decreased the seizure degree and the ADD in thechronic BLA electrical kindling models, increased the expressed levels ofBcl-2and NF-κB and decreased the expressed level of Bax in rat hippocampustissure, suggesting that the DEX can attenuated the severity of chronicepileptic seizure model, and show the protective effect by anti-epileptic,anti-inflammatory and anti-apoptosis action. |
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