| Seizure induced by Ropivacaine central nervous system toxicity is common toxicant adverse reaction. Infants and children may be at increased risk form local anesthetics compared with adults because of their special anatomy and development feature. The previous study confirm that immature rats undergoing long term or recurrent seizure, it can effect the synaptic plasticity, learning and memory.It is the key time for development of synapses, nerve cell differentiationin migration, and fast formation of dendritic synapse in infants. Many investigations describe that other factors induced recurrent or long time seizures induced defect of learing and memory, its mechanism including ion channel, excitatory amino acid (EAA) receptor, neurotransmitter,development of synapse,the p protein synthesis, gene expression, and signal transduction. It is still uncertain if seizure induced by LAs can damage the development of nervous system, learning, and memory.It has been repoted about LAs toxicity seizure in the early1920century. Much has been done about the mechanism and prevention and cure by anesthetists. The mechanism of LAs toxicity seizure including the unbalance of EAA-IAA, NMDA-Ca2+-NOS, dopamine, acetylcholine, M-potassium channels et al. All above receptors, neurotransmitter, ion channels participant the development and maintenance of CNS, synapses, leaning, and memory.Hippocampus is the most important place of learning, and memory, and also the place which LAs toxicity seizure take place.Learning and memory is an higher nervous activity of brain. The two nervous activity intercommunication and influence about learning and memory. Learning means human or biology can change its performance in response to its environment, and memory means the imformation storage and output by learning activity. The nerves base of learning and memory is high plasticity of CNS, its including neural network connection, the plastic relative neurotransmitter, protein, ion channels, and receptors.Synapses is the junction point of shape structure and functional about neuron, it’s the key site of maintenance about information transport. The synapse plastic means when changing of external environment, the synapse take a adaptive change to keep the relative stable condition, the changing including form structure and function changing. The studay consider after recurrent or long time seizures, the synapses form structure was destruction in hippocampal area, the changing impact function of nervous, and its destroy level are positively correlate with learning and memory performance. The studay about LAs CNS toxicity describe that LAs inhibit synapses axon growth, induce nerve impulse attenuated, and impairment the physiologic function.Synaptophysin is a calcium binding glucoprotein which located in presynaptic membrane of axon peripheral. Synaptophysin participate in the release procedure of ACH, glutamic acid,et al, it has a close relation with synaptic plastics. Synaptophysin is a specificity protein of synaptic vesicle, its density and distribution reflect synapsis amount, and distribution. Synaptophysin has a close relation with synaptic restitution and recognition. Synaptophysin can regulate the formation, development, and mature of neurons, and also effect differentiation of neurons by regulate the cytoskeletal protein pack. Synaptophysin has a considerable meaning to axon lengthen, synaptic linkage, and synaptic maturation. The opposite research describe that it is not necessary of synaptophysin to synaptic development, learning, and memory, and it is still uncertain of its mechanism.Ca2+-CaMK Ⅱ-CREB path is one of the main signal of formation synaptic plasticity and learning memory of CNS. CaMK Ⅱ is a protein has a the highest content in the hippocamp, it’s a binding protein of Ca2+ion, has a close correlation of LTP, it can also be called"molecular switch of learning, and memory". Lidocaine. cocaine, and procaine can inhibit LTP in hippocampal by effect of calmodulince. LAs induced CNS toxicity seizure cause calcium overload, if it can effect CaMK Ⅱ, synaptic plasticity, learning, and memory.cAMP-responsive element binding protein (CREB) is a intracellular transcicription factor, which mediates the genetic transcription and the proteinous synthesis, plays a important role in the long term memory. It effect CREB phosphorylation induced learning,memory impairment in with acute or repeat treatment of cocaine.We determine ED95of0.5%Ropivacaine which the dose induced CNS toxicity seizure with postnatal21days(P21) mice, and preparation single and repeated seizure mice model. The learning,memory ability test by Morris water maze (MWM) at time after seizure. Synaptic ultrastructure in hippocamp after seizure transmission electron microscope Immunohistochemical staining and western blotting methods were used in the assays of the expressive levels of synaptophysin in CA1area of hippocamp. Fluorescent quantitation polymerase chain reaction(Q-t PCR) technique was used in the assays of the exprwssive levels of CaMK Ⅱ and p-CREB gene. We detect if LAs CNS toxicity seizure will effect the synaptic plasticity, learning and memory of immature mice, through test ethology; morphology,protein, and gene level, and discuss the possible mechanism about it.ObjectiveWe determine the ED50, ED95of ropivacaine induced seizure through up-down and unit probability regression method.MethodsForty healthy P21Sprague-Dawley (SD) rats. Using sodium chloride configuration0.5%Ropivacaine. Experiment dose divide to5groups, the geometric proportion coefficient is0.8, and the initial dose is24mg/kg. A rat accept intraperitoneal injection only one time. The seizure standard according to Racine V degree (righting reflex loss, generalized tonic-clonic seizures).grade0, normal behavior,1, facial twitches; wet dog shakes, arres t;2, head nodding, chewing;3, forelimb clonus;4, rearing, falling on forelimbs;5, falling on the side or back, hindlimb clonus. The rats seizure degree achieve V bring into experiment.Termination of seizure by means of twitch disappear and recover of self-action(righting reflex recover). Performance according to the up-down method: The first rat treatment with0.5%Ropivacaine intraperitoneal injection dose is24mg/kg, if it appearance seizure,and the next rat will accept the down regulation dose of Ropivacaine, but if the first rat did not appearance seizure, and the next rat will accept the upper regulation dose. Record positive, negative, and all cases in per dose group. Record the latency and duration of rat that undergone seizure.statistics analysisAll the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). The unit probability regression method (probit) was used toanalyze ED50and ED95of seizure induced by ropivacaine toxicity.ResultsThe seizure rats were characterized by free-running decreased, restlessness, run in a hurry aimless, loss of right reflex and generalized tonic-clonic convulsions. From inject time to occurrence of seizure(latency) is4.7±0.94min, and from occurrence to terminal of seizure(duration) is26.4±6.0min. According probit method calculated, equation y=-17.89±12.78(1g dose), the ED50dose is25.12mg/kg(95%confidence interval is22.38-27.54mg/kg). and the ED95dose is33.79mg/kg (95%confidence interval is29.99-48.38mg/kg).ConclusionAccording probit method calculated, the ED50dose is25.12mg/kg(95%confidence interval is22.38-27.54mg/kg), and the ED95dose is33.79mg/kg (95%confidence interval is29.99-48.38mg/kg).ObjectiveUsing the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. The learning,memory ability test by Morris water maze (MWM) at time after seizure.MethodsThirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment. The thirty rats were randomized into three groups (n=10):control group(Cgroup), single seizure group (R group), and recurrent seizure group (FR group). N group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The learning,memory ability test by Morris water maze (MWM) at time (1h～3d;5d～7d; P60～P62) after seizure in each group.Test content①Place navigation, it used to test the learning and memory ability of the rats. The rats swim2min in the pool before the starting formal experiment. The formal test experience three days,and test1time at am and pm per day. Putting the rats into water with face toward barrel wall at different point, the software recorded the swimming image of the rats(the residence time is5s), and analyzing the swimming path, velocity, and latency. If the rats could not find the platform in120s, the operater guide the rats to the platform ang stady for5s, and recording the latency was120s.②Spatial probe:It used to test the space memory ability, spatial probe carried out at the time after thelast place navigationtest, removing the platform, putting the rats at different point into water recording the times of accrosing and the swimming time in the quadrantic which the platform was located.(4d,8d, and P62),statistics analysisAll the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). Repeat measurement F test was ued to analyze escape latency. One-Way ANOVA was used to analyze the latency at different time in different groups or in different groups at the same time. One-Way ANOVA was used to analyze the times of accrosing and the swimming time in the quadrantic which the platform was located. After the equal check of variance the two-two comparisons among the means were done by LSD method. ResultsD1-3Search platform latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C, R and FR groups by train time increased (F=225.09, P<0.001; F=152.614, P<0.001; F=46.603, P<0.001). There were significant deviation of latency time in C, R and FR groups on day1-3(F=7.371, P<0.001; F=111.73, P<0.001; F=172.096, P<0.001). The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C vs R, C vs FR groups at day1-2(P<0.05); and there was significant deviation of latency time between C vs FR, R vs FR groups at day3(P<0.05);Day4spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day4(F=43.246, P<0.001), and there was significant deviation of time accrosing in C vs R, and R vs FR groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=129.646, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not C vs R groups (P<0.05)D5～7Search platfonn latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C and R groups by train time increased (F=14.75, P<0.001:F=17.194, P<0.001),but not in FR group(F=2.777, P>0.05). There were significant deviation of latency time in C, R and FR groups on day5～7(F=169.044, P<0.001; F=235.824, P<0.001; F=285.290, P<0.001). The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C, R vs FR, but not C vs R groups at day5-7(P<0.05). Day8spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day8(F=57.474, P<0.001), and there was significant deviation of time accrosing in C and R vs FR, but not C vs R groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=124.692, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not C vs R groups (P<0.05)P60～P62Search platform latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C, R and FR groups by train time increased(F=16.718, P<0.001; F=20.580, P<0.001; F=14.214, P<0.001)There were significant deviation of latency time in C, R and FR groups on day P60-P62((F=177.218, P<0.005; F=353.348, P<0.001; F=269.125, P<0.001) The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C, R vs FR, but not C vs R groups at day P60-P62(P<0.05). Day P62spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day8(F=25.327, P<0.001), and there was significant deviation of time accrosing in C and R vs FR, but not C vs R groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=120.061, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not N vs R groups (P<0.05)ConclusionIt can effect the learning and memory ability transiently with single seizures induced by Ropivacaine, but the long-term effect by repeated seizures. ObjectiveUsing the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the synaptic morphology in the hippocampal CA1area by transmission electron microscope.MethodsThirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP. which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.The thirty rats were randomized into three groups (n=12):control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days, I/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=3) according to the24h,3d,7d, and P60after covulsion or injection respective.According the time, perfusing the rats with paraformaldehyde and glutaric dialdehyde mixed liquor. After that taking out the hippocampus quickly and putting the glutaric dialdehyde. Using the transmission electron microscope to observe the synaptic cleft, synaptic number, and PSD.statistics analysisAll the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way ANOVA was used to analyze the synaptic cleft, synaptic number, and PSD at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.Results The synaptic number in R group at time24and3d, was less than C group at the same time. And the synaptic number in FR group at time24h,3d and7d, was less than C group at the same time (P<0.05). The synaptic cleft in R and FR groups were reduced than it in the C group at time24and3d (P<0.05).The PSD in R and FR groups were reduced than it in the C group at time24(P<0.05). The synaptic number, synaptic cleft and PSDin Both R and FR groups were the same as in the C at time P60.ConclusionRopivacaine-induced seizures effect the synaptic morphology in the hippocampal CAI area. The synaptic number reduced, synaptic clef widen and PSD thinningz atearlier period (24h,3d, and7d)ObjectiveUsing the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the synaptophysin in the hippocampal by Immunohistochemical stain and western blotting method.MethodsThirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.The thirty rats were randomized into three groups (n=24):control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=6) according to the24h,3d,7d, and P60after covulsion or injection respective.According the time, perfusing the rats with paraformaldehyde liquor. Another rats without perfusing to obtain western blotting sample.After that taking out the hippocampus Using t Immunohistochemical stain and western blotting method to observe the synaptophysin content at different time.statistics analysisAll the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way ANOVA was used to analyze the synaptophysin content at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.ResultsSynaptophysin content expression by Immunohistochemical stainThe synaptophysin content expression in R group was less than it in C group at time24h (P<0.05), and it expression in FR group was less than it in C group at time24h,3d, and7d (P<0.05). Both it in R and FR were same as C group at P60. Synaptophysin content expression by western blottingThe synaptophysin content expression in R group was less than it in C group at time24h (P<0.05), and it expression in FR group was less than it in N group at time24h,3d, and7d (P<0.05). Both it in R and FR were same as N group at P60.ConclusionThe synaptophysin content expression was reduced transiently at time3d after single seizure induced bu Ropivacaine, and it continue reduced until7d after repeat seizures.The synaptophysin content were same as normal in both single and repeat seizures at P60. ObjectiveUsing the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the CaMKⅡand p-CREB in the hippocampal by Fluorescent quantitation polymerase chain reaction technique and Western Blot..MethodsOne hundred and twenty rats healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.The one hundred and twenty rats were randomized into three groups (n=40): control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=10) according to the24h,3d,7d, and P60after covulsion or injection respective.CaMK Ⅱ and p-CREB up, down-stream primer were synthesis. According the time, to obtain hippocampus sample. extracting the sumRNA, reverse transcription, PCR amplification. Using the quantitive method analysis gene expression.statistics analysisAll the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way AN OVA was used to analyze the CaMK Ⅱ and p-CREB at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.ResultsCaMK Ⅱ gene expression The CaMK Ⅱ gene content expression in R group was less than it in C group at time24h and3d (P<0.05), and it expression in FR group was less than it in C group at time24h,3d,7d, and P60.(P<0.05) p-CREB gene expressionThe p-CREB gene content expression in R group was same as it in C group at time24h,3d,7d, and P60; and it expression in FR group was less than it in C group at time24h,3d,7d, and P60.(P<0.05)ConclusionThe CaMK Ⅱ gene expression content expression was reduced transiently at time3d after single seizure induced by Ropivacaine, and it continue reduced until P6o after repeated seizures.The p-CREB gene content expression were same as normal in single group at all the time, but the expression were all reduced significantly in repeated group at all the time. |
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