Experimental Research On The Effect Of Propofol On Spatial Learning And Memory In Rats

PART IEffect of Propofol on Spatial Learning and Memory in RatsObjective To investigate the effect of propofol on spatial learning and memory in rats.Methods 24 rats were randomly assigned to one of the following four groups (n=6): solvent control (intralipid i.p.) and different propofol groups (propofol 10mg/kg, 30mg/kg and 75 mg/kg i.p.). For five training trials, each rat started at one quadrant, on the opposite the platform, with the starting point being fixed through these training trials. 10 or 30mg/kg propofol or intralipid was administered intraperitoneally (i.p.) 20 min before the each training. When rats injected 75mg/kg propofol i.p. returned to a normal righting reflex for 20 min, training began. In each training trial, the latency required escaping onto the hidden platform and swimming path length were recorded. If the rat found the platform, it was allowed to remain there for 30s. If the rat was unable to find the platform within 180s, after it was madeto stay there for 30s, the training trial was terminated and maximum score of 180s was assigned. Training trials were given on 5 consecutive days, and rats underwent training trials each day. On the day after the final training trial, each rat was exposed to an additive training trails test after the starting point was changed to the adjacent left quadrant of the platform. The latency and distance were recorded in the same way as the previous five training trials. On the next day, rat performed a spatial probe test trial with the same starting point as in the five training trails. This trial consisted of removing the platform from the pool and allowing the rat to swim for 60 s in search of it. The time spent in each of the four equal quadrants, into which the pool was divided, of the tank was recorded.Results The escape latencies, swimming distances displayed by propofol (75mg/kg) treated rats were significantly prolonged as compared with the intralipid-treated controls (P<0.01, respectively), whereas swimming speeds were not significantly different between the propofol (75mg/kg) and intralipid-treated groups (P>0.05). In the subsequent additive training trial, escape latencies and swimming distances displayed by propofol (75mg/kg) treated rats were significantly prolonged as compared with intralipid treated controls (P<0.01, respectively), wheres swimming speeds were similar to intralipid treated rats (P>0.05). The results of probe test trial revealed that the propofol (75mg/kg) treated rats spent significantly less time in the Quadrant III than intralipid treated rats (P<0.01) and propofol (10mg/kg) treated rats (P<0.05).Conclusion Multiple repeated anesthesia by propofol induces the impairment of spatial learning and memory in rats.Part II Effect of Propofol on Long-Term Potentiation in CA1 Region of RatHippocampal SlicesExperiment one: Effect of various concentrations propofol on long-termpotentiation in CA1 region of rat hippocampal slices in vitroObjective To observe the effect of different concentration propofol on long-term potentiation (LTP) in CA1 region of rat hippocampal slices in vitro.Methods Extracellular popular spike (PS) was recorded from the CA1 region of rat hippocampal slices. The stimulus intensity was set to evoke 50% of the maximal amplitude of PS. 100μmol/L, 30μmol/L, 10μmol/L or 3μmol/L propofol superfused slices respectively from 60 min before HFS. Intralipid was applied as solvent control and nothing was applied as control. In all experiments, baseline synaptic transmission was monitored for 20 min before HFS. LTP was induced using high frequency stimulation (HFS), which included 400 pluses at 100 Hz, of Schaffer-collateral pathway.Results In control slices, HFS induced LTP (PS 40 min after HFS: 1.501±0.124, P<0.01 vs control) and this degree of LTP was not altered by Intralipid (1.436±0.160, P<0.05 vs control) and 3μmol/L propofol (1.332±0.077, P<0.05 vs control). Administration of 10μmol/L, 30μmol/L or 100μmol/L propofol blocked LTP. The PS was respectively 1.118±0.072, 1.031±0.112, 0.981±0.047 (P>0.05 vs control). In the presence of 100μmol/L or 30μmol/L propofol the PS 40 min after HFS was lower compared with administration of 10μmol/L, 3μmol/L propofol, intralpid or control (P<0.01 or P<0.05). PS 40 min after HFS was depressed significantly by 10μmol/L propofol superfused slices as compared with intralipid superfused slices (P<0.01), control slices (P<0.01), or 3μmol/L propofol superfused slices (P<0.05). However, PS 40min after HFS was not significantly between 3μmol/L propofol superfused slices and intralpid superfused slices or control slices (P>0.05).Conclusion Propofol inhibits the LTP of CA1 region in rat hippocampal slices induced by HFS.Experiment two: The mechanism of propofol-mediated impairment of CA1 long-term potentiation in rat hippocampal slices in vitroObjective To study the role of γ-aminobutyric acid type A (GABA_A) receptor inpropofol-mediated impairment of CA1 long-term potentiation (LTP) in rat hippocampal slices.Methods Extracellular popular spike (PS) was recorded from the CA1 region of rat hippocampal slices. The stimulus intensity was set to evoke 50% of the maximal amplitude of PS. To examine whether the LTP induced by HFS was dependent of NMDA receptor activation, 50μmol/L AP-5, a NMDA receptor antagonist was administered. To determine whether the effects of propofol on LTP involve GABA_A receptors, we examined the effects of propofol on LTP in the presence of 50μmol/L picrotoxin, a GABA_A receptor antagonist. In addition, 30μmol/L propofol or intralipid superfused slices respectively as previous described. All drugs superfused slices respectively from 60 min before HFS. Baseline synaptic transmission was monitored for 20 min before HFS in all experiments. LTP was induced using high frequency stimulation (HFS), which included 400 pluses at 100 Hz, of Schaffer-collateral pathway.Results In slices superfused by intralipid LTP was induced by HFS (PS 40min after HFS: 1.436±0.160, P<0.01 vs control). Administration of 50μmol/L AP-5 LTP was block completely (1.001±0.089, P>0.05 vs control). PS 40 min after HFS was depressed significantly by 50μmol/L AP-5 as compared with intralipid (P<0.01). Administration of 30μmol/L propofol also blocked LTP (1.031±0.112, P>0.05 vs control). 30(μmol/L propofol did not alter the induction of LTP in the prensence of 50μmol/L picrotoxin (1.384±0.102, P<0.01 vs control). PS 40min after HFS was not significantly different between 30mol/L propofol superfused slices in the prensence of pircrotoxin and intralpid superfused slices (P>0.05).Conclusion Propofol inhibits the LTP through modulation of GABA_A receptors.Experiment three: The combined effect of corticosterone and propofol on long-term potentiation in area CA1 of rat hippocampal slices in vitroObjective To observe the combined effect of corticosterone and propofol onlong-term potentiation (LTP) in area CA1 of rat hippocampal slices.Methods Extracellular popular spike (PS) was recorded from the CA1 region of rat hippocampal slices. The stimulus intensity was set to evoke 50% of the maximal amplitude of PS. 3μmol/L propofol, 0.1μmol/L corticosterone, 10μmol/L corticosterone was applied respectively from 60 min before HFS. To study the combined effect of propofol and corticosterone on LTP, slices were superfused with 3μmol/L propofol in the presence 0.1μmol/L or 10μmol/L corticosterone from 60 min before HFS. In addition, intralipid was applied as solvent control and nothing was applied as control. Baseline synaptic transmission was monitored for 20 min before HFS in all experiments. LTP was induced using high frequency stimulation (HFS), which included 400 pluses at 100 Hz, of Schaffer-collateral pathway.Results In slices superfused by intralipid, 3μmol/L propofol, 0.1μmol/L corticosterone LTP was induced by HFS (PS 40min after HFS: 1.436±0.160, 1.332±0.077, 1.385±0.095, P<0.01 or P<0.05 vs control). In control slices, LTP was also induced by HFS (1.501±0.124, P<0.01 vs control). Slices superfused with 3μmol/L propofol in the presence 0.1μmol/L corticosterone, LTP was induced by HFS (1.342±0.031, P<0.05 vs control). Application of 10μmol/L corticosterone depressed LTP (1.180±0.089, P>0.05 vs control). Administration of 3μmol/L propofol blocked LTP completely in the presence of 10μmol/L corticosterone (0.989±0.030, P>0.05 vs control). PS 40 min after HFS was depressed significantly by 3μmol/L propofol in the presence of 10μmol/L corticosterone as compared with 3μmol/L propofol or 10μmol/L corticosterone alone (P<0.05).Conclusion 10μmol/L corticosterone inhibited the LTP in hippocampal slices of rats in vitro. When 10μmol/L Corticosterone applied in the presence of 3μmol/L propofol, inhibitory effect of corticosterone on LTP was facilitated significantly as compared with 10μmol/L corticosterone alone.Part III Effect of Propofol on Glutamate and γ-aminobutyric Acid Releasefrom Rat Hippocampal SynaptosomesExperiment one: Effect of propofol on Ca²⁺-dependent and Ca²⁺-independent release of glutamate and γ-aminobutyric acid release from rat hippocampal synaptosomesObjective To investigate the effect of propofol on Ca²⁺-dependent and Ca²⁺-independen release of Glutamate and γ-aminobutyric acid (GABA) release from rat hippocampal synaptosomes.Methods Synaptosomes was made from hippocampus and incubated with artificial cerebrospinal fluid (aCSF). Propofol was added into aCSF and final concentration of propofol was 3μmol/L, 10μmol/L, 30μmol/L, 100μmol/L or 300μmol/L respectively. Intralipid was added as solvent control and any drugs was added in control. With the experiment of Ca²⁺-dependent release of glutamate and GABA, dihydrokainic acid (DHK) and nipectic acid were added into aCSF. For observation of Ca²⁺-independent release of glutamate and GABA, DHK, nipectic acid and Ca²⁺ were not added into aCSF. Synaptosomes were preincubated in water bath at 37°C for 15-20min. 20μmol/L veratridine or 30 mmol/L KC1 was used to evoke the release of glutamate and GABA. 5min later the reactions were stopped with EGTA (final concentration was 10mmol/L) at 0°C. The mixture in tube was centrifuged at 12000×g for 20min and supernatant fraction was stored at -70°C. The concentration of glutamate and GABA in aCSF was measured using high-performance liquid chromatograph.Results 10, 30, 100 or 300μmol/L propofol significantly inhibited veratridine-evoked Ca²⁺-dependent release of glutamate compared with control (P<0.01 or P<0.05). Administration of 30, 100, 300μmol/L propofol depressed the release of glutamate compared with administration of 3μmol/L propofol (P<0.01). Application of 100 or 300μmol/L propofol decreased the release of glutamate compared with application of 10μmol/L propofol (P<0.01). The release of glutamatewas significantly lower after application of 300μmol/L than that of 30μmol/L propofol (P<0.05). IC50 of propofol for glutamate Ca²⁺-dependent release was 17.2μmol/L. 30, 100 or 300μmol/L propofol significantly inhibited veratridine-evoked Ca²⁺-dependent release of GABA compared with control (P<0.01 or P<0.05). Administration of 30, 100, 300μmol/L propofol depressed the release of GABA compared with administration of 3μmol/L propofol (P<0.01) or 10μmol/L propofol (P<0.01 or P<0.05). IC₅₀ of propofol for glutamate Ca²⁺-dependent release was 20.1μmol/L. However, propofol has no effect on elevated KCl-evoked Ca²⁺-dependent release of glutamate and GABA. Veratridine or elevated KC1 evoked Ca²⁺-independent release of glutamate and GABA was not affected significantly by propofol.Conclusion Propofol inhibits Ca²⁺-dependent release of glutamate and GABA from synaptosomes. However, it has no effect on the Ca²⁺-independent release of glutamate and GABA from synaptosomes.Experiment two: The role of presynaptic GABA_A receptor in the inhibitory effect of propofol on the Ca²⁺-dependent release of glutamate and GABA from synaptosomes in vitroObjective To observe the role of presynaptic GABA_A receptor in the inhibitory effect of propofol on Ca²⁺-dependent release of glutamate and GABA.Methods Synaptosomes was made from hippocampus and incubated with artificial cerebrospinal fluid (aCSF). 30μmol/L propofol, 50μmol/L picrotoxin or both was added into aCSF. Intralipid was administered as solvent control and any drugs was applied in control. To observe Ca²⁺-dependent release of glutamate and GABA, dihydrokainic acid (DHK) and nipectic acid were added into aCSF. The experiment procedure was same as that mentioned in experiment one of part III.The concentration of glutamate and GABA in aCSF was measured using high-performance liquid chromatograph.Results 30μmol/L propofol significantly inhibited the release of glutamate andGABA compared with intralipid (P<0.01). The release of glutamate and GABA from synaptosomes superfused by picrotoxin were similar to that of intralipid application (P>0.05). The release of glutamate and GABA were inhibited by 30μmol/L propofol in the presence of 50μmol/L picrotoxin compared with intralipid (P<0.01).Conclusion Presynaptic GABA_A receptors do not mediate the inhibitory effect of propofol on Ca²⁺-dependent release of glutamate and GABA.