The Effects Of Isoflurane On Memory Function And The Synaptic Long-term Potentiation (LTP) In The Hippocampal Slices Of Different Aged Rats

Back ground:Post-operative cognitive disfunction (POCD) is one of the complicaitons of central nervous system after operation, which will lead to dementia, serious memory injury, when it is severe. Age is the only specific risk factor of POCD, while anesthetics may also contribute to it. Synaptic long-term potentiation (LTP) in the hippocampus, termed as the functional modality of synaptic plasticity of central nervous system and synaptic transmission, is the neural cellular basis of learning and memory. Central cholinergic system plays an important role in recognition such as learning and memory, and central neuronal alpha4beta2 nicotinic acetylcholine receptor subtype are known to be involved in learning and memory. Inhaled anesthetic isoflurane was reported to cause POCD in the aged patient, however, the mechanism is still not clear. Thus, this research tries to investigate the effects of inhaled anesthetic isoflurane on LTP in the hippocampal of rats and the relations between isoflurane and central neuronal alpha4beta2 nicotinic acetylcholine receptor subtype so as to elucidate the mechanisms underlying the effects of inhaled anesthetic isoflurane on POCD.Objective:To investigate the effect of inhaled anesthetic isoflurane on learnig and memory function of rats with different age and try to elucidate the effects of isoflurane on LTP in the hippocampal of rats with different age and the relations between isoflurane and central neuronal alpha4beta2 nicotinic acetylcholine receptor subtype.Methods:Part 1.The effects of isoflurane on memory in rats of different age. Twenty-one adult male Sprague-Dawley (SD) rats weighing 200～250 g (2 months old) were randomly allocated into three groups (n=7 each):Adult control group, breath 30% oxygen and 70% nitrogen mixed gas; Single inhalation of isoflurane adult group were anesthetized with 1.4% isoflurane, breath 30% oxygen and 1.4% isoflurane mixed gas for 2h; Repeated inhalation of isoflurane adult group were anesthetized with 1.4% isoflurane, breath 30%oxygen and 1.4% isoflurane mixed gas 2h a day for five days. Another twenty-one aged male SD rats weighing 500～620 g (20 months old) were randomly allocated into three groups (n=7 each):Aged control group, breath 30% oxygen and 70% nitrogen mixed gas; Single inhalation of isoflurane aged group were anesthetized with 1.4% isoflurane, breath 30% oxygen and 1.4% isoflurane mixed gas for 2h; Repeated inhalation of isoflurane aged group were anesthetized with 1.4% isoflurane, breath 30%oxygen and 1.4% isoflurane mixed gas 2h a day for five days. Learning and memory function were assessed using Morris water-maze test 24 h after anesthesia with isoflurane in each group. Part 2. The effect of isoflurane on the synaptic long-term potentiation (LTP) in the CA1 area of rats hippocampal slices with different age. Transverse hippocampal slices (400μm) were prepared from seventy 2-month-old SD rats weighing 200～250 g and seventy 20-month-old SD rats weighing 500～620 g and maintained in ice-cold artificial cerebrospinal fluid (ACSF), oxygenated with 95% O2～5% CO2. The slices were incubated in ACSF at room temperature for at least 120 min before use. Thirty-five slices of adult rats were randomly divided into 5 equal groups (n=7 each):adult control group; group isoflurane 0.0625; group isoflurane 0.125; group isoflurane 0.25; group isoflurane 0.5. All the slices in each group were perfused with ACSF, 0.0625 mmol/L isoflurane,0.125 mmol/L isoflurane,0.25 mmol/L isoflurane or 0.5 mmol/L isoflurane, respectively. The slices was transferred to a recording chamber for extracellular recording and perfused with ACSF at a rate of 2～3 ml/min (30±1)℃. One recording electrode was positioned in the pyramidal cell layer of the CA1 area of rats hippocampal slices to simultaneously record evoked population spikes (PS). Another thirty-five slices of adult rats were randomly divided into 5 equal groups (n=7 each):group LTP; group isoflurane-LTP 0.0625; group isoflurane-LTP 0.125; group isoflurane-LTP 0.25; group isoflurane-LTP 0.5. All the slices in each group were perfused with ACSF,0.0625 mmol/L isoflurane,0.125 mmol/L isoflurane,0.25 mmol/L isoflurane or 0.5 mmol/L isoflurane, respectively. The slices in each group were performed to record PS for at least 30 min before LTP. For LTP induction, high-frequency stimulation (HFS) conditioning pulses (100 Hz/s) were applied to the Schaffer collateral-commissural pathway of hippocampus using a bipolar stimulating electrode. The changes in PS amplitude of the rats hippocampal slices of each group after HFS were analyzed. Thirty-five slices of aged rats were randomly divided into 5 equal groups (n=7 each):aged control group; group isoflurane 0.0625; group isoflurane 0.125; group isoflurane 0.25; group isoflurane 0.5. All the slices in each group were perfused with ACSF,0.0625 mmol/L isoflurane,0.125 mmol/L isoflurane,0.25 mmol/L isoflurane or 0.5 mmol/L isoflurane, respectively. The slices was transferred to a recording chamber for extracellular recording and perfused with ACSF at a rate of 2～3 ml/min (30±1)℃. One recording electrode was positioned in the pyramidal cell layer of the CA1 area of rats hippocampal slices to simultaneously record evoked population spikes (PS). Another thirty-five slices of aged rats were randomly divided into 5 equal groups (n=7 each):group LTP; group isoflurane-LTP 0.0625; group isoflurane-LTP 0.125; group isoflurane-LTP 0.25; group isoflurane-LTP 0.5. All the slices in each group were perfused with ACSF,0.0625 mmol/L isoflurane,0.125 mmol/L isoflurane,0.25 mmol/L isoflurane or 0.5 mmol/L isoflurane, respectively. The slices in each group were performed to record PS for at least 30 min before LTP. For LTP induction, high-frequency stimulation (HFS) conditioning pulses (100 Hz/s) were applied to the Schaffer collateral-commissural pathway of hippocampus using a bipolar stimulating electrode. The changes in PS amplitude of the rats hippocampal slices of each group after HFS were analyzed. Part 3. The effect of isoflurane on synaptic long-term potentiation (LTP) in the CA1 area of rats hippocampal slices with activation of central neuronal alpha4beta2 nicotinic acetylcholine receptor subtype. Transverse hippocampal slices (400μm thick) were obtained from male SD rats (2 month old) weighing 200～250 g that were ether-anesthetized and decapitated. The hippocampus was rapidly removed, and slices were prepared in ice-cold artificial cerebrospinal fluid (ACSF), oxygenated with 95% O2～5% CO2. The slices were incubated in ACSF at room temperature for at least 120 min before use. Ninety-eight slices were randomly divided into 14 equal groups (n=7 each):group LTP; group isoflurane 0.125; group isoflurane 0.25; group isoflurane 0.5; group A85380 1.0; group A85380 10.0; group A85380 1.0+isoflurane 0.25; group A85380 10.0+isoflurane 0.25; group epibatidine 0.1; group epibatidine 1.0; group epibatidine 0.1+isoflurane 0.25; group epibatidine 1.0+isoflurane 0.25; group DHβE; group DHβE+isoflurane 0.125. All the slices in each group were perfused with ACSF, isoflurane 0.125,0.25,0.5 mmol/L, A853801.0,10.0μmol/L, A853801.0μmol/L+isoflurane 0.25 mmol/L, A8538010.0μmol/L+isoflurane 0.25 mmol/L, epibatidine 0.1,1.0μmol/L, epibatidine 0.1μmol/L+isoflurane 0.25 mmol/L, epibatidine 1.0+isoflurane 0.25 mmol/L, DhβE 0.1μmol/L, DhβE 0.1μmol/L+isoflurane 0.125 mmol/L, respectively. The slices was transferred to a recording chamber for extracellular recording and perfused with ACSF at a rate of 2～3 ml/min (30±1)℃. One recording electrode was positioned in the pyramidal cell layer of the CA1 area of rats hippocampal slices to simultaneously record evoked population spikes (PS). For LTP induction, high-frequency stimulation (HFS) conditioning pulses (100 Hz/s) were applied to the Schaffer collateral-commissural pathway of hippocampus using a bipolar stimulating electrode. The changes of PS amplitude after HFS were analyzed in each group.Results:Part 1. The effects of isoflurane on memory in rats of different age. Pulse Oxygen Saturation (SpO2), heart rate (HR) and blood pressure (BP) varied in the normal range during anesthesia in each group, and there were no significant differences between adult control group and aged control group as to the escape latency (P>0.05). Compared with the adult control group, the escape latency in the repeated isoflurane adult group was longer (P<0.05) but there was no difference with the single isoflurane adult group (P>0.05). When compared with the aged control group, the escape latency was significantly longer in both single isoflurane aged group and repeated isoflurane aged group (P<0.05); the times of accrossing the original platform and the time consumption of staying the original platform quadrant were not significantly different between the adult control group and aged control group (P>0.05). Comparing with adult control group, the times of accrossing the original platform and the time consumption of staying the original platform quadrant were not significantly different in the single isoflurane adult group (P>0.05) while they were shorter in the repeated isoflurane adult group (P<0.05). Compared with the aged control group, the times of accrossing the original platform and the time consumption of staying the original platform quadrant was shorter in both single isoflurane aged group and repeated isoflurane aged group (P<0.05). Part 2. The effect of isoflurane on the synaptic long-term potentiation (LTP) in the CA1 area of rats hippocampal slices with different age. Compared with adult control group, there were no significant differences of the PS amplitude of the rats hippocampal slices in the adult isoflurane 0.0625 group (P>0.05), however, it decreased significantly in adult group isoflurane 0.125. adult group isoflurane 0.25 and adult group isoflurane 0.5 (P<0.05 or P<0.01). Compared with aged control group, there were no significant differences of the PS amplitude of the rats hippocampal slices in the aged isofiurane 0.0625 group (P>0.05), however, it decreased significantly in aged group isofiurane 0.125, aged group isoflurane 0.25 and aged group isoflurane 0.5 (P< 0.01). The PS amplitude of the rats hippocampal slices in adult LTP group after HFS was significantly increased by (52±12)%compared with that of pre-HFS (P<0.01), and thus is the successful induction of LTP. Compared with adult LTP group, the PS amplitude of the rats hippocampal slices in adult group isoflurane-LTP 0.125, adult group isoflurane-LTP 0.25 and adult group isoflurane-LTP 0.5 after HFS decreased significantly (P<0.01). The PS amplitude of the rats hippocampal slices in aged LTP group after HFS was significantly increased by (34±11)% compared with that of pre-HFS (P<0.01), and thus is the successful induction of LTP. Compared with aged LTP group, the PS amplitude of the rats hippocampal slices in aged group isoflurane-LTP 0.0625, aged group isoflurane-LTP 0.125, aged group isoflurane-LTP 0.25 and aged group isoflurane-LTP 0.5 after HFS decreased significantly (P<0.05 or P<0.01). Part 3. The effect of isoflurane on synaptic long-term potentiation (LTP) in the CA1 area of rats hippocampal slices with activation of central neuronal alpha4beta2 nicotinic acetylcholine receptor subtype. Compared with group LTP, the amplitudes of the PS in group A85380 1.0 and 10.0 after HFS increased significantly (P<0.01). The amplitude of the PS after HFS in group A853801.0+isoflurane 0.25 and group A85380 10.0+isoflurane 0.25 was dramatically increased compared with the value of group isoflurane 0.25 (P<0.01). Compared with group LTP, the amplitudes of the PS in group epibatidine 0.1 and 1.0 after HFS increased significantly (P<0.01). The amplitude of the PS after HFS in group epibatidine 0.1+isoflurane 0.25 and group epibatidine 1.0+isoflurane 0.25 was dramatically increased compared with the value of group isoflurane 0.25 (P<0.01). The amplitude of the PS after HFS in group DHβE shows no significant difference compared with the value of group isoflurane 0.125 (P>0.05), but it was significantly lower than that in group LTP (P<0.01). The amplitude of the PS after HFS in group DHβE+isoflurane 0.125 significantly decreased compared with the value of group isoflurane 0.125 (P<0.05).Conclusion:Inhaled anesthetic isoflurane can reduce learning and memory function in both adult and aged rats, but aged rats are particularly significant impacted. The mechanism of isoflurane-induced deficits in memory is involved in the inhibition of LTP induction, and the inhibition of central neuronal alpha4beta2 nicotinic acetylcholine receptor subtype activation in hippocampus of rats contributes to the mechanism of isoflurane-induced LTP inhibition.Innovation point:This study systemically investigated the effects of inhaled anesthetic isoflurane on learning and memory function between adult and aged rats, and observed the effects of isoflurane on the synaptic long-term potentiation (LTP) in the CA1 area of rats hippocampal slices with different age and its relationship with central neuronal alpha4beta2 nicotinic acetylcholine receptor subtype in order to elucidate the effects of isoflurane on learning and memory of rats with different age and on the induction of LTP in hippocampal slices and its underlying mechanisms which demonstrates the mechanism of isoflurane on POCD. This study provides us with new ideas in preventing from awareness in general anesthesia (GA), recall disturbance and cognitive dysfunction after GA, and may push forward for the research of anesthesia mechanism as well as put forward scientific instruction and theoretical foundation for the clinical use of anesthetics and development of new general anesthetics.