| Part I Establishment of a rat model of surgery accompanied with LPS treatmen t-induced postoperative cognitive dysfunctionObjective:We assessed the impact of different dose of LPS given prior to surgery on postoperative cognitive function in order to establish a rat model of postoperative cognitive impairment.Methods:Rats were randomly assigned into the following groups:Control group; 50, 100 and 1000μg/Kg of LPS group by intraperitoneal (i.p.) injection; the surgery group and the surgery plus LPS group. However, the surgery plus LPS group received 50,100 and 1000μg/Kg i.p. injection of LPS 1 h before undergoing surgery respectively. Rats were administered 0,50,100 and 1000μg/Kg i.p. injection of LPS and (or) left unilateral nephrectomy. One hundrent and twenty-eight SD rats were divided into sixteen groups (n=8 for each), Morris water maze test was carried out in the eight groups; others was received fear conditioning test. Behavioral assessment test (locomotor activity test, Morris water maze, and fear conditioning test) were conducted to optimize the dose of LPS for inducing cognitive impairment on the postoperative days 3 and 7 respectively.Results:1. Locomotor activity test Compared with group C (775.35±80.66), motor distance was obviously reduced on postoperative days 3 in the LPS1000ug/Kg group (649.58±91.95,p<0.05)and LPS1000ug/Kg+S group(601.57±67.12,p<0.01). There was no statistical significance among the groups on the postoperative days 7(p>0.05).2. Morris water maze test 2.1 positioning navigation training test. Latency (time that rat find the hidden plat) was significantly shortened after training for 5 days. There was no statistical significance among the eight groups on the same day.2.2 Probe test and working memory test Compared with group C, the Morris water maze test showed cognitive decline in all LPS plus surgery groups, while LPS100ug/Kg+S group showed significant decrease of platform-site crossovers(3.12±0.83; 1.87±0.83, respectively, p< 0.05) and time traveled in the target quadrant during the probe test(19.53±1.28s; 17.55±1.10s, respectively, p<0.05) as well as longer latency in the working memory test on the postoperative day 3(20.85±1.63; 23.41±2.28, respectively,p<0.05), and returned the normal condition on postoperative day 7.3. Fear condition test Compared with group C, LPS 1 OOug/Kg+S group showed significant cognitive impairment, as evidenced by reduction in freezing time percent in the conditional test of the fear condition system on the postoperative day 3 (58.00±24.11; 32.08±7.28, respectively, p<0.01), which persisted until the days 7 (p<0.05); Likewise, there was similar results in the tone test on the postoperative day 3(group C:43.62±14.59; LPS100ug/Kg+S group:32.55±3.87, p< 0.05), but not days 7. Our results showed that LPS (1000ug/Kg, i.p) with surgery or not significantly reduced motor distance in rat on the postoperative day 3, which influenced actional experimental results. Intraperitoneal injection of LPS (100μg/Kg) plus surgery induced cognitive decline in SD rats in the Morris water maze and fear conditioning test, but not affected motor activity of rat, suggesting that LPS (100ug/kg, i.p) when administered before surgery induced cognitive decline, which could serve as POCD model in rat.PartⅡ Effect and mechanism of parecoxib on the postoperative cognitive dysfunction in rat Objective:To investigate effect of parecoxib on LPS administernation combined with surgery-induced cognitive impairment in rat and to explore the possible mechanism. Methods:One hundrend and fourty rats were randomly allocated into the following groups:control group (n=20), model group (n=40), parecoxib therapy group (n=40) and parecoxib group (n=40). The model group was induced by the surgery combined with LPS (100ug/kg)-treatment. Parecoxib (10 mg/kg/day once daily for 3 days before surgery and 2mg/Kg/day for 3 or 7 days postoperatively) was administered i.p. in the rats of parecoxib group and parecoxib therapy group. Postoperative cognitive function in rats was assessed using Morris water maze and fear condition test on the postoperative days 3 and 7. Locomotor activity test was also done to assess motor activity of rat. The status of microglia (Iab-1) and neuron (NeuN), potein levels of COX-2, cleaved-caspase3, iNOS, nNOS, AKT and GSK3p, content of PGE2 as well as expression of EP1-4 mRNA were respectively measured by immunohistofluorescence staining method, Weston blot, ELISA and RT-PCR in the hippocampus and cortex of rats in order to evaluate the neuroprotective effect of parecoxib on the postoperative cognitive function.Results:There was no statistical significance among the groups on the postoperative days 3 and 7 in the locomotor activity test (p>0.05). Compared with group LPS1 OOug/Kg+S, parecoxib treatment obviously improved the cognitive decline in rats underwent LPS i.p. accompied with surgery on postoperative days 3 in the Morris water maze test (time in the target quadrant:17.31±1.27s,19.45±1.99, p<0.05; platform-site crossovers:1.80±0.78,2.10±0.7,P<0.05; latency in the working memory test: 23.36±2.90s,19.94±3.03,P<0.05, respectively). Compared with group LPS100ug/Kg+S, parecoxib administernation significantly alleviated reduction in freezing time percent in the fear condition test on the postoperative day3(conditional test:37.03±6.49,49.56±7.51, p<0.05; tone cued test:24.83±4.35,37.09±7.23, p>0.05, respectively). Treatment of rats with parecoxib significantly attenuated LPS i.p. plus surgery-induced microglial activation (p<0.05) and decrease of NeuN+ number (p<0.05), elevation of COX-2 level (p<0.05), content of and PGE2 (p<0.05). Moreover, parecoxib obviously prevented elevated expression of cleaved-caspase3 (p<0.05); modulated AKT (p<0.05) and GSK3β activity (p<0.05); alleviated elevation of iNOS (p<0.05) and reduction of nNOS level(p<0.05) in the hippocampus and cortex on postoperative day 3. Parecoxib also alleviated LPS plus surgery-induced elevation of EP1 mRNA in the cortex on day 3 after the surgery (p<0.05). Our results suggest that parecoxib reduced LPS i.p plus surgery-induced elevated COX-2, enhanced PGE2 content and EP1 mRNA level, and consequently effectively alleviated the pathological progression such as neuroinflammation, oxidative stress and apoptosis, and eventually improved postoperative cognitive dysfunction.Conclusions:LPS (100ug/kg, i.p) combined with surgery triggered postoperrative cognitive impairment in a rat model. Administration of parecoxib conferred neuroprotection effect against LPS i.p in combination with surgery-induced memory and cognitive decline, possible mechanism of the neuronal beneficial effects of parecoxib was related with reducing microglia activity and neuronal injury, suppressing neuroinflammation, alleviating the oxidative stress,apoptosis and modulating the phosphorylation of the Akt/GSK3P signaling pathway. |
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