| The neurotoxicity of bilirubin has been confirmed by multiple experimental evidences. Obstructive jaundice is a hepatobiliary disease characterized by cholestasis, the suppression or stoppage of bile flow. Other than the damage of the liver function, there are also some pathological conditions, such as hyperbilirubinemia, sepsis, acidosis, hypoproteinemi and others. These factors not only contributed to increase the concentration of unconjugated bilirubin (UCB) in serum, but also changed the status of the blood-brain barrier, so that it is help to accumulation of UCB in the cerebrospinal fluid (CSF) and affect the central nervous system (CNS). Therefore, we want to determine the concentration of UCB in the CSF of rats with obstructive jaundice and and the possible effectiveness of the central nervous system as well as the impact of anesthesia.By doing common bile duct ligation in rats to establish obstructive jaundice animal models, we firstly determined the function of liver and kidney, and the values of UCB in CSF by taking blood and cerebrospinal fluid samples in the postoperative 3 days,5 days,7 days,14 days respectively.And then by using an electronic version of the von frey hair to test the withdrawal threshold of rats with obstructive jaundice to assess the mechanical nociceptive sensitization. When we confirmed the increase in nociceptive threshold in rats with obstructive jaundice, we want to understand further the relationship between bilirubin and nociceptive threshold by intrathecal administration of bilirubin. The rats were divided into saline group, bilirubin 1μM group, bilirubin 10μM group, bilirubin 100μM group, bilirubin 1mM group, and then each group of rats were tested the withdrawal threshold before and after treatments in 0.5h, 1h,1.5h,2h. So that we can observe the changes of nociceptive threshold in different concentrations of bilirubin and at different time points. After that we use the spinal cord patch clamp to determine the impact of bilirubin on the synaptic transmission in spinal dorsal horn neurons. We recorded the spontaneous excitatory postsynaptic currents (sEPSC) and spontaneous inhibitory postsynaptic currents (sIPSC) of substantia gelatinosa (SG) neurons firstly, and then the perfusate was shifted to bilirubin (10μM) containing solution for additional recording. In order to evaluate the relation between the bilirubin and the activities of the synaptic transmission in spinal dorsal horn neurons. Finally we respectively determined the minimum alveolar concentration (MAC) and minimum alveolar concentration for the loss of the righting reflex (MACRR) in the rats of 7 days after bile duct ligation group (BDL7d), sham-operated group (SHAM), intrathecal groups with bilirubin (Bilirubin 1mM) and intrathecal with the saline group (SAL).Through observing MAC and MACRR of the four groups to compare the changes of sensitivity to sevoflurane inhaled anesthetics. To get further understand in the rslationship between bilirubin and sensitivity to inhaled anesthetics.The main results are as follows:1. The assessment of obstructive jaundice model and nociceptive thresholdAfter bile duct ligation, total bilirubin, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase progressively increased, albumin did not change significantly. That means the rats have been in cholestasis and accompanied with liver damage. The obstructive jaundice model is successful. Blood urea nitrogen and creatinine gradually increased on the day after operation 5 days, indicating renal damage is occurred. The concentration of UCB in the CSF samples of rats with obstructive jaundice is between 0.1±0.00 and 1.76±0.57μmol/L comparing the sham group is 0μmol/L. The nociceptive threshold of obstructive jaundice rats began to increase on the 3 day postoperative and maintain on a high level until the end of observation on the 14 day. Compared with the sham group, there is a significant statistical difference (P<0.05). These results suggest that the increase in the concentration of UCB in the CSF and nociceptive threshold in rats with obstructive jaundice. Whether the increasing in nociceptive threshold has some relationship with the UCB, which may impact on the CNS. We need get further experiments to confirm it.2. Intrathecal administration of bilirubin cause the increasing in nociceptive thresholdStatistics showed that the nociceptive threshold of bilirubin 1mM group in 0.5h, 1h,1.5h,2h showed a significant statistical difference compared with others (P<0.05); the nociceptive threshold of bilirubin 100μM group in the 1h,1.5 h,2h showed a significant statistical difference compared with others (P<0.05); Among the saline group, bilirubin 1μM group and bilirubin 10μM group, the nociceptive threshold at different time points was no statistical difference (P>0.05). The results showed that Intrathecal administration of bilirubin cause the increasing in nociceptive threshold. These suggest that UCB is closely related with nociceptive threshold.3. Bilirubin inhibits the excitement of spinal cord dorsal horn neuronsWe use whole-cell patch clamp recording the sEPSC of SG neurons, then through the perfusion bilirubin (10μM,2min), observed that the sEPSC amplitude was significantly inhibition (47%, p<0.01); the sIPSC of SG neurons was observed the frequency was significantly increased (57%, p<0.01). Results indicate that bilirubin inhibited the function of receptors in excitatory post-synaptic neurons and facilitated releasing of neurotransmitter in the inhibition presynaptic neurons. The results suggest that bilirubin inhibits the excitement of spinal cord dorsal horn neurons.4. Bilirubin in CSF make MACRR and MAC smallerBy testing MACRR and MAC, we observed that compared with the control group (SHAM; SAL), MACRR of the experimental group (BDL7d; Bilirubin 1mM) were significantly lower (P<0.05), which BDL7d group lowered by 10% and Bilirubin 1mM group reduced by 13%; MAC also significantly lowered (P<0.05), which BDL7d group reduced by 13% and Bilirubin 1mM reduced by 15%. The between control groups (SHAM; SAL) have no significant difference (P> 0.05); between experimental groups (BDL7d; Bilirubin 1mM) also have no significant difference (P> 0.05). Results indicate that both obstructive jaundice rats and the rats with intrathecal administration of bilirubin increased the sensitivity of the inhaled anesthetics, so that the bilirubin in the CSF is one of the reasons leading to this phenomenon.Conclusion:1. The nociceptive threshold is increased in rats with obstructive jaundice may be related to the effect of UCB in the CSF on CNS.2. The increasing in concentration of bilirubin causes the increasing in nociceptive threshold. 3. The whole-cell patch clamp recorded the changes of the synaptic transmission in SG neurons effected by bilirubin to further comfirmed the bilirubin inhibits the excitement of spinal cord dorsal horn neurons.4. The decreasing of MACRR and MAC in both obstructive jaundice rats and the rats with intrathecal administration of bilirubin, suggested that the bilirubin in the CSF increased the sensitivity of the inhaled anesthetics. |
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