| Background: General anesthesia has been used in clinic for about 170 years, but its underlying mechanism is not fully understood. More and more researchers nowadays tend to use the neural network theory to explain the action of narcotic drugs during general anesthesia. At present, it is believed that a variety of neurotransmitters and neural pathways in the brain involve in the generation and modulation of general anesthesia. Recently, our group has proved that neuropeptide hypocretins(orexins) play an important role in the emergence from general anesthesia. Orexin can activate the histaminergic neurons in tuberomammillary nucleus as well as the dopamine and adrenaline pathways to promote awaking from general anesthesia. Studies have shown that oreixn can activate the serotoninergic neurons in dorsal raphe nucleus(DRN). 5-hydroxytryptamine(5-HT) is a kind of monoamine chemicals which was widely expressed in the body and involved in many physiological functions such as, pain, body temperature, mood, and sleep-wake regulation. There is also evidence proving that 5-HT also has a role in anesthesia. Thepresent studies aimed to demonstrate that the orexinergic signal can regulate the function of serotoninergic neurons to promote awakefulness during isoflurane anesthesia.Objective: To understand the role of serotoninergic neurons in dorsal raphe nucleus in the awaking-promoting circuit of orexin’s awaking loop during isoflurane anesthesia.Experiment One: Effect of microinjection of orexin and its antagonist into DRN on the induction and emergence time of isoflurane anesthesia.Methods: Adult male SD rats, 230-300 g in body weight.(1) The animals were anesthetized by 10% chloral hydrate(1ml/kg, ip) and the guide cannulas for microinjection to the DRN were embedded 5-7 days before experiment. We put the rats in the anesthesia box filled with oxygen at 1.5L/min flow rate and 1 MAC’s(1.4%) isoflurane. Fifteen minutes later, we microinjected orexin-A(30pmol/0.3ul, 100pmol/0.3ul), orexin-B(30pmol/0.3ul, 100pmol/0.3ul) or saline(0.3ul), respectively.The other two groups were microinjected orexin type Ⅰ receptor antagonist SB334867(5ug/0.3ul, 20ug/0.3ul), type Ⅱ receptor antagonist TCS-OX2-29(5ug/0.3ul, 20ug/0.3ul) or its solvent DMSO(0.3ul). After 15 min we stopped anesthetic inhalation and observed the emergence time.(2) To observe the chronic induction time the protocols were the same as above except the reagents were given 15 min before anesthesia and the concentration of isoflurane was changed into 2% during anesthesia. The induction time was recorded.Result:(1) Compared to the saline group(13.91±0.90min), microinjections of 100 pmol(10.05±0.36min) and 30 pmol(11.6±0.36min) orexin into DRN could significantly shorten the emergence time(p<0.05). Microinjection of 100 pmol orexin-B into DRN also has effect on the promoting awaking of the rat from anesthesia(10.59±0.40 min, in comparison to the control)(p<0.05), while 30 pmol orexin-B injection had no impact on the emergence time(p>0.05). Following microinjection of 20 ug orexin type Ⅰreceptor antagonist SB334867 the emergence time(13.81±0.18 min) was prolonged(p<0.05), in comparison to the DMSO control(11.33±0.35 min), while the 5ug injection(11.84±0.46 min)had no such effect(p>0.05 vs the control). Microinjection of 20 ug orexin type Ⅱreceptor antagonist TCS-OX2-29 had no effect on the emergence time either(p>0.05).(2) During the experiment of chronic induction, microinjections of all reagents had no effect on the induction time of anesthesia.(p>0.05).Experiment Two: Effect of microinjection of orexin and its antagonist into DRN on the change of EEG during isoflurane anesthesia.Methods:(1) Guide cannulas for microinjection to the DRN nuclei and 4 stainless screws for monitoring EEG were embedded. 5-7 days later, the rats were anesthetized in the anesthesia box filled with oxygen in a flow rate of 1.5 L/min and 1 MAC isoflurane(1.4%). Microinjection of orexin-A(100 pmol /0.3 ul), orexin-B(100 pmol / 0.3 ul) or saline(0.3 ul) were executed 30 min following anesthesia. EEGs were recorded and the burst suppression ratios were calculated and compared with the control group.(2) The protocols were the same as above except the reagents were changed into orexin-A(30pmol/0.3ul,100 pmol /0.3 ul) or saline(0.3 ul) and the concentration of isoflurane were changed into 0.75 MAC(1%) and observe the EEG changes in rats.Result:(1) Compared to the control(18.72±2.69%), microinjection of orexin-A into the DRN reduced the burst suppression ratio(BSR, 9.55±1.54%) of EEG(p<0.05), and the electroencephalogram was changed to the wave form of wakefulness.(2) After microinjection orexin-A(100pmol/0.3ul) into the DRN, the power of delta wave was reduced compared to that before the drug injection. The electroencephalogram was changed to the awake wave form.Experiment Three: The activity of serotoninergic neurons in the DRN was affected by orexin.Methods: Guide cannulas for microinjection to the lateral ventricles were embedded.5-7 days later, we put the rats into the anesthesia box filled with oxygen at 1.5L/min flow rate, and 1 MAC’s(1.4%) isoflurane concentration. After 15 min we microinjected orexin-A(100pmol/0.3ul) or saline(0.3ul) and after another 15 mins later the rats were sacrificed. The rats in control group were not subjected to anesthesia and sacrificed by infused 10%KCL via intracardiac injection. The brain was immediately removed after perfusion and cut into section for immunofluorescent staining to elucidate the activities ofserotoninergic neurons. The activation of 5-HT neuron was determined by the co-expression of 5-HT and c-Fos.Results: No differences were found for the activation rate of 5-HT neurons between the non-anesthesia group(13.24±1.64%) and the orexin-A group(12.54±1.45%). The rate in the latter group was higher than that in saline group(7.20±0.78%, p<0.05).Conclusion: This experiment proves that orexin can promote the anesthesial emergence, change the EEG pattern to arousal wave, and reduce the delta wave in isoflurane anesthesia by regulating the activities of serotonin neurons in the dorsal raphe nucleus. |
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