A mechanistic investigation on the safety and benefits of nitrous oxide anesthesia.

一氧化二氮，俗稱笑氣，是現代臨床麻醉最為常用的一種麻醉劑。然而，關於一氧化二氮效能及安全性的研究至今仍存在爭議。近來，一個稱為ENIGMA的臨床研究項目對2,050個施行大手術的病人接受麻醉情況及術後併發癥進行了研究。研究發現手術中施行一氧化二氮麻醉的病人術後傷口感染率較之對照組上升了35%。另一方面，對423個ENIGMA病人的長期隨訪研究發現，在術中接受一氧化二氮麻醉的病人中慢性術後痛的發病率相對對照組降低了56%。 對於這些臨床發現的分子機制，目前知之甚少。 因此我們進行了一系列實驗來研究一氧化二氮導致術後感染以及預防慢性痛的分子機制。一氧化二氮對基因穩定性的影響在對93個接受直腸結腸大型外科手術的病人進行的隨機對照試驗中，我們比較了接受一氧化二氮麻醉及其對照組病人的外周白細胞脫氧核糖核酸（DNA）損傷情況和術後傷口感染率。通過單細胞凝膠電泳（彗星實驗），我們發現術中一氧化二氮麻醉顯著增加了手術24小時后病人的DNA損傷情況 （p < 0.001）。且這種變化是劑量依賴的，r = 0.33; p = 0.03。並且，在DNA損傷程度及術後傷口感染率間存在顯著相關。術後DNA損傷程度每增加十個單位，傷口感染率則隨之增加17%。一氧化二氮對DNA損傷應答及修復的影響利用大鼠模型，我們對一氧化二氮導致基因不穩定的機制進行了研究。Sprague Dawley大鼠暴露於一氧化二氮中2小時後，我們對其DNA損傷應答及修復基因的轉錄情況進行了檢測。脂多糖（LPS）注射大鼠模擬了圍手術期的炎癥反應。我們發現LPS刺激的白細胞經一氧化二氮處理后，其編碼DNA連接酶IV的LIG4基因的轉錄量顯著降低（p < 0.05）。LIG4基因的下調導致了一氧化二氮麻醉後圍手術期的免疫抑制效應。NMDA受體抑制在一氧化二氮預防性鎮痛中的作用在大鼠慢性神經痛模型中，我們檢測了一氧化二氮鎮痛作用的機制。我們發現一氧化二氮處理組的機械痛覺過敏相較對照組顯著降低（p = 0.001）。一氧化二氮處理后，神經痛大鼠脊髓背角中的c-Fos表達量也顯著降低，這表明了一氧化二氮對神經元活性的影響。該影響可能是NMDA受體抑制的結果。另外，我們還觀察到一氧化二氮的鎮痛特徵與NMDA受體非競爭性拮抗劑MK-801的鎮痛效果相似。基因表達改變在一氧化二氮預防性鎮痛中的作用一氧化二氮鎮痛效果的遲發性和延續性提示了除受體拮抗之外的其他作用機制的存在。我們發現在大鼠坐骨神經壓迫損傷模型中，一氧化二氮處理顯著降低了同側脊髓背角組織中LIG4基因的轉錄及表達（p = 0.006）。同時一氧化二氮處理降低了星形膠質細胞在同側脊髓背角中的活化。我們的研究表示一氧化二氮影響DNA修復， 抑制脊髓背角基因的表達，從而起到預防性鎮痛的作用。總而言之，一氧化二氮通過抑制鉀硫氨酸合成酶，削弱DNA修復和基因組穩定性，從而成為導致術后傷口感染的危險因素。另一方面，這一機制也阻止了脊髓背角中異常突觸的建立，從而預防了神經損傷導致的慢性神經痛的建立。另外，一氧化二氮的鎮痛機制也和NMDA受體抑制作用有關。Nitrous oxide is a commonly administered anesthetic and analgesic agent in contemporary clinical anesthesia. However, the efficacy and safety of nitrous oxide delivery remains a subject of debate. The recent Evaluation of Nitrous oxide In a Gas Mixture for Anaesthesia (ENIGMA) Trial found that nitrous oxide administration, in 2,050 patients undergoing major surgery, increased the incidence of wound infection by 35%. On the other hand, in a long term follow-up study of 423 ENIGMA patients in Hong Kong, the risk of chronic postsurgical pain was reduced by 56% in patients who received nitrous oxide in the index surgery. Little is known about the mechanisms associated with these clinical observations; we therefore conducted a series of experiments to determine the molecular changes after nitrous oxide administration leading to postoperative wound infection and preventive analgesia.Genomic Instability after Nitrous Oxide AdministrationIn a randomized controlled trial of 93 patients undergoing major colorectal surgery, we compared the changes of deoxyribonucleic acid (DNA) damage in circulating leukocytes and rates of wound infection in patients who were exposed to nitrous oxide or not. Using single cell gel electrophoresis (CometAssay), we found that intraoperative nitrous oxide administration produced significant DNA damage, 24 hours after surgery, compared with controls, p < 0.001. The changes were dose-dependent, r = 0.33; p = 0.03. In addition, there was a significant correlation between DNA damage and postoperative wound infection. For every 10 units increase in the percentage of DNA in tail after surgery compared with baseline, there was 17% increase in the risk of wound infection.DNA Damage Response and RepairIn a rat model, we explored the mechanism of genomic instability after nitrous oxide administration. In Sprague Dawley rats exposed to nitrous oxide anesthesia for 2 hours, we tested the transcription of an array of DNA damage response and repair genes. Lipopolysaccharide (LPS) was added to mimic postoperative inflammation. In the mRNA that were extracted and analyzed by real-time polymerase chain reaction (RT-PCR), we found the transcription of gene encoding for DNA Ligase IV (LIG4 gene) was significantly reduced after nitrous oxide administration in LPS-stimulated leukocytes (p < 0.05). The down regulation of LIG4 gene contributed to perioperative immunosuppression following nitrous oxide exposure.Role of N-methyl-D-aspartate receptor (NMDAR) Blockade for Preventive Analgesia with Nitrous OxideUsing a rat model of chronic neuropathic pain, we tested the mechanisms underlying nitrous oxide analgesia. In Sprague Dawley rats undergoing unilateral constrictive injury to the sciatic nerve, we found that mechanical hyperalgesia was significantly reduced with nitrous oxide compared with controls (p = 0.01). In addition, c-Fos expression was decreased in spinal dorsal horn suggesting that neuron excitability was reduced after nitrous oxide administration which could be caused by blockade of the NMDA receptor. Interestingly, the characteristics of analgesia were similar to that provided by MK-801, a noncompetitive antagonist of NMDA receptors.Transcriptional Changes for Nitrous Oxide AnalgesiaThe onset of nitrous oxide analgesia was delayed and outlasted actual receptor antagonism. We therefore explored mechanisms, other than NMDA receptor blockade, for nitrous oxide analgesia. Specifically, in rats undergoing sciatic nerve constrictive injury, we found that transcription of LIG4 gene was down-regulated in the ipsilateral spinal dorsal horn after nitrous oxide administration (p = 0.006). There was a decrease in DNA ligase IV expression and a reduction in the activation of astrocytes. Our data suggested that regulation of DNA repair and suppression spinal dorsal horn gene transcription is one of the alternative mechanisms for nitrous oxide analgesia.In summary, nitrous oxide administration is a risk factor for postoperative wound infection. This is related to irreversible inhibition of the enzyme methionine synthase, impaired DNA repair and genomic instability. The same mechanism however prevented aberrations of synaptic regeneration in the spinal dorsal horn and could therefore prevent the development of chronic neuropathic pain after direct nerve injury. Nitrous oxide analgesia was also related to NMDA receptor blockade.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Chen, Yan.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 126-157).Abstract also in Chinese.Declaration of origination --- p.IAbstract: --- p.IIAcknowledgements --- p.VIIITable of Contents --- p.XList of Tables --- p.XVList of Figures --- p.XVIList of Abbreviations --- p.XVIIIChapter Part I: --- Literature Review --- p.1Chapter Chapter 1 --- A review of nitrous oxide: Historical, clinical and mechanistic Perspectives --- p.2Chapter 1.1 --- History of Nitrous Oxide --- p.2Chapter 1.2 --- Clinical Pharmacology of Nitrous Oxide --- p.4Chapter 1.3 --- Evaluation of Nitrous oxide In the Gas Mixture for Anesthesia (ENIGMA) Trial --- p.9Chapter 1.4 --- Efficacy and Toxicity of Nitrous Oxide: Biochemical and Molecular Mechanisms --- p.14Chapter 1.4.1 --- Immunosuppression Following Nitrous Oxide Administration --- p.14Chapter 1.4.2 --- Analgesia with Nitrous Oxide - Molecular Mechanisms --- p.17Chapter 1.4.2.1 --- Direct molecular target --- p.17Chapter 1.4.2.2 --- Interaction with γ aminobutyric acid type A (GABAA) receptors --- p.19Chapter 1.4.2.3 --- Regulation of opioid system --- p.20Chapter 1.4.2.4 --- Regulation of noradrenergic neurons --- p.21Chapter 1.4.2.5 --- N-methyl-d-aspartate (NMDA) receptor inhibition --- p.22Chapter 1.4.2.6 --- Long-term Preventive Analgesia with Nitrous Oxide --- p.23Chapter 1.4.3 --- Summary --- p.24Chapter Part II: --- Experiments --- p.26Chapter Chapter 2 --- Study Hypothesis and Objectives --- p.27Chapter 2.1 --- Genomic Instability after Nitrous Oxide Administration --- p.27Chapter 2.2 --- DNA Damage Response and Repair --- p.28Chapter 2.3 --- NMDA Receptor Blockade for Preventive Analgesia with Nitrous Oxide --- p.28Chapter 2.4 --- Transcriptional Changes for Nitrous Oxide Analgesia --- p.28Chapter Chapter 3 --- Genomic Instability After Nitrous Oxide Administration: A Randomized Controlled Trial --- p.32Chapter 3.1 --- Introduction --- p.32Chapter 3.2 --- Methods and Materials --- p.36Chapter 3.2.1 --- Study Participants --- p.36Chapter 3.2.2 --- Study Procedures --- p.36Chapter 3.2.3 --- Randomization --- p.37Chapter 3.2.4 --- Anesthetic Care --- p.37Chapter 3.2.5 --- Postoperative Care --- p.38Chapter 3.2.6 --- Measurement of Genomic Instability --- p.44Chapter 3.2.7 --- Statistical Analysis --- p.47Chapter 3.2.8 --- Sample Size Calculation --- p.47Chapter 3.3 --- Results --- p.48Chapter 3.4 --- Discussion --- p.61Chapter 3.4.1 --- Principal Findings --- p.61Chapter 3.4.2 --- Comparison to Other Studies --- p.61Chapter 3.4.3 --- Strengths and Limitations --- p.63Chapter 3.4.4 --- Implications --- p.64Chapter 3.4.5 --- Conclusions --- p.64Chapter Chapter 4 --- DNA Damage Response and Repair After Nitrous Oxide AdministrationChapter 4.1 --- Introduction --- p.65Chapter 4.1.1 --- DNA Damage Response and Repair Pathways --- p.65Chapter 4.1.2 --- Nitrous oxide and DNA Damage Response and Repair --- p.69Chapter 4.2 --- Materials and Methods --- p.70Chapter 4.2.1 --- Animals --- p.70Chapter 4.2.2 --- Nitrous Oxide Administration --- p.70Chapter 4.2.3 --- Lipopolysaccharide-Induced Infection Model --- p.72Chapter 4.2.4 --- Sample Collection and Preparation --- p.72Chapter 4.2.5 --- Single Cell Gel Electrophoresis (CometAssay) --- p.72Chapter 4.2.6 --- RNA Extraction for Gene Transcription Study --- p.72Chapter 4.2.7 --- Reverse Transcription Polymerase Chain Reaction (RT PCR) --- p.73Chapter 4.2.8 --- Quantitative RT PCR --- p.74Chapter 4.2.9 --- Statistical Analysis --- p.76Chapter 4.3 --- Results --- p.76Chapter 4.3.1. --- Genome Instability in Rat Leukocytes after Nitrous Oxide Administration --- p.76Chapter 4.3.2. --- Effect of Nitrous Oxide on the Transcription of DNA Damage Response Genes --- p.78Chapter 4.3.3. --- Effects of Nitrous Oxide on DNA Damage Response Genes in Animals Treated with Lipopolysaccharide --- p.80Chapter 4.4 --- Discussion --- p.84Chapter 4.4.1 --- Principal Findings --- p.84Chapter 4.4.2 --- Implications --- p.84Chapter 4.4.3 --- Limitation of our Study --- p.85Chapter 4.4.4 --- Conclusions --- p.87Chapter Chapter 5 --- Preventive Analgesia with Nitrous Oxide: Role of NMDA Receptor BlockadeChapter 5.1. --- Introduction --- p.88Chapter 5.1.1. --- The ENIGMA Trial: Long Term Follow-up --- p.89Chapter 5.1.2. --- Nitrous oxide prevents chronic postsurgical pain: putative mechanisms --- p.89Chapter 5.1.3. --- Hypothesis --- p.90Chapter 5.2. --- Materials and methods --- p.91Chapter 5.2.1. --- Animals --- p.91Chapter 5.2.2. --- Chronic constriction injury (CCI) to induce neuropathic pain --- p.91Chapter 5.2.3. --- Behavioral test --- p.93Chapter 5.2.4. --- Nitrous oxide administration --- p.93Chapter 5.2.5. --- Dizocilpine (MK-801) pretreatment --- p.94Chapter 5.2.6. --- Tissue Collection, Preparation and Western Blot --- p.94Chapter 5.2.7. --- Statistical Analysis --- p.96Chapter 5.3. --- Results --- p.96Chapter 5.3.1. --- Preventive Analgesia with Nitrous oxide --- p.96Chapter 5.3.2. --- Nitrous oxide analgesia via NMDA receptors block --- p.99Chapter 5.3.3. --- Preventive analgesia with NMDA receptors Blockade --- p.101Chapter 5.4. --- Discussion --- p.103Chapter 5.4.1 --- Principal Findings --- p.103Chapter 5.4.2 --- Our Findings compared with Other Studies --- p.103Chapter 5.4.3 --- Limitations of the Study --- p.104Chapter 5.4.4 --- Conclusions --- p.105Chapter Chapter 6 --- Transcriptional Changes for Nitrous Oxide Analgesia --- p.106Chapter 6.1 --- Introduction --- p.106Chapter 6.1.1 --- Neuro-immune Interactions in the Development of Chronic Neuropathic Pain --- p.106Chapter 6.1.2 --- Nitrous Oxide Interferes Astrocytes and Glial Responses --- p.107Chapter 6.2 --- Materials and methods --- p.109Chapter 6.2.1 --- Chronic Constriction Injury Pain Model and Nitrous Oxide Administration --- p.109Chapter 6.2.2 --- Lumbar Dorsal Horn Tissue Collection, Preparation and Immunoflurescence --- p.109Chapter 6.2.3 --- RNA Extraction and Quantitative RT PCR for Gene Transcription Study --- p.110Chapter 6.2.4 --- Protein Extraction and Western Blot for protein Expression Study --- p.110Chapter 6.2.5 --- Statistical Analysis --- p.111Chapter 6.3 --- Results --- p.112Chapter 6.3.1 --- Time Course of LIG4 Gene Transcription after Constrictive Nerve Injury --- p.112Chapter 6.3.2 --- Nitrous oxide reduced DNA ligase IV expression in spinal dorsal horn --- p.114Chapter 6.3.3 --- Nitrous oxide Reduced Astrocytes Activation in Spinal Dorsal Horn --- p.116Chapter 6.4 --- Discussions --- p.119Chapter 6.4.1 --- Principal Findings --- p.119Chapter 6.4.2 --- Our Findings in Relation to Other Studies --- p.119Chapter 6.4.3 --- Limitations of Our Study --- p.120Chapter 6.4.4 --- Conclusions --- p.120Chapter Part III: --- Conclusions --- p.122Chapter Chapter 7 --- Conclusions and Future Perspectives --- p.123Chapter 7.1 --- Conclusions --- p.123Chapter 7.2 --- Future Perspectives --- p.124Chapter Part IV --- References --- p.12