DNA Methylation and Perioperative Pain Treatment
Condition: Hyperalgesia; Pain, Postoperative
MeSH terms: Hyperalgesia; Pain, Postoperative
Study type: Observational [Patient Registry]

Summary: 
      In the last few years much attention has been paid to hyperalgesia induced by opioids, which
      represents a clinically significant condition following acute and chronic exposure to
      opioids. It has been suggested that the increase in gene expression of μ-opioid receptors and
      the development of hyperalgesia could be linked to epigenetic mechanisms. In particular the
      use of opioids seems to be related to an increase of the degree of DNA methylation. In the
      literature are not currently available data about the degree of DNA methylation in surgical
      patients, opioid-naive prior to surgery, receiving acute opioids treatment in the
      perioperative period.

      The primary objective of this study is to assess the degree of perioperative DNA methylation
      (extracted from whole blood) in patients undergoing major surgery exposed to opioids
      according to current clinical practice compared to preoperative baseline levels. The timing
      (preoperative vs intraoperative vs postoperative) in which the possible increase of the
      degree of DNA methylation will happen wil be assessed.

      20 consecutive patients undergoing major surgery performed under general anesthesia that have
      expressed their consent to participate to the study will be enrolled. This sample is adequate
      to allow the observation of a possible increase of DNA methylation in opioid-naive patients
      exposed to anesthesia/analgesia based on opiates according to current clinical practice.
    

Detailed description: 
      During perioperative period surgical patients experience profound changes in their
      physiological processes. In addition to surgical stress, these variations are in part due to
      pharmacological treatments commonly used for anesthesia and analgesia during and after
      surgical procedure.

      Opioids are essential drugs for the management of acute perioperative pain. In the last few
      years much attention has been paid to hyperalgesia induced by opioids, which represents a
      clinically significant condition following acute and chronic exposure to opioids. It has been
      suggested that the increase in gene expression of μ-opioid receptors and the development of
      hyperalgesia could be linked to epigenetic mechanisms.

      Epigenetic alterations typically involve changes in chromatin structure, which is composed of
      DNA, histones and non-histone proteins. Main changes in clinical setting are covalent
      modifications of N-terminal sequences of histone proteins and DNA methylation. In particular,
      DNA methylation is carried out with the addition of a methyl group on the fifth carbon atom
      of cytosine residues (5mC), mainly localized in CpG regions.

      DNA methylation is considered as a mechanism of gene silencing. It is a complex process
      regulated by a whole family of enzymes called DNA methyl-transferase (DNMTs: DNMT1, DNMT2,
      DNMT3a, DNMT3B, and DNMT3L) that catalyze the transfer of a methyl group.

      In particular DNMT 1, 3a, 3b were studied during the development of a neuropathic pain model.
      Through immunohistochemical analysis it was observed that in the dorsal root ganglion of an
      adult rat (L4-5) DNMT3B showed an important up-regulation that is observed already 1 week
      after the induction of the model and that persists for 4 weeks; on the contrary, DNMT1 and
      DNMT3a showed a moderate and transient up-regulation. Other studies have shown that in a
      different pattern of pain, acute inflammatory, the expression levels of DNMT3a and DNMT3B are
      particularly high.

      All these findings suggest the important activity of these enzymes in different models of
      pain.

      Opioids admnistration seems to be related to an increase of the degree of DNA methylation. In
      a study published in 2013, Doehring et al. rated the degree of DNA methylation in patients
      heroin addicts undergoing methadone substitution treatment. Patients treated with opioids
      have shown an overall increase in DNA methylation, in particular of μ-opioid receptor. It is
      interesting to note that the degree of DNA methylation correlates with the intensity of pain
      reported by subjects analyzed in the study.

      In the literature are not currently present data related to the degree of DNA methylation in
      surgical patients receiving acute treatment with opiates that are opioid-naive prior to
      surgery.

      The primary objective of this study is to assess the degree of perioperative DNA methylation
      (extracted from whole blood) in patients undergoing major surgery exposed to opiates
      according to current clinical practice compared to preoperative baseline levels. The timing
      (preoperative vs intraoperative vs postoperative) in which the possible increase of the
      degree of DNA methylation will happen will be assessed.

      The secondary objectives are: observe the possible correlation between the degree of DNA
      methylation and the intensity of acute postoperative pain, observe the possible correlation
      between the degree of DNA methylation and the incidence of postoperative nausea and vomiting,
      observe the possible correlations between the degree of DNA methylation and the gene
      expression of μ opioid receptor, observe the possible correlations between the degree of DNA
      methylation and the expression of genes mainly involved in methylation processes, compare the
      degree of DNA methylation in the preoperative phase of the different patients involved in the
      study.

      MEDAPO trial is a monocentric clinical trials with the use of human tissue in vitro. Because
      the lack of knowledge in literature about the increased degree of DNA methylation following
      acute administration of opioids in surgical patients, this study will be explorative.
      Moreover, not being able to avoid exposure to opiates in the intraoperative phase, but being
      possible to guarantee postoperative analgesia without opioid treatments, it will be
      interesting to see if any increase in methylation occurs already during the intraoperative
      phase or whether it can be related to the time of exposure to treatment with opiates.

      This will provide useful information to better plan the timing for randomization into a
      future trial comparing various postoperative analgesic techniques.

      20 consecutive patients undergoing major surgery performed under general anesthesia that have
      expressed their consent to participate to the study will be enrolled. This sample is adequate
      to allow the observation of a possible increase of DNA methylation in opioid-naive patients
      exposed to anesthesia/analgesia based on opiates according to current clinical practice.

      Descriptive analysis related to the primary objective of the trial will be performed. We will
      perform parametric tests for paired data to observe the correlation between the degree of DNA
      methylation and acute postoperative pain.

      After verifying the signature of informed consent, the presence of the inclusion criteria and
      the absence of exclusion criteria, the patient will be considered eligible. Each patient will
      be assigned to a serial number which will identify the samples used for molecular analysis
      making them anonymous.

      The study period will extend from the signing of informed consent up to 48 hours after the
      end of surgical procedure.

      The participation to the study will not change the treatment plan of the patient: surgical
      procedure and anesthetic conduct will represent the normal daily clinical practice without
      any variation regardless the enrollment of the patient.

      At the time of arrival in the operating room the patient will be monitored according to
      standard clinical practice in relation to the type of surgery (ECG, SpO2, EtCO2 and Invasive
      Blood Pressure). After monitoring the patient, it will be placed a peripheral venous catheter
      ≤ 20 G and an arterial catheter (20G) in radial or humeral artery.

      After adequate oxygenation anesthesia will be induced with fentanyl (3 mcg/kg), propofol
      (2-2.5 mg/kg) and Rocuronium (0.6 mg/kg) or Succinylcholine (1 mg/Kg). The maintenance of
      anesthetic level will be obtained by the use of Sevoflurane 0.7 - 1.3 MAC (minimum alveolar
      concentration), and bolus doses of Fentanyl (1mcg /kg) and possibly of Rocuronium (0.15mg/kg)
      every 45 minutes until the end of surgery.

      The acute postoperative pain in the first 48 hours will be treated with a continuous
      intravenous infusion of morphine (1 mg/h), with the possibility of the patient to
      self-administer boluses of 1 mg of morphine repeatable every 8 minutes.

      Continuous infusion of morphine will be preceded by an intraoperative bolus (immediately
      after the last stitch) of 0.1 mg/kg of morphine. Ten minutes after awakening the patient, if
      the pain will be poorly controlled (NRS ≥ 4), will be administered 1 mg of morphine,
      repeatable every 5 minutes, until NRS<3.

      Prophylaxis for postoperative nausea and vomiting will carried out by intravenous
      administration of 0.1 mg/kg of dexamethasone prior to administration of the first dose of
      fentanyl. In case of postoperative nausea and vomiting, it will be administered droperidol
      0.625 mg repeatable every 6 hours.

      During the study period, in addition to the anthropometric parameters, we will collect the
      following data:

      Duration of anesthesia, defined as the time between induction of general anesthesia and
      awakening; Duration of surgery, defined as the time between surgical incision and the last
      stitch; Fentanyl consumption, expressed in mcg/kg /hour; Morphine consumption in the first 24
      and 48 hours after the end of surgery; Pain (NRS) at rest and after deep inhalation 10
      minutes after waking up, 24 and 48 hours after the end of surgery; Number of episodes of
      nausea and vomiting in the first 24 and 48 hours after the end of surgery; Consumption of
      antiemetic drugs in the first 24 and 48 hours after the end of surgery.

      During the study period, in addiction to blood tests carried out according to routine
      clinical practice a blood sample of 5 ml will be collected to perform epigenetic analysis.
      The semple will be added with heparin or EDTA or ascorbate ACD as anticoagulant agents and
      stored at a temperature of - 20 ° C until the delivery to the Department of Pharmacy and
      Biotechnology which will take place after the last blood sampling.

      Samples will be collected according to the following schedule reflects everyday clinical
      practice:

        -  T0: after placement of the arterial catheter, prior to administration of opiates
           (baseline blood gases);

        -  T1: 1 hour after general anesthesia induction (hemogasanalysis verification of
           mechanical ventilation);

        -  T2: after the last stitch, before the intraoperative bolus of morphine (hemogasanalysis
           testing blood loss);

        -  T3: 24 ± 6 hours after after the end of surgery (control blood count);

        -  T4: 48 ± 6 hours after the end of surgery (control blood count). On these blood samples,
           it will be assessed the degree of DNA methylation. The samples will be subjected to
           extraction of RNA and genomic DNA through ZR-Duet ™ kit DNA / RNA Miniprep (Zymo
           Research). The nucleic acids obtained from the samples will be stored at - 80 ° C until
           analysis.

      Since the change in methylation of genomic DNA is observed in various diseases, the need to
      measure the degree of overall DNA methylation has become very important. The 5-mC DNA ELISA
      kit (Zymo Research) offers a method of high-throughput, easy to identify and quantify the
      degree of DNA methylation.

      Total RNA extracted from each sample will be used for gene expression studies using RT-qPCR
      method. This technique will allow making a quantitative assessment of genes' expression
      codifying for the μ opioid receptor and enzymes belonging to the family of DNMTs. Based on
      these results, we will be able to correlate the degree of DNA methylation to the levels of
      expression of the investigated genes.
    

Observational model: Case-Only
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Patient undergoing elective major surgery under general anesthesia
      
Keywords: Hyperalgesia; Opioids; Postoperative pain; DNA methylation