Evaluation of Effects of Melatonin in Reducing Postoperative Pain and Oxidative Stress in Children Undergoing Surgery,. Trought Circulating miR-34 and miR-124a, 4-Hydroxynonenal and Sirtuin 1 (SIRT1) Plasma Concentrations.
Phase: NA
Conditions: Postoperative Pain, Oxidative Stress Response
Sponsor: University of Parma

Analgesic and Antioxidant Effects of Melatonin in Pediatric Surgery  
 
Protocol Version 
v. 1.0 
Date: 
September 15th 2020 
 
 
 
 
1. Introduction 
Pediatric patients undergoing surgery are at significant risk for experiencing postoperative pain. 
Studies have shown a high prevalence of pain and behavioral problems that can persist for several 
weeks after surgery. The memory of perioperative pain in children can be distressing and have lasting 
consequences. Children who develop negatively biased pain memories are more likely to experience 
poorer pain outcomes in the future. Research has shown that children with such memories experience 
increased postoperative pain several days after surgery. In addition, inadequate management of acute 
postoperative pain may contribute to problems such as postoperative bleeding and impaired recovery. 
These findings underscore the importance of effectively managing postoperative pain to potentially 
prevent the formation of negatively biased pain memories in young children. In addition, acute pain 
activates the nociceptive system and triggers a stress response involving the sympathetic nervous 
system and the hypothalamic-pituitary-adrenal axis. This cascade leads to cerebral, autonomic, and 
hormonal responses and induces changes in several biochemical processes. Pain has been shown to 
be associated with oxidative stress (OS) in response to tissue injury, inflammation, and cytokine 
production. 
Melatonin, a neurohormone produced by the pineal gland, plays a role in the nociceptive modulation 
of various types of pain. Melatonin has anti-inflammatory and antioxidant properties. Early oral 

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administration of melatonin in infancy has been shown to significantly reduce plasma levels of OS 
biomarkers such as total hydroperoxides and advanced oxidation protein products.  
High levels of these OS biomarkers have been observed in neonates with high pain scores, 
demonstrating a strong relationship between pain and OS. Melatonin shows a potential role in 
protecting neonates undergoing surgery from the deleterious effects of OS. After administration of 
exogenous melatonin, a significant reduction in lipid and protein peroxidation in the postoperative 
period has been reported in neonates. Recently, Song et al. reported that sirtuin 1 (SIRT1) also plays 
a critical role in the pathogenesis of pain, showing analgesic effects in chronic pain conditions such 
as neuropathic and inflammatory pain. 
Several studies have shown that lipid peroxidation is involved in different types of pain, such as 
cancer pain, fibromyalgia, and neuropathic pain. 4-HNE plasma levels are good indices of free radical 
damage on lipids. Lipid damage from free radical exposure leads to lipid hydroperoxide generation 
from lipids and to carbonyl formation and protein hydroperoxide generation from proteins. Lipid 
peroxidation reaction leads to increased endogenous production of reactive aldehydes and their 
derivatives, such as malondialdehyde and 4-HNE20. Malondialdehyde is the most mutagenic by-
product of lipid peroxidation, while 4-HNE is the most toxic. 4-HNE is often used as a marker of OS 
and has been implicated in the pathogenesis of cancer, neurodegenerative diseases, diabetes, and other 
diseases. 4-HNE is involved in many pathophysiological signaling pathways SIRT1, acting as a 
NAD+-dependent deacetylase, plays a crucial role in critical cellular functions such as energy 
metabolism, genomic stability, inflammation, and immune response. Due to its multifunctional role, 
SIRT1 has been considered a promising target for therapies aimed at treating various conditions, 
including metabolic disorders and age-related diseases. Decreased SIRT1 levels have been observed 
in the spinal cord of various pathological pain models, in which SIRT1 activation helped alleviate 
chronic pain by regulating inflammation, oxidative stress, and mitochondrial dysfunction.  

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Recent animal studies have demonstrated that miRNAs are involved in pain processing pathways. 
Ikuma et al. found the increased extracellular release of microRNAs from dorsal root ganglion cells 
in a rat model of neuropathic pain.  
In this prospective, randomized, double-blind pilot study, we test the hypothesis that melatonin, 
administered as premedication before induction of anesthesia, reduces OS and postoperative pain 
involving the sirtuin pathway in children undergoing minor abdominal surgery.  
The relationship between pain and 4-HNE, between 4-HNE and melatonin levels, and between 
microRNAs and  SIRT1 levels will be investigated.  
 
 
2. Methods 
2.1 Recruitment and Randomization 
The trial is approved by the local Ethics Committee ( LocalNo.125222022) according to the Helsinki 
Declaration of 1964. Patients' legal guardians will provide written informed consent.  
Fifteen Children between 3 and 5 years of age scheduled for elective surgery will be prospectively 
enrolled at the Department of Pediatric Surgery, University Hospital of Messina, Italy. Inclusion 
criteria are the need for elective surgery, in the morning time. Exclusion criteria are children with 
cerebral malformations and/or injuries, or surgery in the afternoon or at night to eliminate conditions 
that could affect melatonin production. Children will be also excluded in cases of withdrawal of 
informed consent, insufficient blood samples, and hemolysis of the blood sample, as hemolysis 
interferes with the biochemical determination of the OS biomarkers. 
Infants matching the clinical criteria for inclusion in the study will then be randomized to be 
premedicated with oral melatonin (Mel group) or 5% dextrose (Control group) 

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Permuted, block randomization will be performed by a biostatistics using computerized sequences, 
and group assignments will be provided in concealed opaque envelopes. Parents of participants will 
be informed of their infant's group allocation at discharge. 
Interventions 
- 
Mel group: Melatonin (Dicoson, Dicofarm, Italy, 5 drops = 1 mg) will be administered orally. 
The product is listed in the Register of Dietary Supplements on the website of the Ministry of 
Health (http://www.ministerosalute.it/alimenti/dietetica) and is classified with the following 
code: 943314283. This product is subject to the European Directive on Foodstuffs according 
to DL n. 169 of May 21, 2004, and not to the European Directive on Medicines 2001/20/EC 
transposed at the Italian level with D.L. n. 211 of June 24, 2003. Melatonin administration 
has a good safety profile, with no known adverse effects. Approximately 1 hour before 
induction of anesthesia, patients will receive oral melatonin or placebo. Mel treated children 
received a single dose of oral melatonin 0.5 mg/kg (for a max 10 mg). Melatonin (Dicoson, 
Dicofarm, Italy) will be prepared by a dedicated resident in a fixed volume of 5 mL by adding 
water to a syringe without a needle.   
- 
Control group: Approximately 1 hour before induction of anesthesia, patients will receive 5 
mL of  5% dextrose (placebo),  used to simulate the sweet taste of the melatonin formulation. 
The contents of the syringe will be blindly administered to the patients by the attending nurse, 
who will not be involved in the study. 
2.3 Perioperative management of children, analgesia and anesthesia 
The induction of general anesthesia will be obtained in all children through bolus doses of 1 mg/kg 
over 20 s of intravenous propofol, followed by similar bolus doses of propofol until the patient will 
be anesthetized, and anesthesia was maintained with inhaled sevoflurane.   

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Children will be considered anesthetized when they are asleep and unarousable, and the eyelash reflex 
disappear. Thereafter, anesthesia will be maintained with inhaled sevoflurane.  
At the end of the surgery, after returning to the ward, all patients will be assessed for postoperative 
pain using the Face, Legs, Activity, Cry, and Consolability (FLACC) scale. If FLACC score is greater 
than 3, the analgesic drug paracetamol will be administered at the dose of 15 mg/kg, every six hours 
at most. Clinical and research personnel will be unaware of the group assignments until the 
completion of data analysis. The number of doses of paracetamol administered to children of both 
treatment conditions within 48 hours after surgery, even if discharged, will be recorded. 
 
2.4 Melatonin, 4-Hydroxynonenal, and SIRT1 Measurements  
Samples of 0.2 mL of plasma will be collected at T0 (pre-hospitalization, 1 day before surgery), T1 
(after anesthesia, immediately before surgery) and T2 (at 1 h after the end of the surgery and 
awakening after anesthesia), and biochemical analyses will be performed.   
Melatonin plasma levels will be assessed at each experimental time point (T0, T1, T2) using a 
competitive enzyme-linked immunosorbent assay (cELISA) kit from Antibodies.com (A87093) 
according to the manufacturer's instructions. T1 and T2 plasma samples from Mel treated children 
suspected of containing concentrations higher than the highest standard (500 pg/mL) will be diluted 
1:100 (v/v) with sample diluent prior to analysis. Color development will be monitored at 450 nm in 
a Thermo Scientific (MultiSkan FC) microplate reader, and a standard curve (range 7.813-500 pg/mL) 
will be generated using a four-parameter logistic (4-PL) curve fit.  
4-Hydroxynonenal (4-HNE) as a marker of lipid peroxidation will be measured to evaluate OS by 
using a cELISA kit from Antibodies.com (A86962). Plasma concentrations will be calculated by 
reading the absorbance at 450 nm and referring to the standard curve (range 31.25-2000 pg/mL).  
SIRT1 will be quantified using an ELISA kit from Invitrogen (EH427RB). Plasma samples will be 
diluted 1:2 as indicated by the Manufacturer before analysis. SIRT1 concentrations will be calculated 
by absorbance reading at 450 nm and referring to the standard curve (range 1.23-300 ng/mL).  

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2.5 Quantitative real-time PCR for mature microRNAs analysis 
MicroRNA analyses will be performed based on the quali-quantitative plasma sample available (N=3 
at each time point, T0, T1, and T2). MicroRNAs (miR-34 and miR-124a) will be isolated from plasma 
using the Norgen total RNA isolation kit1.The Plasma microRNAs and spike-in cel-miR-39 
expressions will be evaluated using the TaqMan miRNA assay. The TaqMan miRNA reverse 
transcription kit will be used to reverse transcribe miRNAs. Subsequently, RT-qPCR will performe 
in 20 μL of PCR mix containing 1 μL of 20× TaqMan miRNA assay, which contained PCR primers 
and probes (5′-FAM), 10 μL of 2×TaqMan Universal PCR Master Mix No Amp Erase UNG and 5 
μL of reverse-transcribed product. The reaction will be first incubated at 95 °C for 10 min followed 
by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. The quantitative real-time PCR (RT-qPCR) 
will be performed on a ABIPRISM 7500 Real Time PCR System. Data will be analyzed by a 7500-
system software (1 1.4.0) with the automatic comparative threshold (Ct) setting for adapting baseline. 
Detection thresholds were set at 35 Ct. The relative amounts of miR-34 and miR-124a will be 
calculated using the Ct method: ΔCt = Ct (miR-34/miR-124a) − Ct (refence miRNA); 2-ΔCt. 
 
2.6 Data analysis 
Data will be expressed as mean (standard error, SE) or median (interquartile range, Q1-Q3) as 
appropriate. Differences between T0, T1, and T2 will be analyzed by repeated measures ANOVA 
followed by Tukey's multiple comparison test for melatonin, 4-HNE, miR34 and miR124a plasma 
levels, while by Friedman test followed by Dunn's test in the case of SIRT1 levels. At each 
experimental time point, comparisons between the Control and Mel treated children will be performed 
using Student's t-test for unpaired data or Mann-Whitney U test as appropriate. Linear regression 
analyses will  also be performed to examine correlations between variables (Pearson correlation). All 
data with P < 0.05 will be considered statistically significant. GraphPad Prism 6.0 (GraphPad 
Software, Inc., San Diego, CA, USA) will be used for statistical analysis. 
 

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