|
 
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| ORIGINAL ARTICLE |
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| Year : 2015 | Volume
: 8
| Issue : 4 | Page : 547-554 |
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Magnesium sulfate as an anesthetic adjuvant for children undergoing adenotonsillectomy
Noha M Elsharnouby1, Mohamed M Elsharnouby2, Nahla F Abou Elezz3
1 Department of Anaesthesia and Intensive Care, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Otorhinolaryngology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
3 Department of Community, Environmental and Occupational Medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt
| Date of Submission | 18-Nov-2013 |
| Date of Acceptance | 23-Dec-2013 |
| Date of Web Publication | 29-Dec-2015 |
Correspondence Address:
Noha M Elsharnouby
Department of Anesthesia and Intensive Care, Faculty of Medicine, Ain Shams University, 3 Ismail Fahmy Street, Seven Building Square, Heliopolise, Cairo
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/1687-7934.172739
Background
This randomized, double-blind, prospective-controlled study was designed to assess magnesium sulfate, an N-methyl-D-aspartate receptor blocker, as an anesthetic adjuvant decreasing emergence delirium/agitation, pain, postoperative analgesic requirement, and adverse events.
Patients and methods
Forty-seven patients undergoing an adenotonsillectomy were included in two parallel groups: the magnesium group received magnesium sulfate 40 mg/kg intravenously after induction of anesthesia, followed by 15 mg/kg/h by a continuous intravenous infusion during the operation. The same volume of isotonic solution was administered to the control group. Primary outcome measures were emergence delirium/agitation assessed by pediatric anesthesia emergence delirium scale. Secondary measures included intraoperative sevoflurane concentration, recovery time, pain assessed by objective pain score, time to first postoperative rescue analgesic, the total dose of rescue fentanyl required, need for rescue antiemetic, postanesthetic care unit (PACU) stay, postoperative total analgesic required, and postoperative adverse events.
Results
In the magnesium group, there was a reduction in the incidence and severity of pediatric anesthesia emergence delirium [(13 vs. 39%) and (8 vs. 14)], objective pain score (1 vs. 3), sevoflurane concentration (0.0001), time to discharge from the PACU (P = 0.04), postoperative analgesic requirement, and adverse events compared with the control group.
Conclusion
Magnesium sulfate as an anesthetic adjuvant decreased the incidence and severity of postoperative emergence agitation and pain, intraoperative sevoflurane concentration, time in PACU, and postoperative analgesic requirement. Keywords: adenotonsillectomy, adverse events, analgesic requirement, emergence delirium/agitation, magnesium sulfate, pediatric anesthesia emergence delirium score
How to cite this article:
Elsharnouby NM, Elsharnouby MM, Abou Elezz NF. Magnesium sulfate as an anesthetic adjuvant for children undergoing adenotonsillectomy. Ain-Shams J Anaesthesiol 2015;8:547-54 |
How to cite this URL:
Elsharnouby NM, Elsharnouby MM, Abou Elezz NF. Magnesium sulfate as an anesthetic adjuvant for children undergoing adenotonsillectomy. Ain-Shams J Anaesthesiol [serial online] 2015 [cited 2023 Sep 24];8:547-54. Available from: http://www.asja.eg.net/text.asp?2015/8/4/547/172739 |
| Introduction | |  |
Adenotonsillectomy is one of the most common surgical procedures in the pediatric population [1] . Sevoflurane provides fast recovery and minimal cardiovascular disturbance [2] , with emergence agitation varying from 18 and 80% mostly in preschool children [3],[4] . Although emergence agitation resolves spontaneously, it still carries the risk of self-injury and mandates extra nursing care and supplemental sedatives and analgesics, delaying patient discharge from the hospital [5] .
The reason for postoperative agitation is unclear [5] and several medications have been suggested to decrease the emergence delirium, [6],[7],[8],[9],[10],[11],[12] including ketamine, [13],[14],[15] an N-methyl-d-aspartate (NMDA) receptor blocker, which was found to be of some benefit. Magnesium sulfate is both an NMDA receptor and a calcium channel blocker with a known antinociceptive effect [16] . In adults, magnesium sulfate is used as an adjuvant in anesthesia, decreasing the anesthetic requirements and postoperative analgesia, but with conflicting results [16],[17],[18] .
The present study was designed to investigate the use of magnesium sulfate as an anesthetic adjuvant in pediatric patients undergoing an adenotonsillectomy, affecting postoperative emergence delirium/agitation, pain, intraoperative sevoflurane concentration, recovery time, postoperative analgesic requirements, and adverse effects.
| Patients and methods | | |
After receiving approval from the institution medical board and obtaining written informed parental consent, 47 children aged 3-10 years, ASA physical status (I-II), undergoing an elective outpatient adenotonsillectomy in the ENT department of Ain Shams University hospital were included in the study. Exclusion criteria were bronchial asthma, cardiovascular disease, renal disease, family history of malignant hyperthermia, mental retardation, any neurological disease potentially associated with symptoms of agitation, coagulation abnormalities, inability to provide informed consent, and children refusal to take premedication.
All patients were fasted and received paracetamol 20 mg/kg syrup orally for perioperative pain relief 60 min before induction of anesthesia. Parents were allowed to accompany their children into the operating room and stay during the induction to alleviate stress response. Before induction of anesthesia, routine monitoring ECG, pulse oximetry, and noninvasive blood pressure monitoring every 5 min during surgery were performed for the patients. Randomization was performed using a computer-generated list, and patients were randomized by opening sequentially numbered opaque envelopes immediately before entering the operating room. The children were assigned randomly to one of the two parallel groups: magnesium group (group M) and control group (group C).
Anesthesia was induced with sevoflurane in 100% oxygen through a facemask, with a gradual increase in sevoflurane concentration with every single breath to a maximum of 8%. After induction of anesthesia, an intravenous cannula was established. The magnesium group (group M) received 10% magnesium sulfate 40 mg/kg over 10 min, followed by 15 mg/kg/h, and the control group (group C) received an equal volume of 0.9% sodium chloride in a double-blinded manner. The solutions were prepared by the coordinator of the study, and the anesthetist who was in charge of the patients during the operation was unaware of the study medication.
Fentanyl (1 mg/kg) and atracurium (0.5 mg/kg) were administered to facilitate tracheal intubation. Supplemental atracurium was administered if indicated by monitoring muscle relaxation with a nerve stimulator (model NS242; Fisher Paykill, New Zealand, Australia). ETCO 2 was monitored and anesthesia was maintained with sevoflurane in oxygen. Sevoflurane concentration during surgery was adjusted to maintain the patient's heart rate (HR) and arterial blood pressure within 20% of the preinduction values with a constant fresh gas flow of 1 l/min using a semiclosed circuit system (Julian, Dräger, Germany). Ventilation was controlled to maintain normocapnia (PetCO 2 32-38 mmHg) and end tidal sevoflurane concentration was recorded. All patients received Ringer's solution in the operating room, 10 ml/kg/h, and completed in the postanesthetic care unit (PACU), 2 ml/kg/h. Standard monitoring included assessment of HR (ST segment was observed), mean arterial pressure, and arterial oxygen saturation (SpO 2 ), end tidal CO 2 tension, temperature, and inspiratory and expiratory gas concentrations (MAC), monitored and recorded every 5 min during anesthesia. Ringer's solution 15 ml/kg was administered as a fluid bolus for more than a 20% decrease in systolic blood pressure from the baseline and atropine 0.01 mg/kg for more than a 20% decrease in HR.
Adenoids were removed with suction cautery and tonsils were excised with electrocautery. No local anesthesia was used during the surgical procedures and the operation was performed by the same surgeon blinded to the anesthetic management. Blood loss was assessed by visual estimation of blood volume in sponges and the suction bottle.
Removal of the mouth gag was considered as the end of surgery. Infusion was stopped; neuromuscular blockade was antagonized with neostigmine 0.05 mg/kg and atropine 0.02 mg/kg at the end of the operation, recovery of neuromuscular function was confirmed with a nerve stimulator, and volatile anesthetics were discontinued. Oropharyngeal secretions were suctioned before extubation, and tracheal extubation was performed when normoventilation was achieved. The patients were then placed in post-tonsillectomy position until they were awake, as defined by eye opening, purposeful movement, or response to command. Time to awakening was defined as spontaneous eye opening or on command from the end of surgery and time from discontinuation of anesthesia to tracheal extubation was defined as the time to extubation; sevoflurane concentration, duration of anesthesia, and surgery were recorded by an observer blinded to anesthetic management.
Thereafter, all patients were transferred to the PACU with supplemental oxygen. Administration of oxygen was continued after extubation until the patient was awake and sustained room air saturations greater than 95% for 5 min. The duration of oxygen requirement was recorded as the time from tracheal extubation to cessation of oxygen supplementation in the PACU.
All patients were observed in the PACU for 2 h by a nurse who was blinded to the anesthetic technique. A quiet environment with one parent staying with the child was provided in the PACU. HR, systolic, and diastolic noninvasive blood pressure, respiratory rate, and SpO 2 were monitored and recorded in the PACU every 5 min for 15 min, and then at 15-min intervals for the next 2 h. Emergence agitation was assessed and recorded on admission and at 5 min, and then every 15 min thereafter using the pediatric anesthesia emergence delirium (PAED) scale [Table 1] [19] in PACU.
Fentanyl 0.5 mg/kg was administered if PAED scale was at least 10 and the agitated patients could not be calmed. Postoperative pain was evaluated using the objective pain score (OPS) [Table 2] [20] . Pain intensity was assessed on admission, 5 min after, and then every 15 min thereafter in the PACU for 2 h. If the OPS was at least 6 in the recovery room, the child received rescue analgesia as a bolus of fentanyl 0.5-1 mg/kg. Any desaturation episode with an SpO 2 below 95% was noted and recorded.
Incidences of adverse events such as nausea, retching, vomiting, shivering, breath holding, considerable secretions, laryngospasm, bradycardia, hypotension, signs of hypermagnesemia (flushing, nausea, retching, vomiting, hypotension, bradycardia, and somnolence), and bleeding were recorded after tracheal extubation and during PACU stay. Patients who had nausea, retching, and vomiting received a rescue antiemetic ondansetron 0.1 mg/kg. Hypotension and bradycardia were defined as a value of at least 15% less than the normal mean value for the child's age. Bleeding was classified as minor if medical attention was required and intravenous fluid or suction of the clot was initiated, and major if electrocautery or reoperation was required. Children were considered ready for discharge from the PACU with a modified Aldrete score of at least 9 [21] [a score of good [2] , fair [1] , or poor (0) for consciousness, respiration, hemodynamic status, skin color, and muscle force] and were free from pain, nausea, retching, and vomiting. Time to first postoperative rescue analgesic, doses of rescue analgesic received, need for rescue antiemetic, and duration of PACU stay were also recorded.
After discharge from the recovery room, every child was evaluated for pain and PAED scale every 2 h for the first 6 h and then every 4 h for 24 h after surgery. Once the child was able to tolerate oral fluids, any child with OPS score ≥6, or PAED score ≥ 10 received rescue medication of paracetamol 15 mg kg -1 orally every 6 hours as needed. If no improvement occurred, the patient received a rescue analgesic ibuprofen 5 mg/kg orally. The total dose of paracetamol and ibuprofen was recorded. Patients were discharged 24 h after surgery when they had no or mild pain, could tolerate clear fluids and soft food, had no bleeding, and no nausea, retching, or vomiting.
Primary outcome measures were emergence delirium/agitation assessed by PAED score. Secondary measures included intraoperative sevoflurane concentration, recovery time, intraoperative blood loss, time of oxygen requirement, pain assessed by the OPS score, time to first postoperative rescue analgesic, the total dose of rescue fentanyl required, need for rescue antiemetic, PACU stay, postoperative total analgesic required (paracetamol dose, ibuprofen dose), and postoperative adverse events.
Statistical analysis
A sample size of 44 patients (22 patients per group) was estimated at a power of study = 80% and a = 0.05 using Power and Sample size calculation 2.1.31 program. A pilot study of 10 patients was carried out and the expected improvement in the incidence of postoperative agitation (PAED score) after adenotonsillectomy among children would be 40% after a magnesium injection was used to calculate the sample size. 10% was added to the sample to cover for dropouts.
Data were analyzed using the computer statistical software system statistical packages for the social sciences, version 15 (SPSS Inc., Chicago, Illinois, USA). Data was presented as mean and SD, median or numbers, and percentages. Analysis of data between the groups was carried out using the Student t-test for independent samples for parametric data (age, weight, duration of surgery, duration of anesthesia, sevoflurane concentration, time to extubation, time to awakening, intraoperative blood loss time of oxygen requirement, time to discharge, fentanyl dose, paracetamol dose, ibuprofen dose, time to first postoperative rescue analgesia) or the Mann-Whitney and the Wilcoxon signed ranked test for nonparametric data (PAED score, OPS score). Categorical data between study groups (sex, ASA physical status, need for rescue antiemetic, episodes of hypotension, bradycardia, shivering, breath holding, incidence of considerable secretions, postoperative nausea, retching, and vomiting, bleeding, laryngospasm, desaturation episodes, and signs of hypermagnesemia) were compared using the c2 or Fisher's exact test. P-value less than 0.05 was considered statistically significant.
| Results | | |
Sixty patients scheduled for adenotonsillectomy from 2011 to 2012 were assigned to the study, seven parents refused to participate, and four patients refused to take the premedication. The 49 patients were then randomized and included in the study to compensate for the possible dropout because of violation of the study protocol. Two patients in group C underwent t-tube placement with the surgery; thus, statistical analysis was carried out for 47 patients: 23 in group C and 24 patients in group M.
The patients in the study group ranged in age 3-9 years, mean age 5(2) years. The groups were comparable with respect to age, sex, weight [Table 3], surgical time, duration of anesthesia, intraoperative blood loss, time to extubation, and time to awakening [Table 4]. All patients underwent the same type of surgery, performed by the same surgeon. However, sevoflurane concentration (P = 0.0001) was significantly lower in group M than in group C. No patient in the study group had any intraoperative episode of hypotension or bradycardia [Table 4].
In the recovery room, oxygen requirements were not significantly different between groups; however, group M had a significantly shorter time to discharge from the PACU (P = 0.04) and a decreased incidence of PAED score >10, median maximum PAED score, median OPS, and median maximum OPS [Table 5].
The median PAED score was significantly reduced in group M than group C, respectively, at T0 (on admission) (P < 0.0001), T1 (5 min) (P < 0.0001), T2 (15 min) (P < 0.0001), T3 (30 min) (P = 0.04), T4 (45 min) (P = 0.04), and T5 (60 min) (P = 0.03), but not significantly differently at T6 (75 min) (P = 0.1), T7 (90 min) (P = 0.9), T8 (105 min) (P = 0.6), and T9 (120 min) (P = 0.07) [Figure 1]. | Figure 1: Median pediatric anesthesia emergence delirium (PAED) on T0 = on admission, T1 = 5 min, T2 = 15 min, T3 = 30 min, T4 = 45 min, T5 = 60 min, T6 = 75 min, T7 = 90 min, T8 = 105 min, and T9 = 120 min postoperatively. *P < 0.05 was considered significant
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Moreover, the median OPS were significantly reduced in group M than in group C, respectively, on T0 (on admission) (P = 0.04), T1 (5 min) (P = 0.01), T2 (15 min) (P = 0.002), T3 (30 min) (P < 0.0001), T4 (45 min) (P = 0.001), T5 (60 min) (P = 0.04) but not significantly different at T6 (75 min) (P = 0.2), T7 (90 min) (P = 0.5), T8 (105 min) (P = 0.3), and T9 (120 min) (P = 0.7) [Figure 2]. | Figure 2: Median objective pain scale (OPS) score on T0 = on admission, T1 = 5 min, T2 = 15 min, T3 = 30 min, T4 = 45 min, T5 = 60 min, T6 = 75 min, T7 = 90 min, T8 = 105 min, and T9 = 120 min postoperatively. *P < 0.05 was considered significant
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In the PACU, time to first analgesic requirement (P = 0.0001) was significantly longer, with significantly reduced doses of rescue analgesic (fentanyl) requirement (P = 0.02) and rescue antiemetic in group M than group C [Table 5].
Postoperatively, there was a significant reduction in episodes of shivering (1 vs. 5, P = 0.02), breath holding (0 vs. 4, P = 0.03), and postoperative nausea, retching, and vomiting [4 vs.11, (P = 0.02)] in group M than in group C. The incidence of considerable secretions was not significantly reduced (2 vs.3, P = 0.6) in group M than group C. No patient had an episode of bleeding, laryngospasm, bradycardia, hypotension, desaturation, or signs of hypermagnesemia.
After discharge from the recovery room, no patient had any episode of agitation defined by PAED greater than 10 or bleeding; only three (13%) patients in group C and one (4.2%) patient in the group M had nausea, retching, and vomiting (P = 0.3), and the incidence of OPS greater than 6 was significantly reduced (P = 0.04) in group M [1 (4.2%)] than group C [6 (26.1%)]. Both the total dose of paracetamol required [447 (146) vs. 578 (216), P = 0.02] and the need for ibuprofen [1(4.2%) vs.6 (26.1%), P = 0.04] were significantly reduced in group M than in group C.
| Discussion | | |
This study evaluated magnesium sulfate 40 mg/kg, followed by magnesium sulfate infusion 15 mg/kg as an anesthetic adjuvant in children undergoing adenotonsillectomy. Magnesium sulfate decreased the intraoperative sevoflurane concentration, incidence, and severity of postoperative emergence agitation and pain, together with increased time to first analgesic requirement, decreased postoperative analgesic requirement, need for rescue antiemetic, time to discharge from PACU, and postoperative adverse events.
Emergence agitation is a complex phenomenon of multifactorial etiology, reported after sevoflurane anesthesia possibly because of central nervous system irritant effects or interaction with other medications [5] . Other risk factors that may cause emergence agitation include young age, preoperative anxiety, pain [5] , otorhinolaryngologic surgery [3] , and shorter time for awakening [3] . Thus, we studied children ranging in age from 3 to 9 years undergoing adenotonsillectomy with sevoflurane anesthesia. Magnesium sulfate was investigated because of its NMDA and calcium channel-blocking effect on central nervous system, causing sedative, anticonvulsant, and affect perioperative analgesia [16] .
In our study, the magnesium group received magnesium sulfate 40 mg/kg over 10 min after the induction of anesthesia, followed by 15 mg/kg/h by a continuous infusion during the operation. This resulted in decreased intraoperative sevoflurane concentration, but had no effect on the duration of anesthesia and surgery, intraoperative blood loss, extubation time, and time to awakening, possibly because opioid potentiation and sedative effect were counteracted by the reduced sevoflurane concentration reported in the magnesium group. The potential risk of using magnesium was the occurrence of episodes of intraoperative hypotension or bradycardia, which did not occur in both groups.
Magnesium possibly decreased the anesthetic requirements and potentiated analgesic action through NMDA receptor antagonism in the central nervous system. The calcium channel-blocking effect prolonged opioid actions and reduced catecholamine released by sympathetic stimulation, which decreased peripheral nociceptor sensitization or the stress response to surgery [16] .
There are major concerns about the assessment tool of emergence delirium/agitation in a pediatric patient group, which is responsible for the wide variation in the incidence reported in various studies [3],[4] . The PAED scale is a comprehensive validated scale available to measure the incidence and severity of emergence delirium/agitation with an appropriate cut-off value greater than 10 to define emergence delirium/agitation [19],[22],[23] . In the present study, we used the cut-off value of greater than 10 to define the incidence and severity of emergence delirium/agitation. In PACU, there was a decreased incidence of PAED score greater than 10 (13 vs. 39%) and median maximum PAED score (8 vs. 14) in the magnesium group (group M) compared with the control group (group C). Moreover, the decreased sevoflurane concentration might have contributed toward the decreased incidence and severity of postoperative emergence agitation. Pain was one of the factors contributing toward emergence agitation; [5] thus, we concurrently assed pain objectively using OPS. The median OPS and median maximum OPS were significantly reduced in group M compared with group C. In the PACU, both PAED and the OPS score were significantly decreased in the magnesium group within the first 60 min, but did not differ significantly later. The decreased incidence of emergence agitation and pain resulted in a shorter PACU stay than group C.
Magnesium as an NMDA receptor and calcium channel blocker had a sedative action with increased production of vasodilator prostaglandins [16] , which could account for the anticonvulsant action that led to a beneficial adjuvant effect, decreased incidence and severity of emergence agitation and pain, as well as decreased episodes of postoperative shivering and analgesic required. The reduced episodes of nausea, retching, vomiting, and the need of antiemetic may be because of the decreased dose of fentanyl in the magnesium group. Episodes of breath holding were also decreased in group M, with no difference in oxygen requirements and incidence of considerable secretions. No patient in our study had episodes of bleeding, laryngospasm, bradycardia, hypotension, desaturation, or signs of hypermagnesemia.
Patients were followed up after discharge from the recovery room. The severity of pain (OPS > 6) was reduced, with a decreased dose of paracetamol and ibuprofen needed in group M than group C. In both groups, no patient had PAED greater than 10, or bleeding, and only one patient in group M and three patients in group C had nausea, retching, and vomiting. Being an inexpensive drug, magnesium sulfate may be a suitable adjuvant in pediatric adenotonsillectomy.
Studies were carried out evaluating the effect of several medications on emergence agitation, with conflicting results [7],[11],[12],[14],[15],[24],[25],[26],[27],[28],[29],[30] . Midazolam [7] and propofol [25] were found to exert no effect [30] . Fentanyl may be beneficial [11],[12] , but may potentially cause delayed recovery [11] , increased nausea and vomiting [31],[32],[33] , and respiratory depression [11] . Clonidine increased sedation [26],[27] , dexmedetomedine increased emergence and extubation time [28],[29] , with concern in terms of episodes of hypotension, bradycardia, postoperative bleeding [28],[29] , and cost [34] . Ketamine produced both analgesic and opioid-sparing effects when used at low doses [14],[15] ; however, studies have reported conflicting results [14],[15],[33],[35],[36] .
The use of different doses of magnesium to reduce emergence agitation has been studied, with conflicting results. Apana and colleagues administered intravenous magnesium sulfate in 110 pediatric patients, 3-16 years of age, undergoing adenoidectomy with or without tonsillectomy. In that study, magnesium sulfate 30 mg/kg or equal volume of saline for controls was started 10 min before and infused until the end of the operation; anesthesia was induced using propofol 2-2.5 mg/kg, vecuronium 0.1 mg/kg, fentanyl 1 mg/kg, and sevoflurane at 1 MAC with nitrous oxide in an oxygen (35%) mixture was administered as maintenance. In the magnesium group, time to open eyes was significantly higher [12.7 (17.5) vs.7.7 (3.5) min], with a lower agitation score at the 60th min [1.0 (0.3)vs. 1.3 (0.7)], and no significant difference in adverse effects [37] . Another dose of magnesium sulfate was studied in 42 children 3-7 years of age undergoing an adenotonsillectomy [38] . Magnesium 15 mg/kg or saline was administered about 20 min after the endotracheal intubation intraoperatively; anesthesia was induced by a mask with 8% sevoflurane in nitrous oxide and oxygen and patients were ventilated with 60% nitrous oxide and sevoflurane at 1-1.5 MAC in oxygen. However, they did not find any significant effect in reducing the incidence of emergence agitation, except increased recovery time [38] . Another study was carried out on 70 children 4-7 years of age undergoing an adenotonsillectomy, where 30 mg/kg bolus of intravenous magnesium sulfate after induction, followed by a continuous infusion of 10 mg/kg/h or an equal volume of saline 0.9% in the control group were used [39] . The incidence of emergence agitation was significantly reduced in the magnesium group (36 vs.72%), but was not associated with a significant difference in postoperative pain scores, delayed recovery, or side effects [39] . The different dose and time of administration of magnesium sulfate, the anesthetic technique used, and pain assessment tool in these studies might have accounted for the difference from our study.
This study, although placebo controlled, has a potential limitation as the perioperative magnesium level was not measured. Further studies are warranted comparing different doses of magnesium sulfate, measuring perioperative magnesium level, and comparing magnesium sulfate with other drugs used to decrease emergence agitation.
We conclude that magnesium sulfate as an anesthetic adjuvant decreased the incidence and severity of postoperative emergence agitation and pain, intraoperative sevoflurane concentration, time in PACU, and postoperative analgesic requirements.
| Acknowledgements | | |
N.M. Elsharnouby, M.M. Elsharnouby, and N.F. Abou Elezz participated in designing the research, and collecting and interpreting the data. N.M. Elsharnouby, M.M. Elsharnouby, and N.F. Abou Elezz participated in writing and reviewing the manuscript. N.M. Elsharnouby and M.M. Elsharnouby are the authors responsible for keeping the research files. N.M. Elsharnouby submitted the final version of the manuscript and is the corresponding author.
Financial support was provided by institution resources only.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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