|Year : 2019 | Volume
| Issue : 4 | Page : 654-662
Comparing effects of intrathecal adjuvants fentanyl and dexmedetomidine with hyperbaric ropivacaine in patients undergoing elective infraumbilical surgeries: A prospective, double-blind, clinical study
TK Shashikala, Sachinkumar S Sagar, Puttaiah Ramaliswamy, Vinod V Hudgi
Department of Anaesthesiology, Mysore Medical College and Research Institute, Mysore, Karnataka, India
|Date of Submission||11-Dec-2018|
|Date of Decision||10-Jan-2019|
|Date of Acceptance||23-Jan-2019|
|Date of Web Publication||16-Dec-2019|
T K Shashikala
No. 156 1st Cross, 4th Main, Bank Employees Colony, Bogadi, Mysore - 570 026, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Spinal anesthesia is most commonly used anesthesia technique for infraumbilical surgeries, and it is cost-effective with decreased hospital stay. Intrathecal isobaric ropivacaine has shorter duration of anesthesia than bupivacaine. By making, ropivacaine hyperbaric will help to achieve dense block with good postoperative analgesia. Materials and Methods: Ninety patients with American Society of Anesthesiologists physical status Classes I and II, aged between 18 and 60 years of either sex, undergoing for elective infraumbilical surgeries were randomly allocated into three groups 30 each (n = 30). Group Ropivacaine + Dexmedetomidine (RD) received 2.5 ml of 0.5% hyperbaric ropivacaine (15 mg) + dexmedetomidine 10 μg (0.5 ml), Group Ropivacaine + Fentanyl (RF) received 2.5 ml of 0.5% hyperbaric ropivacaine (15 mg) + fentanyl 25 μg (0.5 ml), and Group Ropivacaine + Normal saline (RC) received 2.5 ml of 0.5% hyperbaric ropivacaine (15 mg) +0.5 ml of normal saline. The onset, extent, and duration of sensory and motor block, duration of rescue analgesia, hemodynamic parameters, and side effects such as nausea, vomiting, pruritus, and shivering were recorded. Results: Time of onset early in RD (1.673 ± 0.567), in RF (1.73 ± 0.520), and in RC (1.763 ± 0.420) min (P = 0.783). Time to achieve maximum level of sensory block in RD (5.94 ± 1.88), in RF (3.86 ± 1.22), and RC (5.99 ± 0.46) min (P < 0.001). The total duration of analgesia in Group RD (356.67 ± 63.022), in RF (255.10 ± 35.626), and in RC (197.67 ± 37.605) min (P < 0.001). The time onset and duration of motor block in Group RD (1.59 ± 0.59, 319.57 ± 64.752), Group RF (1.59 ± 0.53, 236.83 ± 33.797), and Group RC (2.07 ± 0.20, 183.93 ± 35.252) min both are statistically highly significant (P < 0.001). Conclusion: Addition of glucose to ropivacaine makes the block dense. Addition of adjuvants such as dexmedetomidine and fentanyl further hastens the onset; prolong the postoperative analgesia with minimal hemodynamic and other side effects.
Keywords: Dexmedetomidine, fentanyl, hyperbaric ropivacaine, subarachnoid block
|How to cite this article:|
Shashikala T K, Sagar SS, Ramaliswamy P, Hudgi VV. Comparing effects of intrathecal adjuvants fentanyl and dexmedetomidine with hyperbaric ropivacaine in patients undergoing elective infraumbilical surgeries: A prospective, double-blind, clinical study. Anesth Essays Res 2019;13:654-62
|How to cite this URL:|
Shashikala T K, Sagar SS, Ramaliswamy P, Hudgi VV. Comparing effects of intrathecal adjuvants fentanyl and dexmedetomidine with hyperbaric ropivacaine in patients undergoing elective infraumbilical surgeries: A prospective, double-blind, clinical study. Anesth Essays Res [serial online] 2019 [cited 2022 May 16];13:654-62. Available from: https://www.aeronline.org/text.asp?2019/13/4/654/272982
| Introduction|| |
Spinal anesthesia is the most widely used technique for infraumbilical surgeries providing a fast onset and effective sensory and motor blockade and prolonged postoperative analgesia. A wide variety of local anesthetic drugs are available for spinal anesthesia, namely, bupivacaine, levobupivacaine, and ropivacaine. The most important physical property affecting the level of analgesia after the intrathecal administration of local anesthetic is its baricity., 0.5% hyperbaric bupivacaine hydrochloride is extensively used because of its longer duration of motor and sensory blockade.
Ropivacaine is a optically pure S-enantiomeric form of parent chiral molecule propivacaine, belongs to the pipecoloxylidide group of local anesthetics. By the addition of propyl group to the piperidine nitrogen atom compared to the butyl group in bupivacaine, ropivacaine structurally resembles the bupivacaine with similar anesthetics properties, it has reduced potential for cardiotoxicity and neurotoxicity with improved relative sensory and motor block profile. Ropivacaine being pure S-enantiomer, has low lipid solubility and blocks the nerve fibers which are involved in pain transmission to a greater degree than those involved in motor function, hence it has been used extensively to the local infiltration, epidural, and peripheral nerve block. Addition of dextrose to ropivacaine is to increase the intrathecal cephalic spread, block reliability, duration of useful block, and speed of recovery.
Ropivacaine is well tolerated after intrathecal use and was found to have a shorter duration of action than bupivacaine, making it a possible alternative to lignocaine for ambulatory surgery because of the low incidence of transient neurological symptoms. Spinal hyperbaric ropivacaine may produce more predictable and reliable anesthesia than plain ropivacaine, blocks the nerve fibers involved in pain transmission (Aδ and C fibers) to a greater degree than those controlling motor functions (Aβ fibers). Use of hyperbaric form of local anesthetics is popular among recent anesthesiological practitioners, as their effect is very predictable, but they have a shorter duration of action. To improve the block characteristics of intrathecally administered hyperbaric ropivacaine, adjuvants are added is to hasten the onset and prolongs the postoperative analgesia.
Dexmedetomidine – It is an S-enantiomer of medetomidine with a highly selective α2-adrenergic receptor agonistic activity with a relatively high ratio of α2/α1 activity (1620:1) compared to clonidine (220:1)., Dexmedetomidine acts as an agonist on α2 receptor found in the peripheral and central nervous system (CNS). Stimulation of the receptors in the brain and spinal cord inhibits neuronal firing, causing hypotension, bradycardia, sedation, and analgesia whereas the analgesic action of intrathecal α2-adrenoceptor agonist is by depressing the release of Cfiber transmitters and by hyperpolarization of postsynaptic dorsal horn neurons., Dexmedetomidine's high lipophilicity facilitates rapid absorption into the cerebrospinal fluid (CSF) and binding to spinal cord α2-adrenergic receptor. Dexmedetomidine can be used as adjuvant to local anesthetics, to prolong the duration of both motor and sensory blockade without much side effects.,, They are found to attenuate stress response to surgery and anesthesia. Recent experimental studies indicate that dexmedetomidine produces a dose-dependent increase in the duration of the motor and sensory blocks induced by local anesthetics regardless of the neuraxial route of administration (epidural, caudal, or spinal) without any evidence of neurotoxicity in human volunteers.,
Fentanyl – It is a potent lipophilic synthetic opioid with a rapid onset and duration of action with lesser incidence of respiratory depression. It is a strong agonist at the μ opioid receptor. Fentanyl is most commonly used adjuvant drug for the regional anesthesia. Intrathecal administration of opioids selectively decreases nociceptive afferent input from Aδ and C fibers without affecting dorsal root axons or somatosensory evoked potentials.
Hence, this study is designed to investigate and compare the clinical effects of hyperbaric ropivacaine with additives such as fentanyl and dexmedetomidine on spinal anesthesia for infraumbilical surgeries.
Objectives of study
Comparison of clinical effects of intrathecal 3 ml of 0.5% hyperbaric ropivacaine with additives such as fentanyl 25 μg and dexmedetomidine 10 μg in patients undergoing elective infraumbilical surgeries.
- Onset and duration of sensory blockade
- Maximum sensory blockade attained and time taken for the same
- Time taken for two-segment sensory regression
- Onset and duration of motor blockade
- Quality of motor blockade and time taken for the maximum motor blockade
- Total duration of analgesia
- Rescue analgesia.
- The objective of this study was to study the hemodynamic changes such as hypotension and bradycardia
- Side effects such as pruritus, nausea and vomiting, shivering, urinary retention, and respiratory depression.
| Materials and Methods|| |
Source of data
After approval of the Institutional Ethical Committee clearance, the data were collected in a pretested proforma meeting the objectives of this study. After obtaining approval from the Institutional Ethical Committee and informed written consent from the patients, 90 adult patients of either sex belonging to the American Society of Anesthesiologists (ASA) physical status Class I and Class II posted for elective infraumbilical surgeries at Krishna Rajendra Hospital attached to Mysore Medical College and Research Institute, Mysore, was selected for the study. The study was conducted from April 2017 to April 2018. The study population is randomly divided by shuffled closed envelope technique into three equal groups.
- Group Ropivacaine + Fentanyl (RF) (n = 30): 3 ml of 0.5% hyperbaric ropivacaine with fentanyl 25 μg
- Group Ropivacaine + Dexmedetomidine (RD) (n = 30): 3 ml of 0.5% hyperbaric ropivacaine with dexmedetomidine 10 μg
- Group Ropivacaine + Normal saline (RC) (n = 30): 3 ml of 0.5% hyperbaric ropivacaine with 0.5 ml normal saline.
- Adult patients of either sex, aged between 18 and 60 years
- Patients belonging to ASA physical status Class I and Class II
- Patients without any severe comorbid diseases.
- Patients having any absolute contraindications for spinal anesthesia such as patient not willing, raised intracranial pressure, severe hypovolemia, bleeding diathesis, local infection and cardiac, respiratory, and CNS diseases are excluded from the study
- Pregnant females. chronic diseases such as diabetes and hypertension
- Patients with body mass index >30 kg/m2
- Patients shorter than 150 cm.
The patient received 3 ml of the study drug according to randomization. Preparation of the drug will be done by the senior anesthesiologist who does the randomization but not involved further in the study and drug will be given to the anesthesiologist who performs the spinal anesthesia and he will also be the observer. Hence, the patient and the observer will be blinded to the study drug.
Preparation of the study drug
About 2 ml of isobaric ropivacaine 0.75% was loaded in the 5-ml sterile syringe. To this, 0.5 ml of 50% dextrose will be added using insulin syringe. Total 2.5 ml of study drug will contain 0.5 mg of ropivacaine and 83.33 mg of dextrose plus additives such as 25 μg fentanyl (0.5 ml) and 10 μg of dexmedetomidine (0.5 ml) and 0.5 ml of normal saline in controlled group. Hence, the total volume injected intrathecally is 3 ml, everything was done under strict aseptic precautions.
Preoperative assessment was done for each patient
Patients were kept nil per oral for solids 6 H and clear fluids 2 H before surgery.
Patients were premedicated on the night before surgery with tablet ranitidine 150 mg and tablet alprazolam 0.5 mg.
On the day of surgery, IV line obtained with18G cannula and preloaded with ringer lactate 15 mL.kg-1. Monitoring was done using multiparameter monitor (Edan im 80) having pulseoximetry, electrocardiography (ECG), and noninvasive blood pressure.
Patients were placed in lateral decubitus position with table kept flat horizontally. Under aseptic precautions, lumbar puncture was performed at the level of L2–L3 or L3–L4 through a midline approach using 25G Quincke's spinal needle and prepared study drug 2.5 ml of 0.5% hyperbaric ropivacaine and 0.5 ml of additives such as 25 μg fentanyl and 10 μg dexmedetomidine, and 0.5 ml normal saline was injected into the subarachnoid space after confirmation of free flow of CSF. Patients were made to turn into supine posture immediately after spinal anesthesia, and supplementary oxygen of 4 L was given through simple mask.
The following parameters are noted
- Onset and duration of sensory blockade
- Maximum level of sensory blockade attained and the time taken for the same
- Time for two-segment sensory regression time
- Onset and duration motor blockade
- Total duration of analgesia
- Time of rescue analgesia.
Sensory blockade was tested using the pinprick method with 27G hypodermic needle at every 30 s for first 2 min, every minute for next 5 min, and every 5 min for next 15 min and every 10 min for next 30 min and every 15 min till the end of surgery and thereafter every 30 min until sensory block is resolved.
Onset, quality, and duration of motor blockade were assessed by Modified Bromage Scale (0-3).
All patients were monitored during the surgery and perioperative period employing multiparameter monitor, which displays heart rate, systolic blood pressure (SBP), diastolic blood pressure, mean arterial pressure (MAP), ECG, and arterial oxygen saturation.
Patient was monitored during the postoperative period for requirement of analgesia and side effects such as hypotension, bradycardia, shivering, pruritus, vomiting, urinary retention, and respiratory depression. Post operative sedation was assessed as per modified Ramsay sedation scale [Table 1].
|Table 1: Postoperative sedation will be scored as per the modified Ramsay Sedation Scale|
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Onset of sensory blockade
It is defined as the time taken from the completion of the injection of study drug till the patient does not feel the pinprick at T10 level.
Time taken for maximum sensory blockade
It is defined as the time taken from the completion of the injection of the study drug to the maximum level of sensory blockade attained.
Duration of two-segment sensory regression
It is defined as the time taken from the maximum level of sensory block attained till the sensation has regressed by two segments.
Duration of sensory block
It is defined as the time taken from the completion of the injection of the study drug till the patient feels the sensation at S1 dermatome.
Onset of motor blockade
It is defined as the time taken from the completion of injection of the study drug till the patient attains modified Bromage scale Grade 1 motor blockade.
Quality of motor blockade was assessed by modified Bromage scale.
- Bromage 0 – No paralysis, patients able to flex the hip, knee, and ankle
- Bromage1 – Patients able to move the knees, unable to raise extended legs
- Bromage 2 – Patient is unable to flex the hip and knee but is able to flex the ankle
- Bromage 3 – Patient is unable to move the hip, knee, and ankle.
Duration of analgesia
It is defined as the time taken from the completion of the injection of the study drug till the patient complaints of the pain.
Time of rescue analgesia
It is defined as the time of requests for rescue analgesic in the postoperative period.
Duration of motor blockade
It is defined as the time taken from the time of injection till the patient attains complete motor recovery [Table 1].
It is defined as the reduction of SBP more than 30% below baseline value or fall in SBP <90 mmHg, and it will be treated with increased rate of intravenous (IV) fluids and if needed injection mephentermine 3 mg incremental dose will be given.
It is defined as the heart rate <50 bpm and will be treated with injection atropine 0.6 mg IV.
Patients were monitored for adverse effects such as nausea, vomiting, pruritus, respiratory depression, and also for any hypersensitivity reactions for the drugs.
Statistical sample method
Sampling was purposive sampling, done using the formula S = z2pq/d2 where z is constant, p is prevalence, q is (1−p), and d is significance level. In this study, considering hospital prevalence of 7% and confidence interval of 95%, z was 1.96 and was 0.05 and applying this formula S = sample size was 90 patients.
Data were entered into Microsoft Excel data sheet and were analyzed using SPSS 22 version software. Categorical data were represented in the form of frequencies and proportions. The Chi-square test or Fischer's exact test (for 2 × 2 tables only) was used as test of significance for qualitative data. Continuous data were represented as mean and standard deviation. Analysis of variance (ANOVA) was used as test of significance to identify the mean difference between more than two quantitative variables. If P value was significant, then Tukey's honestly significant difference posthoc multicomparison test was applied to see the significance between each pair of groups. MS Excel and MS Word were used to obtain various types of graphs. P (probability that the result is true) <0.05 was considered as statistically significant after assuming all the rules of statistical tests. Statistical software: MS Excel and SPSS version 22 (IBM SPSS Statistics, Somers NY, USA) were used to analyze data.
| Results|| |
There was no statistical difference in patient's demographic data between the groups as shown in [Table 2]a and [Table 2]b.
[Table 3] shows sensory as well as motor characteristics of spinal block. The time of onset of sensory block is early in RD group when compared to RF and RC groups, which is not statistically significance (P = 0.783) (RD < RF < RC) [Figure 1].
The time for maximal level of sensory block was achieved early in fentanyl group when compared to RD and RC groups which is statistically highly significant (P< 0.001) (RF < RD ≤ RC) [Figure 2].
The time for two-segment sensory regression was early in RC group when compared to RD and RF groups which is statistically significant (P = 0.031) (RC < RF < RD) [Figure 3].
The total duration of analgesia was maximum in Group RD (356.67 ± 63.02) min when compared to RF and RC groups which is statistically highly significant (P< 0.001) (RD > RF > RC) [Figure 4].
The time for rescue analgesia was maximum in Group RD (390.63 ± 84.29) min which is statistically highly significant when compared to RF and RC groups (P< 0.001) (RD > RF > RC) [Figure 5].
Time for onset of motor block was early in RD and RF groups when compared to RC group which is statistically highly significant (P< 0.001) (RD = RF < RC) [Figure 6].
Total duration of motor block was maximum in RD group when compared to RF and RC groups which is statistically highly significant (P< 0.001) (RD > RF > RC) [Figure 7].
Maximal level of sensory block [Table 4] and [Figure 8].
|Table 4: Distribution of the subject according to maximal level of sensory block and group|
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|Figure 8:Graph showing distribution of the subject according to maximal level of sensory block and group|
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[Table 5] shows bradycardia was noted in 7 patients (23.33%) in RD group, 1 patient in RF group (3.33%), and 2 patients in RC group (6.66) which is statistically significant (P = 0.013) [Figure 9].
Hypotension was noted in 7 patients (33.33%) in RD group, 5 patients (16.66%) in RF group (13.33%), and 4 patients in RC group [Figure 10].
Although there was a fall in MAP between 2 and 10 min in all three groups, it was statistically not significant.
Nausea and vomiting noticed in one – one patient in RC and RF groups which is not statistically significant.
Pruritus was noticed in 3 patients in RD group and 5 patients in RF group (10% and 16.66% RD, RF, respectively).
| Discussion|| |
Postoperative pain is a major cause of fear and anxiety in hospitalized patients, which also increase postoperative morbidity and mortality of patients. On the other hand, if the patient is treated well for postoperative pain, patient will cooperate with the circumstances well leading to early recovery. Subarachnoid block with local anesthetics such as bupivacaine and ropivacaine is commonly used anesthetic technique in infraumbilical surgeries, though the patient is having good intraoperative anesthesia, giving only local anesthetic to the subarachnoid block will reduces the sensory analgesia, keeping that in mind various adjuvants have been tried along with local anesthetics such as opioids like fentanyl, sufentanil, remifentanil and morphine and alpha-2 agonist like dexmedetomidine and clonidine to prolong the postoperative analgesia.
Addition of glucose to the isobaric ropivacaine will make the drug hyperbaric; it has proved to increase the speed of onset, block reliability, duration of block, and speed of recovery. Conventionally, hyperbaric bupivacaine was a drug of choice for spinal anesthesia. However, ropivacaine owing to its lower cardio and neurotoxic profile, as evident from number of studies has been emerging as a useful alternative. Intrathecal ropivacaine has been shown to produce local anesthesia with equipotent sensory block but shorter duration of motor block than intrathecal bupivacaine. Hence, the present study was to compare the efficacy of adjuvants such as fentanyl 25 μg and dexmedetomidine 10 μg with hyperbaric ropivacaine 0.5% (15 mg) for elective infraumbilical surgeries.
In the present study, the demographic data are statistically not significant.
In our study, the time of sensory onset in RD group was earlier (1.673 ± 0.567) min when compared to Group RF 1.73 ± 0.520 min and RC group 1.763 ± 0.420 min, which is statistically not significant (P = 0.783). Our study is comparable with Chatterjee et al. study, time of onset of sensory block in hyperbaric ropivacaine group (2.94 ± 0.818) min, and in hyperbaric bupivacaine, it was 1.74 ± 0.443 min.
The time for maximal level of sensory block in the present study was, in RF group 3.86 ± 1.22 min which were earlier than Group RD 5.94 ± 1.88 min and in Group RC 5.99 ± 0.46 min. Which is statistically highly significant (P< 0.001). Our study was comparable with Jagtap et al. study, time to reach the maximal sensory level in Group RF was (6.86 ± 3.73) min, in Group BF (7.07 ± 2.99) min similar to our study, the difference of 1–2 min could be due to baricity of the ropivacaine in our group.
The time for two-segment sensory regression, in our study, which was earliest in RC group (94.03 ± 6.520) min when compared to Group RF (100.00 ± 30.368) min, in Group RD (113.27 ± 38.091) min, which is statistically significant (P = 0.031); our study cannot be comparable with any other study because in other studies two-segment regression parameter was not taken.
In the present study, total duration of analgesia was maximum in Group RD (356.67 ± 63.022) min when compared to Group RF (255.10 ± 35.626) min and Group RC (197.67 ± 37.605) min, which is statistically highly significant (P< 0.001). Our study is comparable with Jagtap et al. study in which they got 234.44 ± 58.76 min in RF group, the small difference in the duration in our study could be due to baricity of ropivacaine. Our study comparable with Makhni et al. study, in their study, they got the total duration of analgesia in Group RD was (414) min the difference could be due to dosage of ropivacaine (isobaric), which was high in Makhni et al. study, that is, 22.5 mg (0.75%). Our study comparable with Gupta et al. study, in their study, the mean total duration of sensory analgesia was 316.40 ± 41.53 min in Group I (isobaric ropivacaine 0.75% 4 ml + 0.4 ml NS) and in Group II, it was 359.80 ± 66.96 min (0.75% isobaric ropivacaine 4 ml + 20 μg fentanyl). The difference in the duration of analgesia in Gupta et al. study could be due to the volume and concentration of ropivacaine.
The time for rescue analgesia in the present study was maximum in Group RD (390.63 ± 84.290) min when compared to Group RF (255.10 ± 35.626) min and Group RC (243.77 ± 41.007) min which is statistically highly significant (P< 0.001). Our study comparable with Makhni et al. study, in their study, in Group D (dexmedetomidine group), the duration of rescue analgesia was 420 min, which is almost similar to our study.
The time of onset of motor block in our study was early in both RD and RF group (1.59 ± 0.59, 1.59 ± 0.53) min, when compared to Group RC (2.07 ± 0.20) min which is statistically highly significant (P< 0.001). Our study can be comparable with Chatterjee et al. study, in their study, onset of motor block was 4.92 ± 0.752 min in Group RP and 4.02 ± 0.553 min in Group BP which was statistically significant (P< 0.001). The difference in the onset time could be due to addition of additives in our study.
Total duration of motor blocked in our study, it was maximum in Group RD (319.57 ± 64.52 min) when compared to Group RF (236.83 ± 33.797) and Group RC (183.93 ± 35.252 min), which is statistically highly significant (P< 0.001). Our study can comparable with Makhni et al. study, in their study, total duration of motor block in Group D (dexmedetomidine group) was 224.2 ± 39.2 min, the difference could be due to baricity of the ropivacaine. Our study can also be comparable with Chatterjee et al. study, in their study, the total duration of motor block in Group RP (hyperbaric ropivacaine) was 112.70 ± 9.96 min, the difference could be due to additives in our study.
The hemodynamic parameters in our study such as MAP, though it was not much fall of MAP among the groups, the fall in MAP in controlled group and in Group RF was statistically significant (P = 0.004); our study can be comparable with Gupta et al. study, in their study, they have noticed significant fall in Group RF compared to Group RC. In our study, hypotension was noted in 7 patients (33.33%) in RD group, 5 patients (16.66%) in RF group, and 4 patients (13.33%) in RC group, which was treated with incremental dose of injection mephentermine.
In our study, bradycardia was noted in 7 patients (23.33%) in RD group, in RF group one patient (3.33%), and in RC group two patient (6.66%) which was statistically significant (P = 0.013), which was treated with injection atropine 0.6 mg IV stat.
In our study, we have noticed nausea in 1 patient each Group RC and RF. Vomiting in 1 patient in each Group RC and RF; pruritus in 3 patients in Group RD and 5 patients in Group RF; and shivering was noticed in 2 patients in Group RC, 3 patients in Group RD, and 2 patients in Group RF, statistically nothing is significant (P > 0.05) [Figure 11].
| Conclusion|| |
To conclude, adding adjuvants such as dexmedetomidine 10 μg and fentanyl 25 μg hastens the onset with adequate intraoperative anesthesia and prolonged postoperative analgesia with decrease requirement of rescue analgesics, with minimal side effects. When compared to both adjuvants, dexmedetomidine is a better choice as an adjuvant to the local anesthetics. Hyperbaric ropivacaine with adjuvant such as dexmedetomidine prolongs the sensory block, faster recovery from motor block with minimal hemodynamic side effects.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]