Re s e arch Article. Keywords: Intrathecal, Ropivacaine, Dexmedetomidine, Clonidine, Lower limb Surgeries. Article History

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IAR Journal of Anaesthesiology and Critical Care

ISSN Print : 2709-1872 | ISSN Online : 2709-1880 Frequency : Bi-Monthly

Language : English Origin : Kenya

Website : https://www.iarconsortium.org/journal-info/IARJACC

61 A Prospective Randomised Double Blind Comparative Study of the Effect of Intrathecal Dexmedetomidine and Clonidine as Adjuvants with Ropivacaine in Lower Limb Surgeries

Abstract: Background & Aim: Sub arachnoid block is most commonly used technique for lower limb sur geries. Various adjuvants are being used with local anesthesia for prolongation of intra operative and post-operative analgesia. Most of the clinical studies abo ut intrathecal alpha-2 agonists are related to clonidine. In view of few evidences of Dexmedetomidine, efficacy as adjuvant to Ropivacaine in Spinal Anesthesia. T his study was conducted to explore its usefulness and a lso to compare this new alpha-2 adrenergic agonist with the previously established and widely use d adjuvants like Clonidine on spinal block characteristics and post - operative analgesia. T his study meant to compare the effects of Dexmedetomidine 5 mcg versus Clonidine 15 mcg on blockade characteristics and postop analgesia when given as adjuvants to 0.75 % Ropivacaine. The objectives were to compare the onset and duration of sensory and the motor blockade, duration of analgesia, hemodynamic parameters and side effect profile in among two groups. Materials

& Methods: 60 adult patients posted for lower limb surgeries were randomly allocated into two groups. Group D received 3 mL 0.75% ropivacaine with 5 mcg of Dexmedetomidine and group C received 3ml of Ropivacaine with 15 mcg of Clonidine. Patients were preloaded with 10mL/kg of Ringer’s lactate. Onset and duration of sensory and motor blockade, peak sensory level, 2 segment regression duration of analgesia and rescue analgesia re quirement were noted and compared.

Results: Patients in group D had earlier onset of senso ry and motor block compared to group C. Mean time of onset of sensory block group D was 3.467 ± 0.507 min and group C wa s 3.900 ± 0.759 min with a p value of 0.012. Mean time for onset of motor block group D 9.033 ± 1.098 min was group C 10.00 ± 1.66 min with a p value of 0.010. Mean time for two segments regression in group D was 135.5 ± 7.925 min and in group C wa s 125.567 ± 5.27 min with a p value of 0.000. Duration of motor and sensory block were also significantly prolonged in group D compared to group C. Mean duration of motor block in group D was 339.367±27.031 min and in group C was 306.600±23.940 min with p value of 0.000. Mean duration of sensory block in group D was 421.133 ±21.968 min and in group C was 360.600±23.940 min with a p value of 0.000. Group D showed a significantly less requirement of analgesia than group C with a p value of 0.000.

Hemodynamic were stable and comparable among both groups. Conclusion: We conclude that dexmedetomidine is a better adjuvant than clonidine in Spinal Anaest hesia as far as patient comfort, stable cardio-respiratory parameters, intra- operative and post-operative analgesia is concerned. Overall the experience with Dexmedetomidine was quite satisfactory as compared to clonidine.

Keywords: Intrathecal, Ropivacaine, Dexmedetomidine, Clonidine, Lower limb Surgeries.

I NTRODUCTİON

Pain is one of the most dreaded effects of the disease and all medical fraternity should take its relief as one of their main duties.

Anaesthesiologists have an important role to play in the fascinating field of pain management.

In view of the wider application of regional anaesthesia procedure in modern anaesthesia practice, there is a need for local anaesthetic with desirable properties like longer duration of sensory blockade and lesser duration of motor paralysis. Ropivacaine is newer amide type of local anaesthetic having a lower potential for cardiac and central nervous system toxicity (Leone, S. et al., 2008;

Simpson, D. et al., 2005; & Wille, M. 2004; Graf, B.), shows a greater differentiation between sensory and motor blockade (McNamee, D. et al., 2002).

Adjuvant drugs are pharmacological agents possessing little pharmacological effect by themselves, but enhance or potentiate t he action of other drugs when given at the same time. Adjuvant drugs modify local anesthetic effects and reduce side effects.

Perioperatively these drugs effect:

Re s e arch Article

Article History

Re ce ive d: 30.03.2021 Re vision : 10.04.2021 Acce pte d: 20.04.2021 Publishe d: 30.04.2021

Author Details

Dr. Tipparaju Varsha¹, Dr. S. Bhupathy² & Dr. C.N.

Chandra Sekhar³

Authors Affiliations

1MBBS (DNB) Resident Anaesthesiologist

2MBBS,MD,Consultant Anaesthesiologist

3MBBS,MD,FCARCSI,MBA, Consultant Anaesthesiologist, Intensivist and Pain Physician, HOD, Department of Anaesthesiology and Pain Medicine, Yashoda Hospital, Somajiguda, Hyderabad.

Corresponding Author*

Dr.C.N.Chandra Sekhar How to Cite the Article:

Tipparaju Varsha, et al; (2021); A Prospective Randomised Double Blind Comparative Study of the Effect of Intrathecal Dexmedetomidine and Clonidine as Adjuvants with Ropivacaine in Lower Limb Surgeries. IAR J Anaes Crtic Cre. 2(2) 61-81 Copyright @ 2021: This is an open-access article distributed under the terms of the Creative Commons Attribution license which permits unrestricted use, distribution, and reproduction in any medium for non commercial use (NonCommercial, or CC-BY-NC) provided the original author and source are credited.

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Tipparaju Varsha; IAR J Anaes Crtic Cre; Vol-2, Iss- 2 (Mar-Apr- 2021): 68-88

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 Latency i.e., time of onset of local anaesthetic block

 Duration of analgesia i.e. duration of sensory and motor block

 Quality of analgesia i.e. patient satisfaction, care provider’s impression of pain relief

Various adjuvants that can be added to local anesthetics and administered in central neuraxial blockade are Opioids like Fentanyl (Biswas, B. N. et al., 2002), Sufentanyl (Dahlgren, G. et al., 1997), Morphine (Culebras, X. et al., 2000), Alpha2 agonists like De xmedetomidine (Al-Ghanem, S. et al., 2009), Clonidine (Sethi, B.S. et al., 2007), Benzodiazepines like Midazolam (Valentine, J. et al., 1996) and other adjuvants like Neostigmine (Jain, A. et al., 2012), Ketamine (Kathirvel, S. et al., 2000), Epinephrine (Abouleish, E. et al., 1993), Magnesium sulphate (Khalili, G. et al., 2011).

Knowledge and use of adjuvant drug therapy has rendered neuraxial analgesia more effective in the management of both acute and chronic pain conditions.

Alpha2 agonists have both analgesic and sedative properties when used as adjuvant in regional anaesthesia (Kamibayashi, T., & Maze, M. 2000;

Scafati, A. 2004; & Gabriel, J., & Gordin, V. 2001).

Dexmedetomidine is a new alpha2-agonist that received FDA approval in 1999 for use as short-term (less than 24 h) sedative, analgesic in the intensive care unit (Gertler, R. et al., 2001).It causes sedation without causing respiratory depression. It has sedative, analgesic, sympatholytic and anxiolytic effect that blunt many CVS responses in perioperative period. It is an S- enantiomer of Medetomidine used in veterinary medicine. It is thought that intrathecal Dexmedetomidine produces its analgesic effect by inhibiting the release of C fibers transmitters and by hyperpolarization of post-synaptic dorsal horn neurons.

The prolongation of motor effect might be caused by direct impairment of excitatory amino acid release from spinal interneurons. Alpha2 agonists produce sedative effect by acting on alpha 2-adrenergic receptors in Locus coeruleus. Alpha2 adrenoreceptors do not have an active role in the respiratory centre, therefore, Dexmedetomidine throughout a broad range of plasma concentration, has minimal effects on the respiratory system.

Clonidine is imidazoline derivative with centrally acting alpha 2 adrenergic agonist activity (Dobrydnjov, I. et al., 2003). Inhibition of substance-P release is believed to be involved in the analgesic effect.

Clonidine partially inhibits voltage gated sodium and potassium channels and supresses the generation of action potential in tonic firing spinal dorsal horn neurons. This may contribute to its analgesic effect. It stimulates central alpha2 adrenergic receptors in the brain stem, blocks the release of noradrenaline from nerve terminals leading to decreased peripheral resistance, renal vascular resistance heart rate and blood pressure.

Dexmedetomidine is a highly selective alpha2 agonist with 8 times greater affinity than Clonidine.

More clinical experience has been gained with regard to usage of Clonidine compared to Dexmedetomidine by intrathecal route. There are less studies available for these drugs as adjuvants to 0.75%Ropivaciane

In our study doses of study drugs were selected based on previous study with other local anaesthetics like Bupivacaine and comparative studies with plain Ropivacaine.

Thus, the present study was undertaken to evaluate clinical effects of adding 5mcg or Clonidine 15mcg to 0.75% Ropivacaine by intrathecal route to study the characteristics of the block, hemodynamic and postoperative pain relief in patients undergoing lower limb surgeries.

M ^ATERIAL A ^ND M ^ETHODS

Hospital ethics committee clearance was obtained for this study. Informed consent was taken from all the patients. Patients who denied participation in study were treated by routine protocol of anaesthesia with dignity.

RANDOMISATION: Randomization was done by using ‘Physical method’, where 60 folded papers were placed in a container, each one labeled either Group D or Group C with each label 30 in number.

BLINDING: The syringes containing the study drug were prepared by the third observer anaesthesia resident to assure a proper blinding procedure. 60 patients of ASA I & II physical status aged 18- 60 years scheduled to undergo elective surgery and satisfying all the inclusion criteria were enrolled in the study.

INCLUSION CRITERIA:

1. Patients of either sex in age group of 18-60 yrs.

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2. Patients with Height > 140 c ms and Weight less than 120 kgs.

3. Patients who are ASA grade I and grade II.

4. Posted for lower limb surgeries

5. Patients who are willing to give consent for participation in the study.

EXCLUSION CRITERIA:

1. Patient’s refusal

2. Patients who are ASA III & above

3. Patients who are hypersensitive to the local anaesthetics

4. Patients on long term analgesic therapy 5. Patients with peripheral neuropathy

6. Patients with local skin infections and spinal deformities

7. Patients having coagulation abnormalities.

A sample size of 30 patients each randomly allocated into two groups using power analysis based on previous studies (Gupta, R. et al., 2011; & Gupta, R. et al., 2011).

STUDY PROCEDURE:

A pre-anaesthetic evaluation comprising of history of previous medical and surgical illnesses, previous anaesthesia exposures, drug allergies; clinical examination; airway examination and baseline investigation of complete blood picture, blood sugar, serum creatinine, chest radiograph and 12 lead ECG will be done. Patients were explained about the procedure of spinal anaesthesia.

 Informed/written consent was obtained from each patient before starting the study. Every patient was supplied with patient information sheet before taking the consent.

 A specially designed proforma was used to collect the data including patient’s particulars, patient’s written informed consent, indication for surgery, the anesthetic details, intra-operative monitoring, post-operative follow up, etc.

 All patients were kept nil by mouth from midnight before surgery. All patients were informed about visual analog scale (Fig.1) (Barash Text book of clinical Anaesthesia. (2013) beforehand.

Fig 1: Visual Analogue Score Anaesthetic Procedure:

Intraoperatively, the following protocol was followed:

1. Intravenous line was secured with 18/20 Gauge cannula. All standard monitors (NIBP, ECG, Pulse oximeter) were attached before giving spinal anaesthesia.

2. Subarachnoid block under strict aseptic conditions was performed in sitting position at L3-L4 or L4- L5 interspines space with 27 Gauge Whitacre spinal needle and drug was administered after confirming the free flow of cerebrospinal fluid.

Study groups received spinal anaesthesia with GROUP D: 3 ml of 0.75% Ropivacaine +

Dexmedetomidine (5 µg) (total vol. – 3.5 ml).

GROUP C: 3 ml of 0.75% Ropivacaine + Clonidine (15 µg) (total vol.- 3.5ml).

The following parameters were observed:- Sensory blockade:

It was subjectively studied by cold swab. The patients were tested every 30 seconds at one fixed dermatome level T10 level. Then highest dermatomal level was noted. Time required for regression of level to S1 is total duration of sensory block.

Motor blockade:

It was assessed by straight leg raising while lying supine and was graded according to modified Bromage scale (Table 1)

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Table 1: Modified Bromage Scale

GRADE CRITERIA DEGREE OF BLOCK

1 Free movement of legs & feet None

2 Just able to flex knee with free movement of feet Partial 33%

3 Unable to flex knee but with free movement of feet Partial 66%

4 Unable to move legs & feet Complete paralysis

The time taken for the onset of motor blockade and the duration of motor blockade were noted. The time from injection of drug to attain bromage 3 was considered as onset of blockade and time to regain bromage score of 0 was considered as duration of motor blockade.

Haemodynamic:

Heart rate & blood pressure were monitored immediately after injection and then after every 5 minutes for first 30 minutes and then every 10 minutes thereafter throughout the surgery and every hour till the complete recovery from block. The anesthesia record was maintained and changes in heart rate, blood pressure were noted. Any decrease in heart rate below 60 beats/min was considered as bradycardia and was treated with 0.3 mg of Atropine. Similarly any fall of systolic blood pressure of more than 30 % of baseline was considered as Hypotension was treated with 6 mg of ephedrine and crystalloids and oxygen as needed.

SEDATION:

Sedation was graded according to Modified Ramsey Sedation Scale.

1. Patient is Anxious, Agitated, Restless or both 2. Patient is Cooperative, Oriented, Tranquil.

3. Patient responds to commands only

4. Patients exhibit brisk response to light glabellar tap or loud auditory stimulus

5. Patients exhibits a sluggish response to light glabellar tap or loud auditory stimulus

6. Patients exhibit no Response.

Pain:

Assessed by visual analogue scale at 6^th hour, 12^th hour, 18^th hour, 24^th hour. Injection tramadol 2mg/kg iv (ma ximum of 100mg) was given as a rescue analgesic when VAS score was greater than or equal to 4.

Side effects:

Intra-operative side effects like sedation, nausea, vomiting, shivering, backache, bradycardia, and hypotension requiring active treatment were noted.

Following variables were observed:

 Sensory blockade – onset & duration.

 Motor blockade – onset & duration.

 hemodynamic parameters

 Sedation

 Maximum level of sensory block & time to reach maximum level.

 Time to reach two segment regression

 Duration of Analgesia.

 Side effects.

 Rescue analgesic requirement.

All the parameters were recorded as per the proforma and statistically analysed.

S TATISTICAL M ^ETHODS

Method of statistical analysis:

The following methods of statistical analysis were used in this study. Data were expressed as Mean + Standard deviation, percentages (%) and numbers (n). The statistical analysis was performed by a statistician using Windostat version 9.2.

1. Proportions were compared using chi-square test of significance.

2. The student t test was used to determine whether there was a statistical difference between two treatment groups in the parameters measured.

3. One-way analysis of variance (Anova)

Differences in parameters such as heart rate, systolic blood pressure, diastolic blood pressure, VAS score, rescue analgesics and adverse effects over time were analysed using ANOVA (analysis of variance). In the entire above test p value <0.05 was accepted as indicating statistical significance.

O BSERVATIONS A ^ND R ^ESULTS

Sixty patients posted for lower limb surgeries were randomly divided into two groups:

Group D: received 0.75%Ropivacaine with 5mcg Dexmedetomidine

Group C: received 0.75% Ropivacaine with 15mcg of Clonidine

Observations and results were obtained from two groups by statistical analysis and calculation the p value

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Table 2: Comparison of mean age in group D and group C GROUP NUMBER OF PATIENTS AGE (IN YEARS)

p value

MEAN SD

GROUP D 30 38.967 11.556

0.879

GROUP C 30 38.533 10.352

Graph 1: Bar diagram showing age distribution

(X axis – Age in years, Y axis – number of patients)

Conclusion: There is no significant difference between mean age (years) in group D and group C as p value is > 0.05 Table 3: Gender wise distribution of patients in group D and group C

GENDER GROUP

p value D C

MALE 17 21

0.873 FEMALE 13 9

TOTAL 30 30

GRAPH 2: Bar diagram showing frequency distribution of gender.

CONCLUSION: there is no significant difference between proportion of gender in group D and group C as p value is

>0.05

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Table 4: Comparison mean weight (kilograms) in group D and group C

GROUP NUMB ER OF

PATIENTS

WEIGHT (Kgs) p MEAN SD

Group D 30 55.267 5.711 0.239

Group C 30 56.833 4.403

GRAPH 3: Bar diagram showing weight distribution

Conclusion: There is no significant difference between the mean weight (kgs.) in group D and group C, as the p value is

>0.05

Table 5: Comparison of mean height (cm) in group D and group C GROUP NUMBER OF PATIENTS HEIGHT (cm)

p value

Mean SD

Group D 30 157.433 5.257

0.191

Group C 30 159.033 4.038

Graph 4: Bar diagram showing height distribution

Conclusion: There is no significant difference in the mean weight of the group D and group C, as the p value is >0.05

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Table 6: Distribution of patients with respect to ASA grade ASA GRADE

GROUP p

D C I 23 20

0.399 II 7 10

TOTAL 30 30

Graph 5: Bar diagram showing ASA Grade

Conclusion: There is no significant difference in the mean weight of the group D and group C, as the p value is >0.05 Table 7: Comparison of duration of surgery between group D and group C

GROUP

NUMB ER OF

PATIENTS

DURATION OF SURGERY

p value

MEAN SD

GROUPD 30 125.90 23.458

0.453

GROUP C 30 133.667 51.238

Graph 6: Bar diagram showing duration of surgery (X axis –duration of surgery, in minutes, Y axis – number of patients)

Conclusion: In our study mean duration of surgery in group D was 125.90+23.458 and group C was 133.667±51.238 and P value was 0.453, thus both groups were comparable with regards to duration of surgery.

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COMPARISON OF STUDY PARAMETERS

Table 8: Onset of sensory and motor blockade

PARAMETER GROUP D GROUP C p- Value

Sensory (min) Blockade

MEAN SD MEAN SD

3.467 0.507 3.900 0.759 0.012

Motor (min) blockade

9.033 1.098 10.00 1.66 0.010

Graph 7: Comparison of Onset of Sensory Block

(X axis –onset of sensory block in minutes, Y axis – number of patients)

Graph 8: Comparison of Onset of Motor Block GROUP C

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(X axis –onset of sensory block in minutes, Y axis – number of patients)

In present study, mean time of onset of sensory block in Group D was 3.467 ± 0.507 min and with Group C was 3.900 ± 0.759 with a (p= 0.012). Mean time of onset of motor block in Group D was 9.033 ± 1.098 and in Group C was 10.00 ± 1.66 min with (p=0.010). This shows that the onset of sensory and motor block was faster in group D compared to group C and was statistically significant.

Table 9: comparison of time to achieve peak sensory level

PARAMETER Group D Group C p-value

Time to achieve peak sensory level

MEAN SD MEAN SD

10.600 1.037 11.413 1.15 0.005

GRAPH 9: comparison of time to achieve peak sensory level X axis –time to achieve peak level in minutes, Y axis – number of patients

Mean time required to reach the maximum level of sensory block in Group D was 10.600 ±1.037 min and in Group C was 11.413 ± 1.15min. (p= 0.005) which was statistically significant, thus the mean time required to achieve a maximum sensory level was lower with group D compared to group C

Table 10: Comparison of 2-segment regression time

PARAMETER Group D Group C p-value

2-segment regression MEAN SD MEAN SD

135.5 7.925 125.567 5.27 0.000

In our study the mean Time required for two segment regression of sensory level in group D was 135.5 ± 7.925 min while in group C this time was 125.567 ± 5.27 min. The P value was 0.00 which is statistically significant.

Graph 10: Comparison of 2 Segment Regressions

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(X axis –time for 2 segment regression in minutes, Y axis – number of patients)

Graph shows that the time required for two segment regression of sensory level in group was greater in group D (Dexmedetomidine and Ropivacaine).

Table 11: Duration of sensory, motor blockade and duration of analgesia

PARAMETER Group D Group C P value

Duration of sensory block

MEAN SD MEAN SD

421.133 21.968 360.600 23.940 0.000

Duration of motor block

339.367 27.031 306.600 23.940 0.000

Duration of analgesia

434.700 27.507 365.767 12.716 0.000

In our study, duration of sensory block in Group D was 421.133± 21.968 min and in Group C was 360.600 ±23.940 min with p value of 0. 000.Thus duration of sensory block is statistically significant (p = 0.000) in Group D as compared to Group C with Dexmedetomidine group having longer total sensory b lockade compared to Clonidine.

Duration of motor block in Group D was 339.367 ± 27.031 min and in Group C was 339.367 ± 27.031 min with p value of 0.000. Thus, duration of motor block was statistically significant (p=0.000) in Group D as compared to Group C with Dexmedetomidine group having longer duration of total motor blockade.

Total duration of Post - operative analgesia in Group D was 434.700 ± 27.507 min and in Group C was 365.767

±12.716 min with p value of 0.000. Thus, duration of Post - operative analgesia was statistically significant with Dexmedetomidine having longer total duration of Post - operative analgesia.

Graph 11: Comparison of Duration of Sensory Block

(X axis –duration of sensory block in minutes, Y axis – number of patients

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Graph 12: Comparison of Duration of Motor Block X axis –duration of motor block in minutes, Y axis – number of patients

Graph 13: Comparision of Duration of Analgesia

(X axis –duration of analgesia in minutes, Y axis – number of patients) Table 12: Comparison of Level of sedation

PARAMETER Group D Group C P value Sedation

MEAN SD MEAN SD

1.900 0.607 2.167 0.699 0.109

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Graph 14: comparison of level of sedation

Mean sedation Group D was 1.900±0.607 and in Group C 2.167±0.699 was with p value of 0.109. Thus, the level of sedation was comparable among both groups.

Table 13: Requirement of rescue analgesics

PARAMETER Group D Group C P-value

MEAN SD MEAN SD

Rescue analgesics 2.433 0.626 3.167 0.461 0.000

Graph 15: Comparison of Rescue Analgesic Requirement

The mean requirement of rescue analgesics in group D was 2.433±0.626 and in group C was 3.167±0.461 and the P value was 0.000 which shows that there was a significantly lower requirement of analgesics in group D compared to group C

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Table 14: Comparision of Systolic Blood Pressure SBP

GROUP D GROUP C

P value MEAN +SD MEAN +SD

BASELINE 125.767 +14.330 131.300 +7.580 0.067 5 MIN 120.667 +11.351 116.100 +10.873 0.117 10 MIN 112.733 +13.172 113.733 +10.389 0.745 15 MIN 113.800 + 9.863 111.033 +7.717 0.231 20 MIN 111.767 + 8.245 107.400 +7.740 0.039 25 MIN 109.233 + 7.664 107.533 +6.202 0.349 30 MIN 111.333 + 6.900 108.833 +8.192 0.206 40 MIN 117.133 + 7.267 112.933 +7.652 0.033 50 MIN 111.667 + 4.880 113.00 +7.439 0.382 60 MIN 113.600 + 5.864 113.200 +7.439 0.818 70 MIN 113.167 + 5.106 114.100 +5.833 0.512 80 MIN 112.033 + 9.550 113.767 +10.441 0.505 90 MIN 113.100 + 6.900 116.433 +10.510 0.152 100 MIN 116.533 +11.175 114.300 +7.429 0.545 110 MIN 114.400 + 7.655 114.300 +7.429 0.959 120 MIN 119.500 + 6.394 117.833 +6.524 0.322 130 MIN 119.500 + 6.421 119.800 +5.088 0.842 140 MIN 118.900 + 5.241 120.200 +4.859 0.323 150 MIN 118.967 + 6.825 118.100 +6.288 0.611 160 MIN 119.767 + 5.283 120.100 +7.218 0.839 170 MIN 119.000 + 4.749 116.733 +5.632 0.097 180 MIN 116.167 +10.171 117.967 +4.979 0.388 Pooled 115.852 +9.154 115.424 +9.076 0.335

Graph 16: Comparision of Systolic Blood Pressure

In our study, a fall in Systolic Blood Pressure of more than 30% was taken as hypotension and was treated with Inj.

ephedrine 6mg iv.

In studied Groups, the mean baseline systolic blood pressure, in Group D was 125.767 mm of Hg and in Group C was 131.300 mm of Hg. (p = 0.067). Both groups were comparable. The mean systolic blood pressure was measured at 0, 5min,10min,15min,20min,25min,30min,40min,50min,60min,70min,80min,90min,100min,110min,120min,130min,140m in,150min,160min,170min,180min period. In our study the systolic blood pressure was comparable in both groups.

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Table 15: Comparision Of Diastolic Blood Pressure

DBP

GROUP D

GROUP C

p value MEAN+SD

MEAN + SD

BASE LINE 77.133+9.895 75.733+9.777 0.584

5 min 72.700+10.242 75.733±8.792 0.223

10 min 71.700±7.979 70.867±5.097 0.632

15 min 68.833±6.696 68.367±6.441 0.784

20 min 69.467±7.642 71.000±5.408 0.373

25 min 70.667±5.554 69.833±4.654 0.531

30 min 69.967±5.592 71.833±6.193 0.225

40 min 69.567±5.569 70.733±4.927 0.394

50 min 73.667±5.542 72.267±6.186 0.360

60 min 75.433±8.924 76.900±8.138 0.509

70 min 72.900±9.960 69.867±8.046 0.200

80 min 68.200±11.109 73.200±7.712 0.047

90 min 74.533±7.519 75.267±8.263 0.720

100 min 73.333±8.527 72.033±10.033 0.591

110 min 68.533±6.580 71.733±8.403 0.106

120 min 68.533±6.580 75.733±7.216 0.000

130 min 74.267±6.085 72.967±5.461 0.387

140 min 71.933±4.025 74.000±4.077 0.053

150 min 71.533±3.866 70.733±3.413 0.399

160 min 71.200±3.398 69.800±3.576 0.125

170 min 71.633±3.285 68.733±5.051 0.011

180 min 70.567±3.775 71.600±6.049 0.431

POOLED 71.650+7.397 72.224+7.074 0.133

Graph 17: Comparison of Diastolic Blood Pressure

In studied Groups, the mean baseline diastolic blood pressure, in Group D was mm77.333±11.595 of Hg and in Group C was78.467±9.961 mm of Hg. (p =0.686). Thus, both groups were comparable in baseline pressures.

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The mean diastolic blood pressure was measured at 0,

5min,10min,15min,20min,25min,30min,40min,50min,60min,70min,80min,90min,100min,110min,120min,130min,140m in,150min,160min,170min,180min period. In our study the diastolic blood pressure was comparable in both groups.

TABLE 16: Comparision of Heart Rate Sr. No HR

Group I Group II

P Value Mean ± SD Mean ± SD

1. Baseline 77.500±7.375 79.700±6.182 0.216 2. 5 min 75.567±9.971 77.167±6.721 0.469 3. 10 min 68.133±9.291 70.000±10.062 0.458 4. 15 min 71.967±10.601 72.267±9.078 0.907 5. 20 min 76.267±6.721 69.800±7.609 0.001 6. 25 min 72.467±7.065 69.733±6.470 0.124 7. 30 min 72.833±5.670 71.433±6.632 0.383 8. 40 min 72.033±6.911 71.933±3.393 0.944 9. 50 min 73.500±8.046 73.400±5.556 0.956 10. 60 min 71.033±5.149 71.333±6.305 0.841 11. 70 min 71.167±7.202 72.933±4.955 0.273 .12. 80 min 73.400±5.661 73.233±6.611 0.917 13. 90 min 71.700±5.724 75.000±5.777 0.030 14. 100 min 73.700±5.161 75.233±4.493 0.225 15. 110 min 74.200±4.701 74.200±5.635 1.000 16. 120 min 72.933±4.185 73.233±3.997 0.777 17 130 min 75.800±3.388 74.400±4.312 0.167 18. 140 min 76.567±2.700 74.233±3.126 0.003 19. 150 min 73.700±4.450 73.667±3.889 0.975 20. 160 min 72.133±3.340 72.300±4.786 0.876 21. 170 min 73.833±2.902 72.300±2.583 0.059 22. 180 min 74.633±5.129 70.867±4.224 0.003 23. Pooled 73.380±6.543 73.138±6.206 0.469

Graph 18: Comparison of Heart Rate

In our study heart rate less than 60 beats per min was taken as bradycardia and was treated with Inj. Atropine. In studied Groups, the mean baseline Heart rate, in Group D is 77.500±7.375. And in Group C was 79.700±6.182 with a p value of 0.216. Baseline heart rates were comparable. The mean heart rates were measured at 0, 5min,10min,15min,20min,25min,30min,40min,50min,60min,70min,80min,90min,100min,110min,120min,130min,140m in,150min,160min,170min,180min. In our study the Heart rate was comparable in both groups with no significant difference.

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Table 17: Comparison of side effects

Parameter Group D Group C P-Value Mean ± SD Mean ± SD

hypotension 0.067±0.254 0.100±0.305 0.647

bradycardia 0.033±0.183 0.133±0.346 0.167

Table 18: Frequency of side effects

Parameter Group D (n=30) Group C (n=30)

Hypotension 2 3

6.66% 10.00%

Bradycardia 1 4

3.33% 13.330%

Nausea/Vomiting 0 0

00% 00%

Shivering 0 0

00% 00%

There incidence of hypotension in group D was 6.66%, the incidence of hypotension in group C was 10% which was promptly treated with Inj. ephedrine 6mg iv stat. Similarly, incidence of bradycardia in group D was 3.0 %, while the incidence of bradycardia in group C was 13.0% which was treated with Inj. Atropine 0.3 mg iv bolus. Incidence of bradycardia and hypotension were comparable among both groups. The Incidence of nausea/vomiting and shivering in group D & C was 0%.

Graph 19: Comparison of Hypotension

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Graph 20: Comparison of Bradycardia

Graph shows that the incidence of bradycardia was higher in group C but that was statistically insignificant with p - Value 0.167

So, from our study we concluded that incidence of hypotension and bradycardia was comparable among both groups.

There was no incidence of nausea/vomiting and shivering in both groups.

Table 19: Comaparison of Vas Score

HOURS GROUP D GROUP C P VALUE

MEAN + SD MEAN + SD

6TH HOUR 0.000 + 0.000 3.467 + 0.507 0.000

12TH HOUR 3.467 + 0.507 5.833 + 0.985 0.000

18 TH HOUR 5.500 + 0.508 7.300 + 0.952 0.000

24 TH HOUR 3.600 + 0.674 7.333 + 0.994 0.000

POOLED 3.142 + 2.051 5.983 + 1.805 0.000

Mean VAS scores in group D were lower than in group C. Mean value in group D was 3.142±2.051 and in group C was 5.983±1.805 with a p value of 0.000.

Graph 21: Comparison of Vas Score

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78 D ^ISCUSSION

The use of Alfa 2 agonists intrathecally are being extensively evaluated as an alternative to opioids due to opioid related side effect profile like respiratory depression, nausea, urinary retention, pruritis.

The pharmacologic properties of α-2 agonists have been extensively studied and have been employed clinically to achieve the desired effects in regional anesthesia like sedation, analgesia, anxiolysis, hypnosis and sympatholysis. Clonidine has been used successfully over the last decade for the said purpose and the introduction of dexmedetomidine has further widened the scope of α-2 agonists in regional anesthesia. The faster onset of action of local anesthetics, rapid establishment of both sensory and motor blockade, prolonged duration of analgesia into the post-operative period, dose-sparing action of local anesthetics and stable cardiovascular parameters makes these agents a very effective adjuvant in regional anesthesia.

We have undertaken a study to evaluate the efficacy of Inj. Dexmedetomidine (5mcg) with 0.75%

Ropivacaine by intrathecal route compared to In j.

Clo n id in e (15mcg) with 0.75% Ropivacaine by intrathecal route in patients undergoing Lower limb surgeries.

The demographic profile of our patients was comparable with respect to mean age, body weight, height, sex distribution, and duration of surgeries.

Onset of sensory and motor blockade:

Onset of sensory block is the time lapse between drug administration and loss of cold sensation at T10 dermatome tested by cold swab. Onset of motor block is the time lapse between drug administration and attainment of Bromage 3.

In our study the mean time of onset of sensory block in group D is (3.467±0.507) and group C is (3.900±0.759) with a p value of 0.012.

Mean time for the onset of motor block in group D is 9.033±1.098, group C 10.00±1.66 with a p value of 0.010, thus indicating a faster onset of sensory and motor block in group D compared to group C.

Dhasmana S et al., (2014) conducted a double-blind randomized control study of intrathecal ropivacaine 7.5mg with dexmedetomidine 5mcg versus ropivacaine 7.5mg with clonidine 15 mcg in patients undergoing TURP. The study showed an earlier onset of sensory and motor blockade.

In another study conducted by Bajwa S et al., (2011) a Comparative evaluation of dexmedetomidine and fentanyl for epidural analgesia in lower limb orthopedic surgeries, Onset of sensory analgesia at T10 was earlier in dexmedetomidine group (7.12±2.44min) compared to fentanyl group(9.14±2.94min).

Peak sensory level and two segment regression:

In our study the mean peak sensory level achieved is comparable in both groups but the mean time to reach the peak sensory level is significantly shorter in group D (10.600±1.037) than in group C (11.413±1.150) with a p value of 0. 005.The mean two segment regression time is also prolonged in group D.

Singh N et al., (2015) conducted a randomized, prospective, double blind study of intrathecal hyperbaric ropivacaine with Dexmedetomidine 5 mcg and hyperbaric ropivacaine with clonidine 50 mcg in lower abdominal surgeries. they concluded that time to two segment regression was significantly greater with dexmedetomidine group (148.16 + 14.42 min) compared to clonidine group (102.16 +14.69 min) with a p value of <0.001.

Duration of sensory and motor blockade:

Mean duration of sensory block in group D is (421.133 ±21.968) and in group C is (360.600±23.940) with a p value of 0.000. Mean duration of motor block in group D is (339.367±27.031) and group C is (306.600±23.940) with a p value of 0.000. Thus, the duration of both sensory and motor block is significantly prolonged in group D compared to group C.

Similar prolongations were observed in a study conducted by Mahendru V et al., (2013). Who compared dexmedetomidine, clonidine, fentanyl as adjuvants to hyperbaric bupivacaine. They concluded that intrathecal dexmedetomidine as adjuvant was associated with a prolonged motor and sensory blockade with a better postoperative analgesia and reduced requirement of rescue analgesia in first 24 hrs.

compared to clonidine and fentanyl groups.

In another study conducted by Gupta R, Verma R et al., (2011)they observed that the total duration of motor blockade was prolonged in dexmedetomidine group as compared to fentanyl group (421±21 min vs.

149.3±18.2 min, P value˂0.0001).

Ravipati P et al., (2017), conducted a randomized, prospective study between intrathecal isobaric ropivacaine 0.75% plus dexmedetomidine and isobaric ropivacaine 0.75% plus fentanyl for lower limb surgeries. They concluded that dexmedetomidine at dose of 5 mcg prolonged the duration of motor

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blockade (136.733 min) as compared to fentanyl (94.866 min) with p value of 0.00.

Duration of analgesia and rescue analgesia:

In our study mean duration of analgesia in group D is 434.700±27.507 and group C is 365.767±12.716 with a p value of 0.000. The mean VAS scores were lower in group D compared to group C. The mean requirement for rescue analgesia in group D is (2.433±0.626) and in group C is (3.167±0.461) with a p value of 0.000. Thus, the duration of analgesia is statistically significantly prolonged in group D with a significant lower requirement of rescue analgesia.

Parmar N et al., (2014) studied the effect of intrathecal ropivacaine and dexmedetomidine and demonstrated that dexmedetomidine significantly prolongs the sensory motor blockade and provides a better analgesia with reduced requirement of analgesics.

Saravana Babu M et al., (2013). conducted a prospective study of ropivacaine with dexmedetomidine versus clonidine by epidural route and concluded that dexmedetomidine is a better neuraxial adjuvant compared to clonidine in providing early blockade and prolonged postoperative analgesia with stable cardiorespiratory parameters.

Gautam B et al., (2018) conducted a randomized double-blind study on the effects of adding intrathecal dexmedetomidine to hyperbaric bupivacaine for saddle spinal block in adults undergoing Peri-anal surgeries.

They concluded that the duration of analgesia was significantly prolonged and analgesic consumption was significantly reduced in group with dexmedetomidine (501.35+ 63.90) with a p value of 0.001.

Sedation Score:

Sedation in our study was assessed by Ramsay sedation scale. The mean Ramsay sedation (RSS) score of Group D was comparable to Group C with p value of 0.109 showing no statistical significance between the two groups.

Mahendru V et al., (2013), Who compared dexmedetomidine, clonidine, fentanyl as adjuvants to hyperbaric bupivacaine showed no significant difference in the sedation scores.

Hemodynamic parameters and side effects:

Average mean systolic blood pressure in group D (dexmedetomidine and ropivacaine) was115.852+9.154 and group C (clonidine and ropivacaine) was 115.424+9.076 with a p value of 0.335. Average mean diastolic blood pressure in group D (dexmedetomidine and ropivacaine) was 71.650+7.397 and group C was 72.224+7.074 with a p value of 0.133. Average mean

heart rate in group D (dexmedetomidine and ropivacaine) 73.380+6.543and group C was 73.138+

6.206 with a p value of 0.469. In our study hemodynamic parameters and side effects are comparable in both the groups.

Gupta R, Bogra J et al., (2011) conducted a study of Dexmedetomidine as an intrathecal adjuvant for postoperative analgesia and demonstrated a no significant change in haemodynamic parameters.

Mahendru V et al., (2013) Who compared dexmedetomidine, clonidine, fentanyl as adjuvants to hyperbaric bupivacaine showed no significant changes in hemodynamic parameters and side effects both were comparable intraoperatively and postoperatively.

Kanazi G, Aouad M et al., (2006) conducted study on Effect of low-dose dexmedetomidine or clonidine on the characteristics of bupivacaine spinal blockade divided 60 patients into 3 groups . Group B received 12 mg of hyperbaric bupivacaine, group D received 12 mg of bupivacaine supplemented with 3 µg of dexmedetomidine and group C received 12 mg of bupivacaine supplemented with 30 µg of clonidine. He observed that the mean arterial pressure, heart rate and level of sedation were similar in the three groups intraoperatively and post-operatively.

Dhasmana S et al., (2014) conducted a double-blind randomized control study of intrathecal ropivacaine 7.5mg with dexmedetomidine 5mcg versus ropivacaine 7.5mg with clonidine 15 mcg by intrathecal route in patients undergoing TURP.

C ^ONCLUSION

From the present study, we draw the following conclusion:

Dexmedetomidine as an adjuvant with ropivacaine intrathecally as compared to clonidine

 Shortens the onset of sensory and motor block

 Prolongs the duration of sensory and motor blockade

 Provides effective postoperative analgesia and prolongs the duration for first rescue analgesia

 Provides desirable sedation intraoperatively

 Does not result in any major adverse effects Thus, intrathecal Dexmedetomidine in a dose of 5mcg as an adjuvant to ropivacaine increases postoperative analgesia without any significant adverse effects.

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80 R ECOMMENDATIONS

Adjuvants used in neuraxial blockade with local anaesthetics prolong the duration of analgesia into postoperative period. Complications associated with neuraxial blockade can be avoided if the adjuvant added to intrathecal local anaesthetic provides adequate analgesia for prolonged periods.

Our experience with dexmedetomidine as an adjuvant to ropivacaine have been very satisfactory in terms of blockade characteristics, post-operative analgesia and smooth and stable maintenance of hemodynamic with no significant side effects as compared to clonidine. So, we recommend the use of dexmedetomidine in a dose of 5 mcg as an adjuvant to ropivacaine for prolonged duration surgeries and those with significant post-operative pain. Further ropivacaine has an added advantage of less cardio toxicity and better differential sensory and motor blockade.

We recommend further studies to evaluate dexmedetomidine as a smaller number of studies are available.

R ^EFERENCE

1. Abouleish, E., Rawal, N., Tobon-Randall, B., Rivera-Weiss, M., Meyer, B., Wu, A., & Rashad, M. N. (1993). A clinical and laboratory study to compare the addition of 0.2 mg of morphine, 0.2 mg of epinephrine, or their combination to hyperbaric bupivacaine for spinal anesthesia in cesarean section. Anesthesia and analgesia, 77(3), 457-462.

2. Al-Ghanem, S., M. Massad, I., Al-Mustaf, M., Al- Zaben, K., Qudaisat, I., Qatawn, A. et al.,. (2009).

Effect of adding Dexmedetomidine versus Fentanyl to intrathecal bupivacaine on spinal block characteristics in gynecological procedures: A Double Blind Controlled Study. American Journal of Applied Sciences. 6(5), 882–887.

3. Babu, M. S., Verma, A. K., Agarwal, A., Tyagi, C.

M., Upadhyay, M., & Tripathi, S. (2013). A comparative study in the post-operative spine surgeries: Epidural ropivacaine with dexmedetomidine and ropivacaine with clonidine for post-operative analgesia. Indian Journal of Anaesthesia, 57(4), 371.

4. Bajwa S, Arora V, Kaur J, Singh A, Parmar S.

Comparative evaluation of dexmedetomidine and fentanyl for epidural analgesia in lower limb orthopedic surgeries. Saudi Journal of Anaesthesia.

2011;5(4):365-370

5. Barash Text book of clinical Anaesthesia. (2013).

paul G Barash, acute Pain Management, Visual analogue scale;7^th ed, 56-1619

6. Biswas, B. N., Rudra, A., Bose, B. K., Nath, S., &

Chakrabarthy, S. (2002). Intrathecal Fentanyl With Hyperbaric Bupivacaine Improves Analgesia During Caesarean Delivery And In Early Post- Operative Period. Indian Journal of Anaesthesia, 46(6), 469.

7. Culebras, X., Gaggero, G., Zatloukal, J., Kern, C.,

& Marti, R. A. (2000). Advantages of intrathecal nalbuphine, compared with intrathecal morphine, after cesarean delivery: an evaluation of postoperative analgesia and adverse effects. Anesthesia & Analgesia, 91(3), 601-605.

8. Dahlgren, G., Hultstrand, C., Jakobsson, J., Norman, M., Eriksson, E. W., & Martin, H. (1997).

Intrathecal sufentanil, fentanyl, or placebo added to bupivacaine for cesarean section. Anesthesia &

Analgesia, 85(6), 1288-1293.

9. Dhasmana, S., Singh, V., Raman, R., & Pal, M.

(2014). De xmedetomidine versus clonidine as an adjunct to intrathecal small dose ropivacaine in patients undergoing transurethral resection of

prostate. Ain-Shams Journal of

Anaesthesiology, 7(4), 534.

10. Dobrydnjov, I., Axelsson, K., Thörn, S. E., Matthiesen, P., Klockhoff, H., Holmström, B., &

Gupta, A. (2003). Clonidine Combined with Small- Dose Bupivacaine During Spinal Anesthesia for Inguinal Herniorrhaphy: A Randomized Double- Blinded Study. Anesthesia and Analgesia, 96, 1496–1503.

11. Gabriel, J., & Gordin, V. (2001). Alpha 2 agonists in regional anaesthesia and analgesia. Curr Opin Anaesthesiol, 14, 751-3

12. Gautam, B., La ma, S. M., & Sharma, M. (2018).

Effects of adding intrathecal dexmedetomidine to hyperbaric bupivacaine for saddle spinal block in adults undergoing peri-anal surgeries. Journal of Nepal Health Research Council, 16(1), 43-48.

13. Gertler, R., Brown, H., Mitchell, D., & Silvius, E.

(2001). De xmedetomidine: a novel sedative analgesic agent. In Baylor University Medical centre Proceedings. 14(1), 13-21

14. Graf, B., Abraham, I., Eberbach, N., Kunst, G., Stowe, D., & Martin, E. (2002). Differences in cardiotoxicity of bupivacaine and ropivacaine are the result of physicochemical and stereoselective properties. Anaesthesiology. 96(6), 1427-1434.

15. Gupta, R., Bogra, J., Verma, R., Kohli, M., Kushwaha, J., & Kumar, S. (2011).

Dexmedetomidine as an intrathecal adjuvant for postoperative analgesia. Indian Journal of Anaesthesia. 55(4), 347.

16. Gupta, R., Verma, R., Bogra, J., Kohli, M., Raman, R., & Kushwaha, J. K. (2011). A comparative study of intrathecal dexmedetomidine and fentanyl as adjuvants to bupivacaine. Journal of anaesthesiology, clinical pharmacology, 27(3), 339.

(21)

81

17. Jain, A., Jain, K., & Bhardawaj, N. (2012).

Analgesic efficacy of low dose intrathecal neostigmine in combination with fentanyl and bupivacaine for total knee replacement surgery. J Anaesthesiology Clin Pharmacol, 28, 486-90.

18. Kamibayashi, T., & Maze, M. (2000). Clinical uses of alpha-2 adrenergic agonists.Anaesthesiology, 93, 1345-9.

19. Kanazi, G. E., Aouad, M. T., Jabbour‐Khoury, S.

I., Al Jazzar, M. D., Alameddine, M. M., Al‐

Yaman, R., ... & Baraka, A. S. (2006). Effect of low‐dose dexmedetomidine or clonidine on the characteristics of bupivacaine spinal block. Acta anaesthesiologica scandinavica, 50(2), 222-227.

20. Kathirvel, S., Sadhasivam, S., Saxena, A., Kannan, T., & Ganjoo, P. (2000). Effects of intrathecal ketamine added to bupivacaine for spinal anaesthesia forum. Anaesthesia. 55(9), 899-904.

21. Khalili, G., Janghorbani, M., Sajedi, P., & Ahmadi, G. (2011). Effects of adjunct intrathecal magnesium sulfate to bupivacaine for spinal anesthesia: a randomized, double-blind trial in patients undergoing lower extremity surgery. Journal of anesthesia, 25(6), 892-897.

22. Krishnappa, M. S., Singh, N. R., Singh, K. M., Doddaiah, D. B., Narasimha, P. V., & Fatima, N.

(2015). A comparative study of analgesic effects of intrathecal hyperbaric ropivacaine with dexmedetomidine and hyperbaric ropivacaine with clonidine in lower abdominal surgery. Journal of Medical Society, 29(3), 164.

23. Leone, S., Di Cianni, S., Casati, A., & Fanelli, G.

(2008). Pharmacology, toxicology and clinical use of new long acting local anaesthetics, ropivacaine and levobupivacaine. Acta Biomed. 79(2), 92- 105.

24. Mahendru, V., Tewari, A., Katyal, S., Grewal, A., Singh, M. R., & Katyal, R. (2013). A comparison of intrathecal dexmedetomidine, clonidine, and fentanyl as adjuvants to hyperbaric bupivacaine for

lower limb surgery: A double blind controlled study. Journal of anaesthesiology, clinical pharmacology, 29(4), 496.

25. McNamee, D., Mc Clelland, A., Scott, S., Milligan, K., Westman, L., & Gustafsson, U.

(2002). Spinal anaesthesia: Comparison of plain ropivacaine 5mg/ml with bupivacaine 5mg/ml for major orthopaedic surgery. British Journal of Anaesthesia. 89(5), 702-706.

26. Parmar, N. K., Bhandari, G., Shahi, K. S., Chand, G., Rani, D., Sharma, G., & Kumar, S. (2014).

Effect of intrathecal ropivacaine with dexmedetomidine for operative and post operative analgesia: A prospective randomized study. Journal of Evolution of Medical and Dental Sciences, 3(11), 2917-2926.

27. Ravipati, P., Isaac, G. A., Reddy, P. N., Krishna, L., & Supritha, T. (2017). A comparative study between intrathecal isobaric Ropivacaine 0.75%

plus Dexmedetomidine and isobaric Ropivacaine 0.75% plus fentanyl for lower limb surgeries. Anesthesia, essays and researches, 11(3), 621.

28. Scafati, A. (2004). Analgesia and alpha agonists 2.Medens Rev, 4-7

29. Sethi, B.S., Sa muel, M., & Sreevastava, D. (2007).

Efficacy of analgesic effects of low dose intrathecal clonidine as adjuvant to bupivacaine. Indian J Anaesth, 51, 4159.

30. Simpson, D., Curran, M., Oldfield, V., & Keating, G. (2005). Ropivacaine. Drugs. 65(18), 2675- 2717.

31. Valentine, J., Lyons, G., & Bella my, M. (1996).

The effect of intrathecal midazolam on postoperative pain. European Journal of Anaesthesiology. 13(6), 589–593.

32. Wille, M. (2004). Intra thecal use of ropivacaine: a review. Acta Anesth Belg, 55, 251-9.

Re s e arch Article. Keywords: Intrathecal, Ropivacaine, Dexmedetomidine, Clonidine, Lower limb Surgeries. Article History

IAR Journal of Anaesthesiology and Critical Care

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A Prospective Randomised Double Blind Comparative Study of the Effect of Intrathecal Dexmedetomidine and Clonidine as Adjuvants with Ropivacaine in Lower Limb Surgeries

I NTRODUCTİON

Re s e arch Article

62

M ATERIAL A ND M ETHODS

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S TATISTICAL M ETHODS

O BSERVATIONS A ND R ESULTS

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D ISCUSSION

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C ONCLUSION

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R ECOMMENDATIONS

R EFERENCE

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M ^ATERIAL A ^ND M ^ETHODS

S TATISTICAL M ^ETHODS

O BSERVATIONS A ^ND R ^ESULTS

D ^ISCUSSION

C ^ONCLUSION

R ^EFERENCE