α2-adrenergic agonists enhance central [1-3] and peripheral neural blockade [4-6] when they are added to local anaesthetics in human beings and animals. The admixture of clonidine prolongs analgesia after brachial plexus block from 40% to 100%, depending on the local anaesthetics used [4-6], and a dose dependent relationship for clonidine when added to lidocaine has been shown . This effect of clonidine admixture is beneficial in clinical practice for two main reasons: (a) to provide adequate analgesia for time consuming surgical procedures, especially in patients who are at high risk of developing complications associated with general anaesthesia and (b) to enable a reduction of potentially dangerous high doses of local anaesthetics that are required for long procedures [8-10]. A dose reduction of the local anaesthetic is especially desirable when bupivacaine is used, because of its greater potential to cause toxicity compared to other drugs [8-10]. The admixture of epinephrine and clonidine to local anaesthetics for brachial plexus block has been shown to prolong analgesia [4,5,7]. However, no study is available which tests the combined admixture of clonidine and epinephrine to bupivacaine for brachial plexus block.
Indirect evidence that clonidine enhances analgesia by means of a local effect when added to the local anaesthetic was reported in patients undergoing intercostal nerve blockade for thoracotomy  and brachial plexus block . Nevertheless, a central nervous system effect due to systemic absorption or retrograde axonal transport cannot yet be completely excluded. The objective of this study was to test whether the admixture of clonidine + epinephrine to bupivacaine enhances and/or prolongs brachial plexus block in healthy volunteers. Additionally, the study was designed to provide insight into the site of action of clonidine for possible enhancement and/or prolongation of brachial plexus block.
After approval by our Institutional Review Board, seven young and healthy male and female volunteers gave written informed consent to participate in the study. Subjects were considered to be in good health on the basis of physical examination, electrocardiogram (ECG) and routine laboratory tests. Subjects were not allowed to take any kind of drug for 1 month before the study and during the time period for which they were taking part in the study. None of the volunteers had abnormalities of the sensory and motor examination in the radial, median, ulnar and musculocutaneous nerve regions of the hand and forearm. Furthermore, all subjects with a history of injuries (fracture, burns, deep cuts, etc.) or/and surgery of the hand and forearm were excluded from the study.
On the study days, subjects were randomized in a double-blind and cross-over fashion to one of three treatment groups: (a) control group, receiving bupivacaine 0.25% (1 mg kg−1) + epinephrine 1 : 200 000 with 0.9% sodium chloride (saline) for brachial plexus block together with an intramuscular (i.m.) injection of saline; (b) i.m. group, receiving bupivacaine 0.25% (1 mg kg−1) + epinephrine 1 : 200 000 with saline for brachial plexus block together with an i.m. injection of clonidine 2 μg kg−1 and (c) block group, receiving bupivacaine 0.25% (1 mg kg−1) + epinephrine 1 : 200 000 with an admixture of clonidine 2 μg kg−1 for brachial plexus block and an i.m. injection of saline. The volumes of clonidine and saline were adjusted to be of equal magnitude by the individual who prepared the solutions, who was not further involved in the study. During the study all volunteers were assigned to all three treatment groups, thus each volunteer participated in 3 study days. More than 21 days elapsed between single study days of the volunteers.
Brachial plexus block
Volunteers were monitored using an ECG lead II, an automated sphygmomanometer and a pulse oximeter. A catheter was inserted into a peripheral vein of the unblocked forearm. Axillary perivascular brachial plexus block was then performed by the same and experienced anaesthesiologist (EMT) aided by a nerve stimulator (Stimuplex®; Braun Melsungen AG, Germany). A 22-G needle (Stimuplex®) of 5 cm was inserted high in the axilla while the subject was supine, with the arm abducted at 90°, externally rotated and the elbow flexed at 90°. The radial, median and ulnar nerve were stimulated consecutively. When a current of less than 0.5 mA elicited visible motor response one-third of the prepared drug admixture was injected into the region of the nerve. Thereafter, the same procedure was performed with the two remaining nerves. The subject was excluded from the study with failure to achieve the above-mentioned conditions or arterial puncture. An ultrasonic picture was regularly taken immediately after injection to document the distribution of the injected drugs.
Methods of evaluation
Sensory function, motor function and temperature sensitivity were evaluated before 5, 10, 15, 20, 30, 40, 50 and 60 min after injection. Thereafter, blocks were evaluated at 30 min intervals until complete recovery from sensory block, motor block and temperature block occurred. Sensory block was evaluated using a pinprick test on a three point scale (0: normal sensation; 1: blunted sensation; 2: no perception) in the four regions of nerve distribution (median, ulnar, radial and musculocutaneous nerves) in the forearm and hand. Complete sensory block was defined as no perception in all four regions of nerve distribution. For assessment of motor blockade a modified Bromage score was used (0: normal motor function with full extension and flexion of elbow, wrist and fingers; 1: decreased motor strength, with ability to move all fingers; 2: ability to move one finger or two fingers, whereas the other fingers, the wrist and the elbow cannot be moved; 3: complete motor block with inability to move the elbow, wrist and fingers) . Additionally, a temperature test to ether (0: normal; 1: loss of temperature perception) was performed. Complete temperature insensitivity was defined as loss of temperature perception in all four regions of nerve distribution.
Sedation (0: wide awake; 1: drowsy; 2: dozing intermittently; 3: asleep but awakening when addressed by name; 4: only aroused by tactile stimulation) was assessed before block evaluation. Additionally, heart rate (HR), systolic arterial pressure (SAP) and diastolic arterial pressure (DAP) were recorded automatically (Careview®; Hewlett Packard, Endover, MA, USA) at the measuring time points after evaluation of the sedation score.
For determination of clonidine plasma concentrations, 10 mL of whole blood was drawn from the venous cannula before and at 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, 150 and 180 min after the brachial plexus block. Blood samples were immediately centrifuged at 4°C with 3000 rpm for 15 min. Thereafter, plasma was harvested and immediately deep frozen at −20° to −25°C pending analysis. Analysis of plasma clonidine concentrations was performed by means of combined gas chromatography mass spectrometry after extraction. A similar method was used as described and evaluated previously by using only 1 mL plasma .
Sample size (number of subjects tested) was determined on the basis of a power analysis prior to initiation of the study. The smallest difference to be detected was defined as 100% with a standard deviation of 20%. With a two-tailed α error of 0.1 and β error of 0.2, the sample size was determined to be 2.47. A sample size of seven subjects studied in a cross-over design was found to be adequate to detect clinically relevant differences between treatment groups.
Results were reported as mean ± SEM if data were normally distributed. Whenever the data set revealed no normal distribution values were given as median and range. The Wilcoxon matched pairs signed rank sum test for correlated samples was used to detect differences in duration and onset of complete sensory blockade, complete motor blockade and complete temperature blockade. Two-way ANOVA for repeated measurements was used for comparison of the time course of the median number (#) of nerves revealing complete blockade between groups. P < 0.05 was considered significant.
Healthy volunteers were 26.6 ± 1.3 yr of age, 174.6 ± 3.5 cm in height and weighed 68.7 ± 4.1 kg. The presented data were derived from four males and three females.
Characteristics of brachial plexus blockade
Complete sensory blockade was found in five of the seven volunteers in the block treatment group compared to three volunteers in the i.m. treatment group and three volunteers in the control treatment group (Table 1). The median onset of complete sensory blockade was comparable between the three treatments (Table 1). The median duration of complete sensory blockade was significantly longer for block treatment compared to i.m. treatment (P < 0.05) and control treatment (P < 0.05) (Table 1). The number of sensory blocked nerves with no perception to pinprick was significantly higher for block treatment compared to i.m. treatment (P < 0.001) and control treatment (P < 0.001) (Fig. 1). No difference in the number of sensory blocked nerves was observed between the i.m. treatment and the control treatment (Fig. 1).
Complete motor blockade was observed in five of the seven volunteers in the block treatment group compared to two volunteers in the i.m. treatment group and two volunteers in the control treatment group (Table 1). The median onset of complete motor blockade (modified Bromage grade 3) was comparable between groups, whereas the median duration of motor blockade Bromage grade 3 was significantly shorter in the control treatment (P < 0.05) and i.m. treatment groups (P < 0.05) compared to the block treatment group (Table 1). The median Bromage grade reached at distinct observation time points was significantly higher for block treatment compared to i.m. treatment (P < 0.001) and control treatment (P < 0.001) (Fig. 2). No difference in the median Bromage grade was observed between i.m. treatment and control treatment (Fig. 2).
Complete loss of temperature perception was observed in seven volunteers with the block treatment compared to four volunteers with the i.m. treatment and three volunteers with the control treatment (Table 1). The median onset of complete loss of temperature perception was comparable between treatment groups (Table 1). The median duration of loss of temperature perception was significantly higher in the block treatment group compared to the i.m. treatment (P < 0.05) and the control treatment groups (P < 0.05) (Table 1). The median number of nerves insensitive to temperature at distinct observation time points was significantly higher for the block treatment (P < 0.001) compared the i.m. and control treatments (P < 0.001). No difference in temperature perception was observed between i.m. and control treatments.
Sedation and haemodynamics
The median sedation score was significantly lower in the control treatment group (P < 0.0001) compared to the two other treatment groups (Fig. 3), whereas no significant difference could be observed between block and i.m. treatments (Fig. 3). No grade of sedation deeper than intermittent drowsiness was observed in any of the volunteers at all measuring time points.
SAP was significantly lower in the block treatment group (P < 0.01) and the i.m. treatment group (P < 0.01) compared to the control treatment group after brachial plexus block was performed (Fig. 4a); no significant difference in SAP was observed between block and control treatments (Fig. 4a). The maximum reduction in SAP was −17% of baseline following the i.m. treatment and −15% of baseline after the block treatment within the first 240 min after the block. In contrast, the maximum reduction in SAP was −2% for the control treatment. DAP changes showed a similar results to SAP (Fig. 4b). HR fell significantly in the block treatment (P < 0.01) and the i.m. treatment groups (P < 0.01) compared to the control treatment. The maximum fall in HR was −10% of baseline for the i.m. treatment and −11% of baseline for the block treatment within the first 240 min after block. In contrast, the maximum fall in HR from baseline was −1% after the control treatment.
Plasma clonidine concentration
Mean plasma clonidine concentrations were considerably higher in the i.m. treatment group compared to the block treatment group at all time points (Fig. 5), though the difference was not significant.
Our objective was to evaluate the effects of a combined mixture of clonidine and epinephrine to bupivacaine with respect to the enhancement and prolongation of brachial plexus blockade. Additionally, the study was designed to provide insight into the site of action of clonidine for enhancement/prolongation of brachial plexus blockade.
Critique of the study design
Brachial plexus blockade was performed in healthy volunteers and each subject was assigned to all three treatments to overcome the great inter-individual variability of blockade  and avoid confounding clinical factors such as differences in injury and variability of surgery. The cross-over design was chosen to detect relevant differences in a relatively small group of experimental subjects. Furthermore, the lowest doses of bupivacaine plus 1 : 200 000 epinephrine resulting in complete sensory and complete motor blockade in healthy, young volunteers was used to test for enhancement of blockade due to additional administration of clonidine. This dose was determined from the results of a dose finding pilot trial in four other healthy subjects (unpublished observations). This dosage scheme, using the lowest possible dose, was preferred to higher doses used in clinical practice to avoid dense anaesthesia, which would have hampered detection of any clonidine effect. In the study by Culebras and colleagues the lack of clonidine effect detection could possibly be attributed to the high dose of bupivacaine (0.5%, 40 mL) additional to a combination with general anaesthesia . Additionally, electrical stimulation of the median, ulnar and radial nerves was preferred because of its greater success rate compared to sole stimulation of the median nerve . However, it must be noted that this study design using healthy, uninjured volunteers might not reflect typical clinical situations due to absence of injury and pain before blockade.
Comparison with other studies
The admixture of clonidine 2 μg kg−1 to bupivacaine 0.25% (1 mg kg−1) + epinephrine 1 : 200 000 resulted in significantly enhanced duration of complete sensory blockade, complete motor blockade and complete insensitivity to temperature compared to adding only epinephrine to the bupivacaine in our study. However, no difference in onset of blockade was observed. The enhancement of median duration of complete blockade was not observed for the i.m. administration of clonidine.
The admixture of clonidine to bupivacaine + epinephrine for brachial plexus block has not been tested before. However, the admixture of clonidine to mepivacaine + epinephrine for brachial plexus block revealed comparable results in previous studies [6,17]. Nevertheless, the study design using outpatients and data collection via telephone interviews [6,17] as well as differences in baseline data between groups  left considerable doubts about the validity of the results. However, these results were confirmed by our study. Similar findings to ours were detected by Erlacher and colleagues when adding clonidine to bupivacaine for axillary brachial plexus block in trauma patients . Prolonged duration of motor and sensory blockade was also observed when clonidine was added to ropivacaine for plexus blockade . Since ropivacaine becomes more and more popular in clinical routine this information seems of special importance.
In a previous clinical study by Gaumann and colleagues  neither the onset time nor the efficacy were improved by the admixture of clonidine to lidocaine compared to admixture of epinephrine to lidocaine. However, a mixture of clonidine + epinephrine to the bupivacaine solution indirectly suggested an additive effect of clonidine and epinephrine in our study. Bernard and Macaire described a dose dependent effect of clonidine added to lidocaine on the efficacy of block  comparable to our study, but epinephrine was not used in that study.
Mechanism of prolonged blockade with clonidine
A variety of mechanisms may be responsible for our results:
• Vasoconstriction of adjacent vessels: The vasoconstrictor effect of clonidine is inferior compared to the vasoconstrictor effect of epinephrine [5,20]. We observed enhanced duration of blockade in the block treatment group in the presence of epinephrine (Table 1, Figs 1 and 2), which is not compatible with a mechanism of local vasoconstriction.
• Action of clonidine on the central nervous system: In the present study clonidine could have acted centrally due to systemic absorption. This mechanism is well characterized for oral administration of clonidine and results in a prolonged nerve block . Additionally, slow, retrograde axonal transport  or diffusion along the nerve could directly influence the central nervous system. Systemic absorption is very unlikely responsible for enhancement and prolongation of blockade in our study, because clonidine plasma concentrations were lower in the block treatment group compared to the i.m. treatment group (Fig. 5). Retrograde axonal transport of clonidine is extremely slow (<13 mm h−1), and thus very unlikely responsible for our findings (Table 1, Figs 1 and 2).
• Direct action on local nerve fibres: Very low dose clonidine increases the C-fibre blockade from lidocaine in an isolated nerve model . Therefore, a drug interaction between bupivacaine and clonidine might offer an explanation for our results. Furthermore, the effect could be mediated by direct action of clonidine on peripheral α2-receptors . Our study design does not allow to distinguish between the above mentioned possibilities of local action. However, a local action (drug interaction, action on peripheral α2-receptors, or both) is supported by our low clonidine plasma concentrations in the block treatment group compared to the i.m. treatment group (Fig. 5). The plasma concentrations of clonidine were comparable to other studies in a similar setting .
Haemodynamic effects of adding clonidine to bupivacaine + epinephrine
In several clinical studies that tested the admixture of clonidine to the local analgesic a dose dependent incidence of adverse effects such as bradycardia or hypotension has been observed [26-28]. In accordance with these results, we observed increased sedation, decreased arterial pressure and decreased HR when clonidine was administered either locally or systemically (Figs 3 and 4). Nevertheless, these side-effects were not severe and did not result in discomfort or any endangerment of our subjects. Our study population consisted of young healthy volunteers who may be better at compensating for haemodynamic instabilities than a typical patient population . Thus, our observations on side-effects of an admixture of clonidine cannot necessarily be extrapolated to a clinical population.
To conclude, our study suggests that small doses of clonidine added to bupivacaine 0.25% (1 mg kg−1) + epinephrine 1 : 200 000 leads to prolonged and enhanced brachial plexus blockade in healthy volunteers. No relevant side-effects due to clonidine admixture were observed. This clonidine effect is likely mediated locally.
The study was funded by the Department of Clinical Pharmacology, University of Vienna, Austria.
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