Animal models are commonly used in orthopedic research1,2. Experimental procedures in this field frequently involve severe postoperative pain, and researchers have focused on refining such procedures by developing minimally invasive surgical approaches3,4 and multimodal analgesia regimens to prevent and treat postoperative pain5. The combination of several classes of parenteral analgesic drugs with regional anesthesia should be considered routinely in animal surgical proce-dures5. Regional anesthesia and nerve blocks are widely used in human anesthesia, and the use and evaluation of these techniques in dogs6–8, cats9, sheep10,11, goats12, pigs13 and rabbits14 have been described. Ultrasound guidance is an emerging aspect of peripheral regional anesthesia. Sonographic visualization enables the opti-mal distribution of local anesthetic around the nerve structures and the avoidance of complications such as intraneural and intravascular injection. Use of this technique requires a thorough understanding of the anatomical structures involved15.
Work in our laboratories involves a rabbit experi-mental model of radius osteotomy for studying allogenous bone grafts. The use of non-steroidal anti-inflammatory drugs is contraindicated in this project because these drugs can have adverse effects on bone healing. Therefore, regional analgesia is a crucial com-ponent of our multimodal pain management strategy. The main objectives of this study were to describe the relevant anatomical references and to develop an ultra-sound-guided technique for axillary brachial plexus nerve block in the rabbit. Our secondary objective was to describe the duration of sensitive and motor nerve block using ropivacaine in rabbits.
TECHNIQUERabbitsThe experimental protocol was reviewed and approved by the Animal Ethics Committee of Vall d’Hebron Research Institute. In the first phase of the study, we used a rabbit cadaver to examine the anatomy of the
1Experimental Surgery Unit, Vall d’Hebron Research Institute, Barcelona, Spain. 2Department of Anesthesiology, Vall d’Hebron Universitary Hospital, Barcelona, Spain. 3Bioengineering, Orthopaedics and Surgery in Pediatrics, Vall d’Hebron Research Institute, Barcelona, Spain. 4Hospital Sant Joan de Deu, Barcelona, Spain. Correspondence should be addressed to C.F. ([email protected]).
An ultrasound-guided technique for axillary brachial plexus nerve block in rabbitsCarla Fonseca, DVM, PhD1, Anna Server, MD2, Marielle Esteves, DVM, MS1, David Barastegui, MD3, Marta Rosal, DVM, MS1, Cesar G. Fontecha, MD, PhD3 & Francisco Soldado, MD, PhD3,4
Regional anesthesia techniques, such as nerve blocks, are routinely used in humans and can contribute to multimodal approaches to pain management in research animals. Ultrasound guidance is an emerging aspect of regional anesthesia that has the potential to optimize local delivery and distribution of anesthetic agents, thereby reducing the amounts of these agents that must be administered. The authors developed an ultrasound-guided technique for effective block of the axillary brachial plexus in rabbits. They used this technique to carry out nerve block in 14 rabbits. The procedure was accomplished in a relatively short amount of time and achieved successful nerve block in all rabbits with no adverse effects. Sonographic visualization of the distribution of the local anesthetic ropivacaine led to administration of smaller anesthetic doses in eight of the rabbits without affecting the duration of nerve block. The authors conclude that their technique is feasible and safe and provides effective analgesia of the thoracic limb in rabbits. They recommend that this technique be integrated into multimodal approaches to pain management in rabbits undergoing thoracic limb surgery.
brachial plexus. This rabbit had been euthanized at the endpoint of another experiment that did not involve any manipulation of the thoracic limb. In the second phase of the study, we used 14 New Zealand white rab-bits purchased from Granja San Bernardo (Navarra, Spain). The rabbits were 10–12 weeks old, six males and eight females, with a mean ± standard deviation (s.d.) body weight of 2.7 ± 0.2 kg.
Rabbits were singly housed in cages (R-type Cage; Tecniplast, Varese, Italy) with an overall dimension of 74 cm width, 72 cm depth and 47 cm internal height each. Each cage contained an elevated resting platform measuring 29.5 cm wide, 68.5 cm deep and 25 cm high, which served also as a shelter. All animals had ad libitum access to water and food during the experi-mental period. Food consisted of a commercial dry pellet diet formulated for rabbits (Sanders R-01; Agroalimentaria Aznar SA, Pamplona, Spain), and water was supplied by water bottles. As environmental enrichment, hay complement was supplied every day and a small amount of parsley, cabbage, carrot, apple or pear was given once per day.
Animals were housed under controlled environmen-tal conditions, with a room temperature of 20–21 °C, relative humidity of 45–55% and 15 fresh air changes per hour. Photoperiod consisted of a 12-h:12-h light:dark cycle, with a progressive transition period of 30 min simulating sunrise and nightfall.
Anatomical studyWe studied anatomical references and nerve locali-zation after dissecting the brachial plexus in a rabbit cadaver (Fig. 1). We made a skin incision in the axillary area and carefully dissected the muscles and adipose tissues using microsurgery techniques and a surgical microscope (Universal S3, Zeiss, Jena, Germany).
Anesthesia and analgesiaFor the nerve block procedure, each rabbit was placed under general anesthesia and the procedure was carried out using sterile materials and aseptic techniques. Each rabbit was premedicated with a subcutaneous injection of 50 mg ketamine (Ketalar; Pfizer, Istanbul, Turkey) and 2 mg midazolam (Hospira, Synthon Hispania, Sant Boi de Llobregat, Spain) per kg body weight. We then established intravenous (i.v.) access in the ear marginal vein and arterial access in the ear marginal artery. Each rabbit was pre-oxygenated by facemask with 100% oxygen, and anesthesia was induced by i.v. injection of 4 mg propofol (Fresenius Kabi, Homburg, Germany) per kg body weight. Endotracheal intuba-tion was carried out, and general anesthesia was main-tained with 2% isoflurane (Aerrane; Baxter, Valencia, Spain) using a fraction of inspired oxygen of 60%. Anesthesia equipment included an anesthesia system (Aespire; Datex-Ohmeda, Inc., Madison, WI) and a
multiparametric monitor (S/5 Anesthesia Compact Monitor; GE Datex-Ohmeda, GE Healthcare Finland Oy, Helsinki, Finland). Each rabbit received a con-tinuous infusion of lactated Ringer’s solution (Grifols Laboratories SA, Barcelona, Spain) at a rate of 10 ml per kg body weight per h during general anesthesia. Intra-operative monitoring consisted of electrocar-diography, pulse oximetry, invasive blood pressure and capnography. For parenteral analgesia, each rab-bit received a transdermal fentanyl patch (25 µg/h, Fendivia; Nycomed, Roskilde, Denmark) and 25 mg magnesic metamizol (Nolotil; Boehringer Ingelheim España, Barcelona, Spain) per kg body weight.
Brachial plexus nerve blockWe used a portable ultrasound imaging device (Vivid Q, GE Healthcare, Belford, UK) and a 12-MHz linear ultrasound transducer for sonographic visuali-zation. We used a 23-gauge, 35-mm needle with an electroneurostimulation port and an extension tube (Locoplex; Vygon, Ecouen, France) and a nerve stimu-lator (Stimuplex HNS 12; B.Braun Melsungen AG, Melsungen, Germany) for nerve stimulation.
We placed each rabbit in lateral recumbency, with the thoracic limb in which we planned to carry out the nerve block placed uppermost and in abduction (Fig. 2). After thorough clipping of the fur, the axillary area was prepared aseptically by topical application of 10% povidone-iodine dermic solution (Iodina; Lab. Reig Jofré SA, Barcelona, Spain). We placed the transducer on the axilla, allowing visualization of the axillary artery and vein and of hyperechoic structures just dorsal to the vessels identified as the brachial plexus (Fig. 3). Once the target nerves were centered in the visual field, we inserted the nerve stimulation needle longitudinally to
12
3
A
V
FIGURE 1 | Macroscopic dissection of the brachial plexus of the rabbit, showing close proximity between the axillary vessels (A, axillary artery; V, axillary vein) and the brachial plexus. The proximal brachial plexus is organized into cranial (1), intermediary (2) and caudal (3) trunks that provide the fascicles and terminal branches distally.
the probe. It appeared on the ultrasound image as a hyperechoic line (Fig. 4). We advanced the neurostimu-lation needle under ultrasound guidance and attempted to elicit a thoracic limb twitch by applying a stimulus of 1 mA, decreasing progressively to 0.5 mA. We car-ried out neurostimulation using a sequential electrical nerve stimulation (SENSe) technique. The stimulation pattern consisted of a sequence of two fixed impulses with durations of 0.1 ms followed by a third longer impulse with a duration of 0.26 ms . The third, longer impulse stimulated the nerve at a distance: a thoracic limb twitch indicated that the needle was directed at the nerve. We advanced the neurostimulation needle
toward the nerve while continuing SENSe to maintain the motor response. Adjustments to the current strength led to reductions of the third impulse. At the thresh-old level of 0.5 mA, three thoracic limb twitches were visible as all three impulses elicited a motor response, confirming proximity of the needle tip to the nerve. At this point, we repeatedly injected 0.2 ml of 0.4% ropi-vacaine (made by diluting 0.4 ml of a 10 mg/ml solution of Naropin (AstraZeneca AB, Södertälje, Sweden) in 0.6 ml of 0.9% saline solution (Grifols, Barcelona, Spain)) around the nerve plexus, moving the needle to different points around the brachial plexus until it was completely surrounded by ropivacaine as visualized in the ultrasound image (Fig. 5). We used the mini-mum volume of ropivacaine necessary to surround the brachial plexus with a pre-established maximum dose of 0.8 ml of 0.4% ropivacaine per kg body weight. We recorded the total amount of ropivacaine administered to each rabbit. We recorded the time required for sur-gery and the time required for nerve block in each rab-bit; these data are reported as mean ± s.d.
After the nerve block, animals underwent thoracic limb surgery (radius osteotomy). General anesthesia was stopped when the surgical procedure was finished, and the surgical wound was completely sutured. After the procedure animals were monitored with pulse oxi-metry until their oxygen saturation was >95% with-out oxygen supplementation via facial mask. Once their oxygen saturation reached 95% and they showed an interest in walking, they were returned to their home cages.
Nerve block assessmentWe assessed the effectiveness of the nerve block on sensitive function and motor function in the affected thoracic limb. We evaluated sensitive function in each rabbit by applying a noxious stimulus (pinching each digit using Halstead-mosquito straight hemo-static forceps; Aesculap AG&Co.KG, Tuttlingen, Germany) every 30 min after recovery from general anesthesia until the stimulus elicited withdrawal of the thoracic limb. We evaluated motor function in
FIGURE 2 | A rabbit that has been prepared and positioned for ultrasound-guided axillary brachial plexus nerve block.
FIGURE 3 | Imaging the axillary area of the rabbit. Ultrasound image of the axillary vein (blue circle), axillary artery (red circle) and brachial plexus roots (hyperechoic structures near the vessels).
each rabbit by testing its ability to bear weight on the affected limb every 30 min. We recorded the dura-tion of sensitive nerve block and duration of motor nerve block in each rabbit; these data are reported as mean ± s.d.
Statistical analysisWe analyzed differences in duration of sensitive and motor block in rabbits that received different doses of ropivacaine using Student’s t-tests with the software OpenEpi version 2.3.1 (Andrew G. Dean and Kevin M. Sullivan, Atlanta, GA). We considered P values <0.05 to be statistically significant.
OUTCOMEAnatomyWe visualized the roots of the four ventral nerves (C6, C7, C8 and T1) that contribute to the brachial plexus of the rabbit. The brachial plexus emerges in the ventral border of the scalenus muscle, covering the origin of the axillary artery, which passes in the mid-dle of brachial plexus branches16. The proximal bra-chial plexus is organized into three trunks—cranial trunk, intermediary trunk and caudal trunk—which result from the union of the nerve roots and provide the fascicles and terminal branches distally (Fig. 1). We observed that the nerve roots were located dorsal to the axillary vessels.
Nerve blockSurgery time was 47 ± 22 min. Rabbits recovered smoothly from general anesthesia without any adverse effects. The amount of time required for the ultrasound-guided axillary brachial plexus nerve block technique was 7.6 ± 2.9 min. To obtain complete distribution of ropivacaine around the nerves, doses of 0.6 ml ropi-vacaine per kg body weight were required in four rabbits, 0.7 ml ropivacaine per kg body weight were required in four rabbits and 0.8 ml ropivacaine per kg body weight were required in six rabbits. We observed sen-sitive nerve block in all 14 rabbits, lasting 319 ± 51 min, and we observed motor nerve block in 8 rabbits, last-ing 274 ± 14 min (Table 1). Duration of sensitive and motor nerve block were not significantly different in rabbits that received different doses of ropivacaine (P > 0.05; Table 2).
CONCLUSIONSOne key requirement for successful regional anes-thetic blocks is to ensure optimal distribution of local anesthetic in close proximity to the nerve structures15. This goal can be more effectively achieved in humans by using sonographic visualization rather than using neuro stimulation to guide administration of the anesthetic agent15,17. Furthermore, use of ultrasound guidance together with nerve stimulation can reduce the time necessary for peripheral nerve blockage in
FIGURE 4 | Introduction of the neurostimulation needle under sonographic guidance. The needle (yellow arrows) is approaching the brachial plexus dorsally to the vein (blue circle) and artery (red circle).
FIGURE 5 | Injection of ropivacaine around the brachial plexus. Final ultrasound image of ropivacaine (hypoechogenic fluid, yellow outline) surrounding the brachial plexus (hyperechoic structures in straight relation to axillary vascular structures).
humans18. Studies comparing neurostimulation and ultrasound localization for peripheral nerve blocks in animals are lacking, but our results suggest that com-bining ultrasound guidance and neurostimulation to carry out brachial plexus nerve block enables successful nerve block in a short time period in rabbits.
One advantage of ultrasound guidance is reduc-tion of the anesthetic dose needed for effective nerve block15,19. Our results show that sonographic visuali-zation enables researchers to determine when the bra-chial plexus of the rabbit is adequately surrounded by local anesthetic. This visualization allowed us to reduce the volume of anesthetic injected in some rabbits by 12.5–25%, without reducing the duration of sensitive or motor nerve block. Dose reduction is advantageous as it can minimize the risk of adverse reactions to local anes-thetics. In addition, ultrasound guidance minimized disruption of surrounding structures during the nerve block procedure.
Ropivacaine is a relatively new long-acting local anesthetic, developed as an alternative to bupivacaine, with less toxic effects on the central nervous and cardio-vascular systems20. Little information is available regarding use of ropivacaine in rabbits21, and so we used the same doses reported in clinical settings22. Rabbits had no adverse reactions to ropivacaine in our study. Ropivacaine administration resulted in a long duration of sensitive nerve block, allowing good recovery in the
immediate postoperative period. Some rabbits began voluntary ingestion of food within a few hours after surgery (data not shown). More studies should be done to investigate the minimal dose of ropivacaine required to achieve nerve block in rabbits.
Nerve blocks in laboratory rabbits have been frequently described as experimental procedures for testing local anesthetics23–26 but not as refinements in experimental surgery. We believe that regional anesthesia techniques should be routinely included in anesthesia protocols, as they can improve pain relief and animal welfare.
To our knowledge, this is the first description of an ultrasound-guided technique for nerve block of the axillary brachial plexus in the rabbit. We conclude that this technique is feasible, reproducible, safe and not time-consuming. Ropivacaine provided good and safe analgesia of the thoracic limb in the rabbit. Our tech-nique could be used routinely in integrated multimodal analgesia approaches for rabbit thoracic limb surgery.
ACKNOWLEDGMENTSWe thank Dr. Bruno Fonseca and Dra. Dolores Garcia Olmo for their critical review and helpful comments on the original manuscript and Angel Lorente and Albert Cored for their careful assistance to animal management. The project was funded by a grant from the Fundación Mutua Madrileña.
COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.
Received 30 June 2014; accepted 26 September 2014Published online at http://www.labanimal.com/
1. Pearce, A.I., Richards, R.G., Milz, S., Schneider, E. & Pearce, S.G. Animal models for implant biomaterial research in bone: a review. Eur. Cell. Mater. 13, 1–10 (2007).
2. Ahern, B.J., Parvizi, J., Boston, R. & Schaer, T.P. Preclinical animal models in single site cartilage defect testing: a systematic review. Osteoarthritis Cartilage 17, 705–713 (2009).
3. Fonseca, C. et al. An arthroscopic approach for the treatment of osteochondral focal defects with cell-free and cell-loaded PLGA scaffolds in sheep. Cytotechnology 66, 345–354 (2014).
4. Caminal, M. et al. Use of a chronic model of articular cartilage and meniscal injury for the assessment of long-term effects after autologous mesenchymal stromal cell treatment in sheep. Nat. Biotechnol. 31, 492–498 (2014).
5. Auer, J.A. et al. Refining animal models in fracture research: seeking consensus in optimising both animal welfare and scientific validity for appropriate biomedical use. BMC Musculoskelet. Disord. 8, 72 (2007).
TABLE 1 | Duration of sensitive and motor nerve block in rabbits
Rabbit
Dose of 0.4% ropivacaine (ml/kg body
weight)
Sensitive block
duration (min)
Motor block duration (min)
1 0.6 270 270
2 0.6 300 –
3 0.6 390 270
4 0.6 420 240
5 0.7 390 330
6 0.7 270 270
7 0.7 330 –
8 0.7 330 330
9 0.8 270 270
10 0.8 270 210
11 0.8 270 –
12 0.8 300 –
13 0.8 330 –
14 0.8 330 –
Mean ± s.d. 319 ± 51 274 ± 41
TABLE 2 | Duration of sensitive and motor nerve block (mean ± s.d.) in rabbits that received different doses of ropivacaine
6. Campoy, L. et al. Ultrasound-guided approach for axillary brachial plexus, femoral nerve, and sciatic nerve blocks in dogs. Vet. Anaesthes. Analg. 37, 144–153 (2010).
7. Costa-Farré, C., Blanch, X.S., Cruz, J.I. & Franch, J. Ultrasound guidance for the performance of sciatic and saphenous nerve blocks in dogs. Vet. J. 187, 221–224 (2011).
8. Echeverry, D.F. et al. Ultrasound-guided block of the sciatic and femoral nerves in dogs: a descriptive study. Vet. J. 186, 210–215 (2010).
9. Adami, C., Dayer, T., Spadavecchia, C. & Angeli, G. Ultrasound-guided pudendal nerve block in cats undergoing perineal urethrostomy: a prospective, randomised, investigator-blind, placebo-controlled clinical trial. J. Feline Med. Surg. 16, 340–345 (2014).
10. Estebe, J.-P. et al. Motor blockade by brachial plexus block in the sheep. Anesthesiology 93, 292–294 (2000).
11. Wagner, A.E., Mama, K.R., Ruehlman, D.L., Pelkey, S. & Turner, A.S. Evaluation of effects of sciatic and femoral nerve blocks in sheep undergoing stifle surgery. Lab Anim. (NY) 40, 114–118 (2011).
12. Adami, C. et al. Sciatic-femoral nerve block with bupivacaine in goats undergoing elective stifle arthrotomy. Vet. J. 188, 53–57 (2011).
13. Royal, J.M. et al. Assessment of postoperative analgesia after application of ultrasound-guided regional anesthesia for surgery in a swine femoral fracture model. J. Am. Assoc. Lab. Anim. Sci. 52, 265–276 (2013).
14. d’Ovidio, D., Rota, S., Noviello, E., Briganti, A. & Adami, C. Nerve stimulator–guided sciatic-femoral block in pet rabbits (Oryctolagus cuniculus) undergoing hind limb surgery: a case series. J. Exotic Pet Med. 23, 91–95 (2014).
15. Marhofer, P., Greher, M. & Kapral, S. Ultrasound guidance in regional anaesthesia. Br. J. Anaesth. 94, 7–17 (2005).
16. Barone, R. & Simoens, P. Anatomie Comparée des Mammifères Domestiques. Tome 7: Neurologie. II: Système Nerveux Périphérique; Glandes Endocrines; Esthésiologie (Vigot, Paris, 2010).
17. Abrahams, M.S., Aziz, M.F., Fu, R.F. & Horn, J.L. Ultrasound guidance compared with electrical neurostimulation for peripheral nerve block: a systematic review and meta-analysis of randomized controlled trials. Br. J. Anaesth. 102, 408–417 (2009).
18. Orebaugh, S.L., Williams, B.A. & Kentor, M.L. Ultrasound guidance with nerve stimulation reduces the time necessary for resident peripheral nerve blockade. Reg. Anesth. Pain Med. 32, 448–454 (2007).
19. Marhofer, P. et al. Ultrasonographic guidance reduces the amount of local anesthetic for 3-in-1 blocks. Reg. Anesth. Pain Med. 23, 584–588 (1998).
20. Leone, S., Di Cianni, S., Casati, A. & Fanelli, G. Pharmacology, toxicology, and clinical use of new long acting local anesthetics, ropivacaine and levobupivacaine. Acta Biomed. 79, 92–105 (2008).
21. Malinovsky, J.-M. et al. Intrathecal ropivacaine in rabbits: pharmacodynamic and neurotoxicologic study. Anesthesiology 97, 429–435 (2002).
22. Rosenberg, P.H., Veering, B.T. & Urmey, W.F. Maximum recommended doses of local anesthetics: a multifactorial concept. Reg. Anesth. Pain Med. 29, 564–575 (2004).
23. Boogaerts, J.G., Lafont, N.D., Luo, H. & Legros, F.J. Plasma concentrations of bupivacaine after brachial plexus administration of liposome-associated and plain solutions to rabbits. Can. J. Anaesth. 40, 1201–1204 (1993).
24. Pere, P., Watanabe, H., Pitkänen, M., Wahlström, T. & Rosenberg, P.H. Local myotoxicity of bupivacaine in rabbits after continuous supraclavicular brachial plexus block. Reg. Anesth. 18, 304–307 (1993).
25. Boogaerts, J., Lafont, N., Donnay, M., Luo, H. & Legros, F.J. Motor blockade and absence of local nerve toxicity induced by liposomal bupivacaine injected into the brachial plexus of rabbits. Acta Anaesthesiol. Belg. 46, 19–24 (1995).
26. Richard, B.M. et al. The safety of EXPAREL® (bupivacaine liposome injectable suspension) administered by peripheral nerve block in rabbits and dogs. J. Drug Deliv. 2012, 962101 (2012).
