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A Model of Anterior Cruciate Ligament Injury in Cynomolgus Monkeys Developed Via Arthroscopic Surgery

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A Model of Anterior Cruciate Ligament Injury in Cynomolgus Monkeys Developed Via Arthroscopic Surgery EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 2239-2246, 2018 A model of anterior cruciate ligament injury in cynomolgus monkeys developed via arthroscopic surgery LEI ZHANG1*, TAIYUAN GUAN1*, JI QI2*, SHAOQUN ZHANG2*, XIN ZHOU1, YANG LIU1 and SHIJIE FU1 1Department of Orthopedics, The Affiliated T.C.M Hospital of Southwest Medical University, Luzhou, Sichuan 646000; 2Department of Orthopedics, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China Received August 4, 2017; Accepted January 5, 2018 DOI: 10.3892/etm.2018.5722 Abstract. The anterior cruciate ligament (ACL) is an impor- Introduction tant structure that maintains the stability of knee joints. Animal models of ACL injury are helpful to explore its under- The anterior cruciate ligament (ACL) is among the important lying mechanisms, and strategies for prevention, treatment structures that maintain the stability of knee joints (1,2). Partial and rehabilitation. Therefore, the aim of the present study was ACL injury and complete ruptures of the ACL are associated to develop an efficient model of ACL injury in cynomolgus with high energy injuries and sports trauma. ACL injury is monkeys via arthroscopic techniques. In the present study, commonly divided into three types: Mild, moderate and 18 cynomolgus monkeys were randomly divided into a model severe injury, According to the severity of the injury. In terms group (n=6), a sham operation group (n=6) and a blank control of the severity of injury, conservative treatment or ligament group (n=6). One-quarter of the ACL was removed under reconstruction can be recommended. Whichever treatment is arthroscopy in the model group. In the sham operation group, selected, developing an animal model of ACL injury is one of only arthroscopic exploration was performed as a control. In the effective methods of studying the developmental mecha- the blank control group, monkeys were housed under the same nism, prevention, treatment and rehabilitation of ACL injury. conditions for the same length of time. Magnetic resonance Severe injury or complete ruptures of the ACL have more imaging examination was performed pre- and post-operatively, impact on the stability of knee joints, and ligament reconstruc- as well as measurements of the circumference of the thigh tion is often considered as a clinical treatment (3-8). Injury and calf, and of the maximum flexion degree of the knee. to one-quarter of the ACL is referred to as a mild injury. In Anterior drawer test, Lachman test and pivot-shift tests were cases of mild injury, if the other ligaments and muscles around also performed. The results revealed that the injured side of the knee joint still maintain the stability of the joint, ligament the knees in the model group became unstable, as determined reconstruction surgery is not necessary, and non-surgical from evaluation of the physical tests. In conclusion, based on treatment is usually applied. In this aspect, related research these findings, the modeling method of ACL injury was effec- using animal models of partial ACL injury are helpful and tive, and may contribute to the associated research concerning necessary to improve the efficiency of non‑surgical treatments ACL injury. and the recovery from ACL injury. Many animal models of ACL injury have been reported, some of which have been widely used in related research (9-11). Generally speaking, there is still no suitable animal model that exactly replicates the human knee and ACL injury. On the one Correspondence to: Professor Shijie Fu, Department of hand, it is not easy to develop an ACL injury model in small Orthopedics, The Affiliated T.C.M Hospital of Southwest Medical animals, such as rats, despite the decreased modeling time, University, 182 Chun Hui Road, Luzhou, Sichuan 646000, P.R. China as the small size of the knee joint increases the difficulty of E-mail: [email protected] surgery and nursing, leading to an increased risk of infection * or even accidental death. On the other hand, for some models Contributed equally of large animals such as rabbits and dogs, it is easier to perform surgery due to the larger surgical visual field of the knee joint. Abbreviations: ACL, anterior cruciate ligament; SPF, specific pathogen free; AAALAC, Association for Assessment and However, a longer duration of modeling and rehabilitation are Accreditation of Laboratory Animal Care International; PCL, needed, which is costly in terms of experimental funding. In posterior cruciate ligament particular, previous studies have been reported that the stifle joints of sheep and the knees of pigs may be more similar to Key words: cynomolgus monkeys, anterior cruciate ligament, human knees than other animals (such as rats, rabbits or dogs) animal model, arthroscopic, injury based on the size and anatomy of the knees (12,13). However, sheep or porcine models are different from humans in terms of their standing physics. By contrast, primates represent valuable 2240 ZHANG et al: AN ANIMAL MODEL OF ANTERIOR CRUCIATE LIGAMENT INJURY models for human disease research, since they are phylogeneti- 22‑24˚C and relative humidity of 45‑65%. The monkeys were cally close to humans, sharing more similarities in physiology, fed daily with formula feeds. Water was available ad libitum anatomy and genetics. From previous observations made via water bottles. Additionally, all animals were moved out during feeding of cynomolgus monkeys, it was noted that the of the cages to another spacious activity room, approximately monkeys had the ability to stand or walk on their hind limbs measuring 4 m (H) x 12.5 m (W) x 8 m (D), equipped with alone, and that the length of time spent in such a position was resting shelves, a small rockery, wooden branches and a greater compared with the aforementioned animals, thereby swing, for 6-8 h of free time per day. Additionally, videos and showing more similarity with the human body in terms of music were occasionally played to relax the monkeys as well. the physics of standing or walking. These physiological char- (Fig. 1A and B). acteristics allow the knee joints of primates to better mimic the human model. Moreover, the instability of the knee joint Animal modeling. The animals were randomly divided into could be more obvious in the state of standing on hind limbs three groups, including a model group (n=6), a sham opera- alone than on four limbs and may, therefore, be more similar tion group (n=6), and a blank control group (n=6). Based on to clinical manifestation of ACL injury in humans. Therefore, previous experiments and related statistical estimations, the the ACL injury model of cynomolgus monkeys may be helpful sample size of 6 per group can be statistically significant, in studying injury and reconstruction of the ACL. However, to especially for experiments on primates, such as cynomolgus date, few studies using an ACL injury in primates have been monkeys. In the model group and the sham operation group, reported. On the basis of this, the current study mainly focused the unilateral side of injury was chosen at random for each on modeling ACL injury in cynomolgus monkeys. animal. Furthermore, the previous methods of developing animal Cynomolgus monkeys in the model group were subjected models of ACL injury have mainly involved open surgery, with to unilateral knee surgery to induce ACL injury. The the risk of blood loss and trauma. With injury to the soft tissues arthroscopic instruments (Smith & Nephew Endoscopy, and anatomical structures around knee joint at the time of Andover, MA, USA; 72200616) were prepared before surgery surgery, rather than injury to the ACL alone, these models are and the equipment was strictly sterilized by the operators. different from common spontaneous sports injury to the ACL Then, the monkeys were anesthetized using Zoletil 50 (Virbac, in the human body. Besides, these methods may have negative Carros, France; 5 mg/kg, intramuscular) and were fixed in a effects on certain follow-up experiments involving structures supine position with the surgical area shaved. Marking of the around the knee joint. Considering aforementioned points, it incision and the application of an ipsilateral lower extremity is necessary to seek a modeling method to overcome these proximal tourniquet was performed. After all the preparatory shortcomings. In this aspect, arthroscopic technique could steps, anterior medial and anterior lateral approaches of the be a better solution. Based on these considerations, we found knee joint were built with 0.5-cm long incisions to explore that the size of the knee joint in monkeys was large enough the knee joint (Fig. 2A and B). Exploration indicated that the to operate arthroscopic technique as well, in comparison with articular cartilage, ACL, posterior cruciate ligament (PCL) and other animals. meniscus were intact. Approximately, one-quarter of the ACL In this study, we developed a useful alternative model was transversely cut (Fig. 2C and D) under a clear arthroscopic of ACL injury in cynomolgus monkeys via arthroscopic field of vision. At the end of the surgery, the incision was techniques, which may provide a basis for research into ACL closed with 3-0 absorbable sutures (Alcon Laboratories, Inc., injury. Fort Worth, TX, USA). The operation finished. The modeling operation was completed by the same group of doctors. Soft Materials and methods padded bandages were placed and maintained on the operated limbs for 2 weeks. For the first 3 postoperative days, levo- Ethical statement. All procedures were approved by the floxacin hydrochloride and sodium chloride injection (Heng Ethical Inspection Committee of Animal Experiments of Ao, Shanghai, China; 8 mg/kg, one dose/12 h, intravenous) Yunnan Yinmore Biological Technology Co., Ltd. (Yunnan, were used to prevent infection. The animals were monitored China; no. 2016001). Animal care was in accordance with the daily and tramadol hydrochloride for injections (QiMaiTe, ‘Guide for the Care and Use of Laboratory Animals’ (Office of Shijiazhuang, China; 2 mg/kg, one dose/day, intramuscular) Science and Health Reports CPRR/NIH 1996).
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