Keywords

Introduction

The study of injuries of the anterior cruciate ligament (ACL) makes up a large body of research into the etiology of PTA in both an animal and clinical setting. Currently, animal models using ACL transection (ACL-T) include dogs, sheep, cats, rabbits, guinea pigs, rats, and mice [1–3]. The first reported ACL-T model was developed using a stab incision in a canine model by Pond and Nuki [4]. Subsequent studies in dogs and other animals have examined the effects of ACL-T on articular cartilage, synovium, gene expression, biomarkers, and pain, and have been used in a variety of settings to test various therapeutic interventions. This section focuses on the use of ACL-T animal models as a method for studying PTA, with the advantages, disadvantages, and relevant studies for each animal described below.

Dog Model

The canine model has been used extensively for studying the effects of ACL-T, and there are a variety of advantages to using dogs for osteoarthritis (OA) research. For instance, they have a slow disease progression after injury, allowing for long-term observation of changes that occur as a result of PTA. Dogs also have thick articular cartilage, have larger joints, are trainable, and have well-documented outcomes to injury models, with a pathology that mimics naturally occurring arthritis. However, the high cost and public perception of using dogs are drawbacks to this model [1–3]. The first use of dogs for an ACL-T model was reported by Pond and Nuki in 1973, which utilized a stab incision into the knee joint to induce ACL-T [4]. Subsequent studies followed using the stab incision model, focusing on areas such as osteophyte formation [5], biochemical changes and gene expression [6–9], mechanical properties [10], and imaging techniques [11]. Therapeutic studies examined the effect of inhibiting nitric oxide (NO) [12] or delivering licofelone [9, 13] as a chondroprotective agent. A summary of the studies utilizing the stab incision model are given in Table 6.1.

Table 6.1 Canine ACL-T models (closed induction)
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After the introduction of the Pond-Nuki transection model (stab incision), other methods of ACL-T were studied. Brandt published a review validating the use of the canine ACL-T model for the study of arthritis [16], and open induction models were implemented, where the ACL was visualized and transected either through an arthrotomy or arthroscopically. A wide range of studies followed, looking at aspects of open-induction ACL-T such as biochemical changes and gene expression [17–24], bone morphological changes [25–28], biomarkers [29–31], and imaging techniques [32, 33]. O’Connor and coworkers published two studies looking at the combined effect of nerve removal and ACL-T on the development of PTA [34, 35]. Two therapeutic studies used the open-induction ACL-T model, including a doxycycline therapy study [36] and an MMP inhibitor study [37]. Doom and coworkers published a review of immunopathological mechanisms that result from the ACL-T model, leading to PTA [38]. A summary of the studies using the open-induction canine ACL-T model are listed in Table 6.2 below.

Table 6.2 Canine ACL-T model (open induction)
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Sheep Model

The use of sheep has not been widely utilized for the study of PTA; however, sheep may provide advantages because of their large joint size, which allows for the analysis of biochemical and biomechanical measures that may not be able to be performed in human subjects [39–41]. As with other large animals, sheep can readily undergo arthroscopic surgery and MRI observation, allowing for more direct translation of studies to the clinic. However, there are limited reagents and antibodies available, and until recently, a limited mapped genome for sheep, making it difficult for genetic studies [1–3]. Furthermore, their large size is a disadvantage in testing novel pharmacologic interventions. Most studies utilizing the ACL-T model in sheep have focused on radiographic tracking and kinematics of PTA. O’Brien and coworkers examined the effects of immediate reconstruction of the transected ACL on cartilage degeneration and osteophyte formation [41], while Atarod and coworkers examined the kinematic loads placed on soft tissue after ACL-T in the sheep [39]. A summary of the use of ovine ACL-T models is in Table 6.3 below.

Table 6.3 Ovine ACL-T models
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Cat Model

Neuromuscular control has been extensively studied in cats, as well as muscle mechanics and locomotion [42]. Logically, cats would be well suited to study interventions towards musculoskeletal diseases, such as PTA that results from an ACL-T injury. Cats are advantageous to use because of their large size and known genome. However, like dogs, cats can be costly to house during experiments, and public perception and their role as companion animals discourage the use of cats for research [1–3].

Herzog and coworkers first studied the effect of ACL-T in cats on hindlimb loading and changes in articular cartilage [42]. Khalsa and coworkers studied the effect of severing the nerves associated with the joint capsule after ACL-T [43]. Herzog and coworkers monitored cats for a year, using force testing plates and radiographs to track kinematic and radiographic changes due to OA [44, 45]. Boyd and coworkers studied the changes to the periarticular bone as a result of ACL-T, while Clark and coworkers studied the adaptive response of cartilage after ACL-T [46, 47]. A summary of the studies utilizing feline ACL-T models follow in Table 6.4.

Table 6.4 Feline ACL-T models
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Rabbit Model

Rabbits have been a popular model for use with both ACL-T and meniscus injury models because of their low spontaneous joint degeneration, large joint size, and ease in use for testing new therapeutic agents. Rabbits preferentially load their lateral side, unlike rodents, and have the capability to spontaneous regenerate transected menisci with fibrous tissue, which can cause disadvantages for some studies. Similarly, rabbits have altered joint biomechanics, potentially resulting in a change in disease pathology compared to what may be expected in other animals. However, rabbits have been widely used as a model for OA because they form lesions similar to those seen in clinical OA [1–3].

The ACL-T model has been used in rabbits to study many aspects of PTA development. Studies have examined articular cartilage and meniscus properties [48–50], gene expression and surface receptors [51–53], osteophytes [54], bone properties [55, 56], and imaging techniques [57, 58]. The rabbit ACL-T model has also been used to test out therapeutics, such as HA therapy [59] and oral glucosamine supplements [60]. Furthermore, one study compared surgically induced ACL-T versus a blunt trauma ACL-T, which closely resembles clinical ACL-T in humans [61]. Studies using rabbit models of ACL-T are summarized in Table 6.5.

Table 6.5 Rabbit ACL-T models
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Guinea Pig Model

Guinea pigs have been used to study OA because the Hartley strain, among others, develops spontaneous OA beginning at 3 months of age [1, 62–65]. Other advantages of using guinea pigs for the study of PTA include the fact that their histopathology is very similar to humans and that they are easy to manage during long studies. Disadvantages for their use include the fact that they preferentially load the medial side of the knee joint, are mainly sedentary animals, and are too small to allow for use of arthroscopic techniques for injury induction and observation [1–3].

Recently, guinea pigs have been used to study the effects of PTA as well as spontaneous OA by looking at the effects of ACL-T such as osteophytes and histopathologic changes [66], coefficient of cartilage [67], levels of lubricin in the joint [67, 68], and levels of biomarkers in synovial fluid including C2C, GAG, IL-1β, MMP-13, and SDF-1 [68]. The use of guinea pig ACL-T models in PTA studies is summarized in Table 6.6.

Table 6.6 Guinea pig ACL-T models
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Rat Model

Rats have been increasingly used for ACL-T studies due to their small size, rapid speed of OA symptom onset, ability for pharmacological testing, translational potential to human PTA, and low spontaneous degeneration of their knee joints [1, 3, 69–71]. Rats also have thick enough cartilage to allow for both partial and full cartilage defects, which allows for a low-cost defect model for OA research. Disadvantages include their small size for injury induction, and the rapid onset of disease [1–3].

Rats have been used to examine a variety of different PTA outcomes. One group of studies focused on the articular cartilage destruction, subchondral bone changes, and osteophyte production after ACL-T [71–73]. Another group introduced exercise as a therapy for reducing the symptoms of PTA after ACL-T [74]. Three other studies focused on the addition of supplements or inhibitors, including alendronate, which inhibits bone resorption, lubricin, hyaluronic acid (HA), and etanercept, an inhibitor of tumor necrosis factor alpha [75–77]. Finally, one group examined gene expression of different groups of OA progression markers, including matrix degradation, chondrocyte differentiation, and osteoclastic bone markers as a way to track disease progression [69]. Studies utilizing rat ACL-T models are summarized in Table 6.7 below.

Table 6.7 Rat ACL-T models
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Mouse Model

Mice provide a number of important advantages for studying OA and PTA. They are relatively inexpensive and easy to manage during studies, can incorporate genetic modifications, and are easy to use for pharmacological studies because of the low dosage required for efficacy [1–3]. However, relatively few murine models have been developed using ACL-T, likely due to small size and difficulty of the surgical approach. Mice also have fairly rapid onset of severe OA changes after surgery [1, 78]. Mice also have thin articular cartilage, which has limited the use of certain techniques, such as MRI or gene expression studies, to study PTA.

One example of the use of surgical ACL-T for the study of PTA was in a study published by Glasson and coworkers. When they compared the effects of ACL-T and destabilization of the medial meniscus (DMM) on the development of OA, the DMM model resulted in a slower and less severe progression of OA. However, as an alternative to surgical ACL-T, recent studies have examined the effect of cyclic loading [79] or a single loading cycle [80–82] to induce ACL-T in mice. The use of murine ACL-T models in studies is summarized in Table 6.8. A more detailed description of the single loading cycle to create ACL transection in a mouse is presented in the next chapter.

Table 6.8 Murine ACL-T models
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Conclusions

In summary, transection or rupture of the ACL provides a reproducible model of PTA. This procedure can be performed surgically or noninvasively and has been demonstrated in a variety of different animals that range in size from the mouse to the sheep. The changes occurring in the joint appear to parallel the degenerative changes that occur in clinical PTA, and appear to affect all of the joint tissues including the cartilage, meniscus, bone, and synovium.