41 General Considerations for an In Vivo Model
The American National Research Council Committee on Animal Models for Research and Aging drafted the following definition: “An animal model for biomedical research is one in which normative biology or behaviour can be studied, or in which a spontaneous or induced pathological process can be investigated, and in which the phenomenon in one or more respects resembles the same phenomenon in humans or other species of animals.”
The three Rs is a principle aimed at trying to reduce the unnecessary use of animals in research while still recognizing the need for animal models: reduction of animals, replacement of animals, and refinement of procedures (Table 41.1). It is also essential that the design of any animal experiment is optimized, as discussed in Chapter 2.
According to Einhorn, 1 there are three fundamental aspects that a bone repair or restoration model must comply with:
It must closely reflect the mechanics and physiology of the parallel human clinical scenario.
It must fail to heal of its own accord.
It must not heal by the application of simpler strategies.
There are several hundreds of animal models described in the scientific literature. Broadly, they can be divided into three types: exploratory, explanatory, and predictive. Other ways that animal models have been classified are as induced, spontaneous, transgenic, negative, and orphan (Table 41.2). These terms are more for academic discussion than practical implication but are worth considering when choosing the most appropriate model for a specific piece of research. In orthopedic research, the animal models most likely to be required will be induced or spontaneous. Another term used in the literature is fidelity, which refers to how closely the model reflects the human scenario.
41.1 Mode of Interrupting Normal Healing/Physiology
Numerous factors contribute to delayed repair. They include host, local, mechanical, biological, and infective factors, which need to be taken into account when selecting a model of impaired healing.
41.1.1 Control Positive/Negative
All animal models require a positive and/or negative control. The requirements will depend on various factors including the hypothesis of the study, the model type (e. g., induced or transgenic), the type of bone healing scenario (nonunion, delayed union, etc.), and whether the model is well validated in the literature.
41.1.2 Time Scale of Model
The model (simple fracture, delayed union, nonunion), type of fixation, choice of animal (for its natural time to healing and life expectancy), and aims of study will all directly influence how long the model should be run. Clearly, if the model is not allowed to run for a sufficient period of time, the data will not be valid, especially in the case of a nonunion. However, it is a humane and fundamental principle in the use of animals in research that they should not be exposed to any unnecessary or prolonged periods of stress or suffering. The time scale of the model must be discussed with the departmental head veterinarian and is often a clause in the project license that has been granted.
41.1.3 Numbers Required
For an animal study to show a significant result, a certain number of animals will need to be used. The correct number should be decided upon using power calculations and allowing for potential complications prior to undertaking the study. The number will depend upon the species, model type, and controls. It is vital that such calculations are done, otherwise the study may be invalid or conclude with a false result (type II/beta error). This topic is considered further in Chapter 52.
41.1.4 Regulatory Aspects
Each institution will have to adhere to strict rules for performing in vivo studies. The project and experimental procedures will have to meet ethical standards and be fully justified. It may be necessary to obtain a project license from the local regulatory authority. The individual performing the procedures also has to meet certain requirements. It may be essential for this person to have attended animal welfare and handling courses and to have a personal license. Obtaining the necessary project and personal licenses can take a number of months, and it is essential to have early discussions with the local animal unit.
41.1.5 Animal Housing and Care
Each housing unit will have its own rules and guidelines, and there should be at least one veterinarian overseeing the unit. It is important to have several meetings with the staff of the housing facility so that aspects such as bedding, water, chow, and analgesia can be discussed. The study animals need to acclimatize to their environment and typically should be in their new housing for at least a week prior to undergoing surgery.
Some species can be housed in groups (e.g., mice), whereas others require separate housing due to their territorial behavior (male rats); this has both financial and space implications. The housing should be in a suitable environment that is overseen by a senior veterinarian. The team looking after the animals needs to be aware of what the model will entail, the presence of surgical wounds, and possible side effects to be aware of afterwards.
In considering animal welfare in the postoperative recovery period, consider the need for subcutaneous fluid to compensate for blood loss and anesthesia, warming lamp and mat to prevent hypothermia, subcutaneous or intravenous analgesia, and separation from the rest of the group.
In the subsequent postoperative period, animals should be weighed daily initially then twice weekly thereafter. Weight loss is an important sign of animal distress; up to 10% weight loss can be expected postoperatively, but more than this or ongoing weight loss is concerning. Analgesia may be required for a few days or weeks postoperatively depending on the model, again this can be decided in collaboration with the veterinarian, an opiate dissolved into dessert jelly and cut into cubes works well in smaller animals. The data available regarding the effect that postoperative use of anti-inflammatory agents for pain relief is mixed but the overall recommendation is that it should be avoided if possible.
Any animals showing signs of distress or wound infection should be urgently discussed with the on-call veterinarian for the unit.
41.1.6 Handling
Depending on the age, species, and exposure that the animals have had to humans, they will respond very differently to being handled. Rats in particular will become more tame with increased handling and attention.
Anyone who is involved in the handling of animals should attend an animal handling course to learn how to handle the relevant species correctly. This is usually a compulsory part of the personal license.
41.1.7 Anesthesia
The license will outline when an animal can be given a general anesthetic. If an anesthetic is required for purposes other than the initial surgery (e.g., delayed introduction of a chemotherapeutic agent into the fracture site, debridement of the fracture, removal of sutures or taking of radiographs), ensure that it is included in the project license.
The anesthetic required should be discussed with the senior veterinarian. In some institutions, there will be a veterinarian to perform the anesthetic, in others the surgeon will be expected to perform it, in which case it is vital that the surgeon has a good understanding of the anesthetic agent and equipment. During the procedure, warmth (warming mat and lights), analgesia, and fluids (volume will be dependent upon species type and weight) will be required.
41.1.8 Perioperative Antibiotics
Antibiotics are not routinely used in small animal models, even in the case of metalware implantation; the emphasis to reduce the risk of infection is on aseptic techniques and appropriate tissue handling. If the operation is prolonged, there maybe justification for a single intravenous shot of clindamycin. In large animals, the AO Institute recommends antibiotics only if the procedure time exceeds 90 minutes, metal implants are used, or extensive soft tissue injury is present, in which case it should be administered prior to the initial surgical incision or inflation of a tourniquet if being used.
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