LAAC system classifies various procedures into 12 classes and three main categories with an overall increasing level of AA from LAAC1 to LAAC12 (Fig. 1). Category A comprises non-surgical procedures with discomfort up to the discomfort threshold (DT). Category B, mainly includes non-surgical procedures, and category C includes surgical procedures. Below each of these classes are discussed. The choice of the medication in each class depends on several factors, such as: 1) the species and strain of the animal; 2) type and extent of the procedures; 3) clinical state of the animal; 4) effect of the medication on the research parameters; 5) familiarity of the researcher with a medication/technique; and 6) availability of the medication to researchers. The aim of this paper is not to provide a comprehensive list of medications for various species of animals and research protocols. Rather, it provides a guide for proper selection of potential drugs and techniques that could be considered in each class. It should also be noted that some medications may have contradictory effects depending on the animal species, interspecies differences, and dose. For example, a sedative drug like diazepam may cause excitement in some species (dogs and cats) 11. Readers are encouraged to consult a relevant textbook for more details on the properties of a specific drug 12, or anesthetic/analgesic method for various species of animals 11,13,14.
Category A
LAAC1
Sample procedures
Handling and minimal restraint of trained animals; sexing; displacing habituated animals; inserting a hypodermic needle; oral gavage.
Intraoperative anesthesia/analgesia
No anesthesia is required. Analgesia is not essential. However, topical anesthetics may be used to reduce the pain of needle insertions. Distraction techniques may be used for reducing the discomfort of the animal.
Post-operative analgesia
Post-operative analgesia is not necessary.
LAAC2
Sample procedures
Using an agitated animal for minimal restraint, sexing, displacing, injecting; pre-anesthetic preparations in distressed animals.
Intraoperative anesthesia/analgesia
No anesthesia is required. Occasionally, analgesia may be provided as discussed for LAAC1. Tranquilizers may be used to calm down the animal. In this regard, low dose phenothiazines (chlorpromazine, acepromazine, promazine), benzodiazepines (diazepam, midazolam), α2‐adrenergic agonists (xylazine, detomidine, romifidine, medetomidine, dexmedetomidine), or butyrophenones (droperidol, fluanisone, azaperone) may be used 11,13.
Post-operative analgesia
Post-operative analgesia is not necessary.
LAAC3
Sample procedures
Short-term immobilization in a non-stimulating environment; pre-anesthetic preparations in agitated animals.
Intraoperative anesthesia/analgesia
No anesthesia is required. Analgesia may be achieved as discussed in LAAC1. Sedatives may be used to create a state of sedation and immobilization. In this regard, higher doses in the therapeutic window of LAAC-2 drugs may be used. Additionally, opioids (alfentanil, buprenorphine, butorphanol, etorphine, fentanyl, hydromorphone, methadone, morphine, nalbuphine, oxymorphone, pentazocine, pethidine (meperidine), remifentanil, sufentanil) may also be used to induce a state of moderate sedation accompanied with varying levels of hypoalgesia 11.
Post-operative analgesia
Post-operative analgesia is not necessary.
LAAC4
Sample procedures
Immobilization and sleep in a moderately stimulating environment; non-invasive imaging.
Intraoperative anesthesia/analgesia
Unconsciousness can be induced by hypnotics or anesthetics. These include alpha-chloralose, the barbiturates (e.g., thiopental, thiamylal, and methohexital, thiobutabarbital, and pentobarbital), chloral hydrate, etomidate, metomidate, propofol, the steroid anesthetics (alphaxalone/alphadolone), tribromoethanol, inhalational anesthetics, and injectable anesthetics.
Post-operative analgesia
Post-operative analgesia is not necessary.
Category B
LAAC5
Sample procedures
Conditions with non-somatic origin causing discomfort more than the DT and psychosomatic conditions; procedures leading to anxiety, fear, distress, depression; activity anorexia; developing thin sow syndrome; compulsive behaviors (stereotypical behaviors); addiction and withdrawal.
Intraoperative anesthesia/analgesia
Anesthesia may not be required. However, depending on the cause of the condition, various types of medications or non-medical methods may be used to alleviate the symptoms. For example, a highly distressed animal may receive sedatives and placed in a quiet and dim environment, or an animal in depression may benefit from anti-depressants.
Post-operative analgesia
Since there is no somatic pain in this class, the use of analgesics is questionable.
LAAC6
Sample procedures
Non-surgical procedures that involve visceral organs; procedures through natural body orifices (e.g., endoscopies); intravisceral injection or sampling (not thorax; since it induces discomfort more than the DT); models of sepsis, neoplasia, and inflammation that are induced non-surgically and involve visceral organs.
Intraoperative anesthesia/analgesia
The use of anesthesia may or may not be necessary. For endoscopies and intravisceral injection/sampling, depending on the type and length of the procedure, deep sedation or hypnosis may be used. Therefore, LAAC3 or LAAC4 provisions may be undertaken.
Models of sepsis in this class can be induced by injection of bacterial toxins, purified live bacterium, or fecal-derived bacteria (polymicrobial infection), which do not require anesthesia. Neoplastic models in this class may also be developed by inoculation of neoplastic cell lines and may not require anesthesia.
Post-operative analgesia
Visceral analgesia may be required if any of the aforementioned procedures lead to visceral pain. In this regard, local anesthetics (e.g., lidocaine) may be used for inhibiting pain transduction, transmission, or modulation. Opioids and tramadol may be used for inhibiting pain modulation or perception 10.
There are controversies regarding the need of analgesia for non-surgical models of sepsis 15. However, the latest findings recommend considering analgesics (such as nalbuphine, buprenorphine, morphine, fentanyl) and closely monitoring the clinical signs of pain in animals 15,16. The use of non-steroidal anti-inflammatory drugs (NSAIDs) in sepsis models is discouraged due to the risk of bleeding and renal dysfunction. Moreover, the anti-inflammatory effect of NSAIDs could interfere with the pathobiology of the sepsis model and negatively influence the study 16.
Analgesic treatment for inflammatory and neoplastic visceral pains depends on the tissue involved, the pathogenesis and severity of pain, and the species of the animal. For example, opioids such as buprenorphine, butorphanol, hydromorphone, meperidine, or methadone are recommended for pancreatitis pain in dogs 17. Other analgesic modalities may be found in related references 18.
Neoplasia may influence the animal by pain and general ill-being (sickness syndrome) resulted from the paraneoplastic syndrome 19,20. The neoplastic pain has several components as: pain resulted from the tumor, pain caused by disease progression, pain due to side effects of treatments, pain from other sources not related to malignancy 21. Few studies have investigated the neoplastic pain in laboratory animals 13, but as a general rule, neoplastic pain evolves quite differently than acute procedural pain. Neoplastic pain may be acute, chronic, or intermittent 21 but in general it may start gradually, and may last for a longer time and may involve coping mechanisms that could suddenly fail 13. Opioids and tramadol are used to alleviate cancer pain in animal models 19, and it is shown that certain nonsteroidal anti-inflammatory drugs may have synergy with opioids 13. Antiepileptics (such as gabapentin and pregabalin) and antidepressants have used for managing chronic pain in humans 19. Occasionally, NMDA receptor antagonists and local anesthetics have also been used for managing chronic pain 19.
LAAC7
Sample procedures
Intraosseous access; infection or neoplasia that involves hard tissues.
Intraoperative anesthesia/analgesia
The procedure of intraosseous access or induction of bone infection/neoplasia may be performed without general anesthesia. However, category A modalities may be undertaken to reduce the animal distress and facilitate the procedure. For intraosseous access, infiltration with local analgesics (e.g., lidocaine, bupivacaine) should also be used 22.
Post-operative analgesia
Analgesia is required. Bone infection models 23–25 leading to osteomyelitis may induce inflammatory pain. In this regard, NSAIDs such as celecoxib (a selective COX-2 inhibitor) has been used to alleviate the pain of the condition 26.
Bone cancer is a very painful condition and is one of the most common models of cancer pain in laboratory animals 27. The mechanism of bone cancer pain are partially understood 28 and are described elsewhere 29,30. Since bone cancer pain results from different mechanisms than bone inflammation pain, a higher dose of analgesics may be required for alleviating it 28. The choose of analgesics depends on the species of the animal, extent of the neoplastic lesions, the severity of pain, and the objectives of the study. Morphine is shown to produce short-term analgesia (2-3 hr.) for bone cancer pain in mice. Concurrent use of acetaminophen and morphine is shown to produce even higher level of analgesia in this animal model 13. Other complementary approaches include eradicating the tumor, stabilizing the affected bones, reducing bone loss, and using NSAIDs 31. Analgesic therapy should be devised such that it does not negatively influence the study objectives, as described elsewhere 32.
LAAC8
Sample procedures
Sepsis or neoplastic conditions that involve the hard tissues and viscera.
Intraoperative anesthesia/analgesia
Anesthesia may not be required to induce the neoplastic or septic condition. However, category A modalities may be applied to reduce the distress of the animal.
Post-operative analgesia
A combination of LAAC6 and LAAC7 analgesic modalities may be used. In this regard a number of new modalities are also under investigation 21.
Category C
LAAC9
Sample procedures
Biopsies from skin, muscle, mucosa, or conjunctiva; incisional or excisional wound models; models of dead space, flap surgery, or burns; catheterization in major blood vessels (carotid artery, jugular vein, femoral artery and vein); vascular ligature models; neoplasms involving skin, muscle, mucosa, or conjunctiva.
Intraoperative anesthesia/analgesia
- Short term general anesthesia may be used. In this regard, inhalation anesthetics may be more appropriate due to their rapid onset of action and fast recovery. Analgesia should be provided by administration of systemic opioids, NSAIDs, or local anesthetics.
Or
- Category A modalities may be applied along with local anesthesia at the site of tissue injury.
Or
- LAAC3 may be applied along with systemic opioids to induce neuroleptanalgesia.
Post-operative analgesia
Depending on the extent of the injury, one or a combination of the below modalities may be used:
- For limited injuries, a long-acting local anesthetic (e.g., bupivacaine) may be used short-term 33 by injecting at the injury site before termination of the procedure.
- One dose 33 of NSIADs (e.g., carprofen, meloxicam) or opioids may be used for minor injuries. In more severe cases, less than one week use of NSAIDs may be warranted 33.
- Systemic or topical opioids may be used short-term (less than 10 d) without considerable effect on wound healing 33
- Local infiltration of tramadol at the incision site may be used for analgesia without negative effect on wound healing 33.
- Gabapentin may be used as an adjunct analgesic in wound models 33
- Acetaminophen may be used as a part of the post-surgical analgesia for wound models 33.
- For neoplastic lesions, LAAC6 and LAAC7 modalities may be used.
Systemic or topical steroids should not be used in wound healing models 33.
LAAC10
Sample procedures
Procedures on visceral organs in the abdominal cavity; thoracic interventions that do not involve hard tissues (ribs or sternum); thoracoscopies; ophthalmic procedures; neoplasms involving skin, muscle, mucosa, conjunctiva, and visceral organs.
Intraoperative anesthesia/analgesia
- For abdominal cavity procedures, injectable general anesthesia along with opioids, NSAIDs, and/or local anesthetics may be used.
- For cardiac puncture (as a terminal procedure), deep general anesthesia may be achieved using higher doses of injectable or inhalational anesthetics.
- For thoracic surgeries and thoracoscopies, deep general anesthesia with positive pressure ventilation, and proper multimodal analgesia (opioids, NSAIDs, and/or local anesthetics) should be considered.
- For ophthalmic procedures, one should consider the length of the procedure; goal of the research; depth of anesthesia; effect of the anesthetic on intraocular pressure, extraocular muscles relaxation, retinal ultrastructure, ocular position during surgical plane of anesthesia, and intraocular air volume 13. Topical ophthalmic anesthetics may be used as an adjunct to general anesthesia for corneal procedures. Oculocardiac reflex may be inhibited using anticholinergic drugs such as atropine or glycopyrrolate 13. To maintain a central position of the eyeball during intraocular surgeries, neuromuscular blocking agents (e.g., atracurium) may be used by people with special training on the use of these agents.
In large ruminants, enucleation may be performed by regional anesthesia (retrobulbar block of deep orbital nerves) or Peterson block 13.
Post-operative analgesia
- For survival procedures, a combination of opioids and NSIADs (8-72 hr. for non-thoracic procedures) depending on the species of the animal and the extent of the tissue injury may be considered 11.
- For thoracic procedures, LAAC12 postoperative analgesia should be applied.
- Combination of local anesthesia (e.g., lidocaine, bupivacaine) with systemic analgesics may be used 34.
- For neoplastic conditions, LAAC6 and LAAC7 modalities may be applied.
LAAC11
Sample procedures
Orthopedic procedures; surgically induced osseous conditions (e.g., osteotomy); chemically induced skeletal conditions such as arthritis; neoplasms involving skin, muscle, mucosa, conjunctiva, and bones.
Intraoperative anesthesia/analgesia
Deep general anesthesia along with potent analgesia is required. Furthermore, muscle relaxants (i.e., neuromuscular blockers or spasmolytics) may be used to facilitate orthopedic surgeries in animals with large muscular mass. Neuromuscular blockers must only be used by people specially trained in using these agents. Local and regional anesthesia may be used to provide preemptive analgesia and relaxing the muscles.
Post-operative analgesia
NSAIDs are excellent analgesics for orthopedic pain. The efficacy of the propionic acid derivatives (e.g., ibuprofen, fenoprofen, flurbiprofen, flunoxaprofen, ketoprofen, naproxen, and carprofen) to ameliorate orthopedic pains, is comparable with some opioids (such as buprenorphine, butorphanol, pethidine, and codeine) 13. Overall, there are controversies regarding the effect of NSAIDs on bone healing 33. There is some evidence that NSAIDs have a potential to alter fracture healing in microscopic levels 13, though it seems a short term use of some NSAIDs does not affect the long term healing rate of the bones 33,35. However, the use of nonselective COX inhibitors (indomethacin, ibuprofen, ketorolac, parecoxib) are not recommended in orthopedic models of rodents, because of the negative effect of these drugs on bone healing 33. Also, long term use (10 days- 4 months) of selective COX2 inhibitors (diclofenac, carprofen, rofecoxib) have negative effect on bone healing, but this has not been observed by their short term use (less than 10 days) 33. Acetaminophen may be used along with other analgesics for treating the pain of orthopedic models in rats 33.
Orthopedic models usually cause chronic pain 7,33. Therefore, a 2-3 week analgesic therapy using opioids may be warranted following extensive orthopedic surgeries without negatively affecting bone healing 33. In this regard, sustained release medications such as fentanyl patch, fentanyl gel, or sustained release buprenorphine injection may be considered 35. It should be noted that buprenorphine seems to have a ceiling analgesic effect, meaning that increasing the dose of the drug could not provide more analgesia after a ceiling point of analgesic effect is achieved 36. Long term use of opioids (i.e., 8 weeks in rats), may negatively affect the remodeling and resorption of the fracture callus 33. Combining NSAIDs with opioid agonists provide a synergistic effect. Therefore, if NSAIDs are omitted from the postoperative analgesic regimen, the dose of the opioid should be considerably increased.
Postoperative local and regional anesthesia may also be achieved by placing catheters for continuous anesthetic drug infusion (e.g., ropivacaine, bupivacaine). However, this should not be considered as the sole method of postoperative analgesia 13. Low dose ketamine may be used as a preventive analgesic modality in orthopedic models 33.
Multimodal analgesia in shown to effectively reduce postoperative pain in spine surgery. It may comprise gabapentinoids, acetaminophen, regional anesthesia by placing local anesthetics close to the spinal nerves, and slow-release local anesthetics 35.
Steroids (e.g., prednisolone, methylprednisolone, dexamethasone) should be avoided in orthopedic models 33. Gabapentin is also not a suitable analgesic for orthopedic models in rats 33. Information on the general anesthetic medications used in orthopedic research on large laboratory animals are provided elsewhere 37. For neoplastic pains, LAAC7 provisions should be applied.
LAAC12
Sample procedures
Thoracic surgeries involving sternum (sternotomy) or ribs (costotomy); brain surgeries through craniotomy; metastatic neoplasms involving skin, muscle, mucosa, conjunctiva, bone, and visceral organs.
Intraoperative anesthesia/analgesia
Deep general anesthesia and potent analgesia are required. Thoracic surgeries require positive pressure ventilation. If the anesthetic depth, analgesia, and ventilator settings are adjusted properly, the ventilation could usually be performed without the need of neuromuscular blocking agents 13. When using neuromuscular blocking agents, special care should be paid to the monitoring of the anesthetic depth 11.
Various anesthetic protocols are described for thoracotomy in larger animals 14,38 and pets 39. However, available literatures usually do not report specialized anesthesia/analgesia for thoracic surgeries in smaller animals such as mice and rats. For example, there are reports of using common anesthetics such as ketamine/xylazine 40–42, or isoflurane/carprofen 43, and narcotics such as sodium pentobarbital 44,45. However, these agents could not provide an adequate depth of anesthesia and enough analgesia for thoracic surgery as one of the most painful operations. The same holds true for rabbits, in which sodium pentobarbital 46,47 or sodium pentobarbital/isoflurane 48 are used. There are also reports of proper anesthesia but inadequate perioperative analgesia 49,50 and inadequate postoperative analgesia 51 on rabbits.
Therefore, for this class of procedures, I recommend anesthetic protocols that incorporate cardiac-safe balanced anesthesia with multimodal analgesia (i.e., concurrent use of opioids, NSAIDs, and intercostal nerve block) as it could be exemplified in some previous works 52–54. Cardiac effects of various anesthetics and analgesics are discussed elsewhere 55. Nitrous oxide should be avoided due to its effect on developing pneumothorax after the surgery 13. Craniotomy may be performed under general anesthesia and multimodal analgesia, along with a splash block of lidocaine on the skull periosteum.
Post-operative analgesia
Thoracic surgeries are accompanied with severe post-operative pain. Left untreated, this pain could lead to difficult breathing, hypoxia, and pneumonia. Pain of thoracotomy procedure may last longer than 48 hours 7. Therefore, pure agonist opioids must be used parenterally for the first 48 hours after the surgery. During which time, the respiratory rate and quality should be monitored to identify and correct opioid-mediated respiratory depression. Other analgesics such as NSAIDs, intercostal nerve block with local anesthetics or intrapleural local anesthetics, epidural opioids, and ketamine may be used in combination with systemic opioids to provide multimodal analgesia. This multimodal analgesic therapy reduces the required dose of opioids and their side effects on respiration 13,56. Some post-operative analgesic provisions could be adopted from companion animal practice 39.
Neuropathic pain (+)
Neuropathic pain (NP) may develop as a side effect of an intervention on laboratory animals 57, or it may be intentionally modeled on animals 57–60. Proper detection of NP is a challenge in veterinary patients and laboratory animals 61–63. Currently, there is no efficient analgesic drug to fully counteract NP 58,64. However, it is believed that the same concepts of preemptive analgesia and multimodal analgesia also apply to NP 61.
The first line drugs for treatment of NP includes certain anticonvulsants (e.g., gabapentin and pregabalin), some tricyclic antidepressants (e.g., imipramine, amitriptyline), and topical lidocaine 61. Other analgesics such as opioids, tramadol, and NSAIDs, if used alone, are usually less effective in treating NP. However, they may be considered as part of a multimodal analgesic approach in this condition 61. A number of nonchemical approaches are also suggested as an adjunct therapy for alleviating NP, such as: providing environmental enrichment 65, using long particles as bedding material, reducing the soy and adding the taurine content of the feed, group housing the animals to encourage social buffering 66, massage, and thermotherapy 61. It is shown that in animal models of NP, postoperative analgesic therapy may not adversely affect the development of NP 67.