Models of Acute Pain

Animal models of acute pain allow the evaluation of the effects of potential analgesics on pain sensation/transmission in an otherwise normal animal. In addition, the same tests may be used to measure stimulus-evoked pain in animals with chronic inflammation or nerve injury. Usually, these tests rely on an escape behavior/withdrawal reflex or vocalization as an index of pain. The animals have control over the duration of the pain, that is, their behavioral response leads to termination of the noxious stimulus. Acute thermal pain

Models have been developed to interrogate acute thermal pain sensitivity, using various means of applying a noxious heat stimulus to the paw or the tail of rodents. These models have been widely used in the characterization of opioid analgesics. Usually, 'latency to behavioral response' is recorded and a cutoff is set to avoid any tissue damage to the animal. The tail-flick test involves the application of a focused heat (usually light) source on the tail until a tail-flick (rapid removal of the tail) reflex occurs. This test has an advantage in that it does not involve repeated assessments of animal behavior, i.e., animals learning with time when the stimulus is going to be applied and anticipating the test.

The hot plate assay uses a hot plate set at a fixed temperature, usually 50-55 °C. Latency to licking, shaking of hind limbs or fore limbs, in addition to latency to jump can be recorded and statistically analyzed for groups of animals. This assay can be difficult to standardize since the heat stimulus is not delivered in a controlled fashion. Possible sources of variability include differential exposure to the heated plat depending on how much weight the animal puts on each limb.

Another approach to assess acute thermal pain is the use of a radiant heat source.27 Using this methodology, the temperature of the heat source applied to the hind paw increases over time until it reaches a painful threshold. Latency to hind paw withdraw is recorded and analyzed. In each test session, each animal is tested in three to four sequential trials at approximately 5 min intervals to avoid sensitization of the response. One of the advantages of this method versus the tail-flick assay is that both paws can be tested. This important control has proven a useful behavioral assessment in models of unilateral inflammation or nerve injury, the contralateral paw serving as control for the injured paw. In addition, in this assay, rats are confined in plastic chambers but not manually restrained as in the tail-flick assay or in the immersion tests (see below), decreasing the stress level of the test subjects. This method also uses a heated (30 °C) glass test surface to prevent paw cooling and to minimize sensitization artifacts.

Acute thermal pain can also be evaluated using a fixed temperature (45-50 °C) water bath and assessment of latency to withdraw of a hind limb or tail from the hot water. One of the advantages of this method is that the water bath can be set at various temperatures and it can be less sensitive to environmental conditions. However, this assay requires handling of the animals when testing for nociceptive behavior, making this measure highly dependent on experimenter experience/comfort handling/restraining animals by hand.

This method can also be used to test for reactivity to cold, using a 4 or 10 °C water bath and recording latency to withdraw as an index of pain. Another method uses a cold plate cooled by cold water circulating under it. Latency to nociceptive behavior or duration of guarding behavior can be recorded. As for the hot plate assay, the cold plate test has the advantage of not requiring animal restraint. However, depending on the position of the animal paw on the plate (or just above it), the cold stimulation can be very variable. Another widely used method is application of a drop of cold acetone on the plantar skin of animals resting on an elevated mesh floor. Acetone produces a distinct cooling sensation as it evaporates. Normal rats will not respond to this stimulus or with a very small response (in amplitude and duration) while nerve-injured rats will almost always respond with an exaggerated response. Acute mechanical pain

Models have been developed to interrogate acute mechanical pain/sensitivity, using various means of stimulating the paw or the tail of a mouse or a rat. A common method for the assessment of acute mechanical pain is determination of withdrawal thresholds to paw/tail pressure using the Randall Selitto test.28 This apparatus allows for the application of a steadily increasing pressure to the dorsal surface of the hind paw/tail of a rat via a dome-shaped plastic tip. The threshold (in g) for either paw/tail withdrawal or vocalization is recorded. Usually, two or three measurements are conducted on each paw or tail. This apparatus was designed originally for measuring mechanical sensitivity of inflamed paws and its use in normal noninflamed paws can produce great variability in the response, depending on the location of the stylus (soft tissue between the metatarsal/bone/joint). It is worth noting that training helps generate a more stable response with this assay.

Another approach for assessment of acute mechanical pain is to use a pinprick, applying painful pressure to the plantar surface of the hind paw. This is similar to the pricking pain test done during the neurological examination in patients. The behavior can be measured by the duration of paw lifting following the pinprick application or recorded as a frequency of withdrawal (percentage of response to the pinprick in 10 trials).

Finally, mechanical hypersensitivity can also be tested with von Frey monofilaments. These are a series of hairs/ nylon monofilaments of various thicknesses that exert various degrees of force when applied to the planter surface of the hind paw. Responses can be quantified as percentage response or duration of response to a given monofilament force applied several times, or mechanical threshold can be determined using the up-down method.29 Acute chemical pain

Usually, when studying acute chemical pain, behaviors such as flinching, biting, or licking the injected paw are recorded at various time points following the injection of a chemical irritant (capsaicin, formalin, PGE2, mustard oil, ab-methylene ATP). The duration of the nociceptive behavior as well as the number of behaviors can be quantified and analyzed. Two models are mostly used to study acute chemical pain: nocifensive behaviors following injection of capsaicin or formalin into the hind paw.30 Doses of capsaicin can vary from 1 to 10 mg per 10 mL injected to the dorsal surface of the rat hind paw. The injection of capsaicin is immediately followed by an intense period of nocifensive behaviors that are usually recorded for 5 min following capsaicin injection. Following formalin injection (usually 5% per 50 mL) into the dorsal or sometimes plantar surface of the rat hind paw, a biphasic behavioral response can be observed. Phase I of the formalin response is defined as the period of time immediately following injection of formalin until 10 min after the formalin injection and corresponds to acute thermal pain by direct activation of nociceptors by formalin. Following a 'quiet' period of little or no nocifensive behavior, the second phase of the formalin response can be observed (20-60 min post formalin injection) that corresponds to a more persistent inflammatory state. Models of Nociceptive Pain

Models of nociceptive pain are defined as models of pain following tissue injury induced by trauma, surgery, inflammation, and cancer. As stated above, spontaneous pain in these models is difficult to measure. However, evoked pain behaviors have been well characterized and can be induced by the methods described above in the 'acute pain' section. The focus of this section will be on models of nociceptive pain, mimicking as closely as possible rheumatoid arthritis and osteoarthritis clinical conditions since they have been the most studied and widely used. Models of postoperative pain or cancer pain will not be described, as they are recent and still under validation. Adjuvant-induced arthritis

Experimental arthritis is generated by an intravenous injection of complete Freund's adjuvant (CFA) at the base of the tail. The development of the joint inflammation is progressive and dramatic, leading to a multijoint arthritis with dramatic swelling and permanent joint tissue destruction.31 In this model, it is clear that the animals are in chronic pain, all their joints are swollen, they have decreased appetite, they limp, and have lower threshold for limb withdrawal or vocalization to paw pressure/joint manipulation. This model is rarely used today as the polyarthritic rat has significant systemic disease with abnormal hunchback posture and piloerection. Unilateral inflammation

To further study inflammatory pain, various models have been developed to induce a localized inflammatory reaction by injecting various substances, e.g., formalin, carrageenan, or CFA into the paw or the joint. Following the initial injection, pain can be measured minutes to days later, at the site of inflammation or away from the primary site of injury. Usually, the inflamed paw/joint becomes very sensitive to both thermal and mechanical stimuli while the contralateral paw remains 'normal.' Sometimes, secondary mechanical hypersensitivity can also develop on the contralateral side as observed 2 weeks following carrageenan injection into the knee joint when testing on the contralateral paw. These models of more localized inflammation/inflammatory pain have been widely used in pain research to test the effects of potential analgesic compounds but also in electrophysiological and gene expression studies to determine the plastic changes that initiate/maintain chronic inflammatory pain. Models of osteoarthritic pain

More recently, models have been developed to mimic osteoarthritic (OA) pain observed in the clinic. Contrary to rheumatoid arthritis (RA) and the models of inflammatory pain, OA in the clinic and in animal models is not associated with a large amount of inflammation. In addition, to mimic more closely the clinical situation, pain evaluation in OA pain models relies on functional measures such as weight bearing or grip force of the affected limb rather than evaluation of withdrawal latencies to thermal or mechanical stimuli. Two models have been widely used, intraarticular administration of sodium monoiodoacetate (MIA) into the knee and partial meniscectomy.32 Contrary to what is observed in the polyarthritic rat, no changes in body weight were observed over a 4-week period after either iodoacetate injection or partial medial meniscectomy. In addition, the general health of the animals is good with no signs of spontaneous nociceptive behavior, impaired locomotion, or distress. Furthermore, both iodoacetate injection and partial medial meniscectomy in the knee joint of the rat induced histological changes and pain-related behaviors characteristic of clinical OA. Although the behavioral changes and histology both worsened over time, the majority of the pain responses were apparent within one week of surgery or iodoacetate injection. It is important to note that the pain behaviors are less pronounced in the surgery model than in the MIA model and that these findings agree with the clinical situation. Indeed, magnetic resonance imaging (MRI) studies have shown that although meniscal lesions in humans are common, they are also rarely associated with pain. Models of Neuropathic Pain Direct trauma to nerves

To mimic nerve injury observed in the clinic, a number of different animals models have been developed. One of the most studied models is the L5-L6 spinal nerve ligation (SNL, Chung model) (Figure 4) model.33 In this model, following sterilization procedures, a 1.5 cm incision is made dorsal to the lumbosacral plexus, the paraspinal muscles are separated from the spinous processes, the L5 and L6 spinal nerves are isolated, and tightly ligated with 3-0 silk thread. Usually the animals are allowed to recover from surgery for 7 days before being tested for mechanical allodynia using von Frey monofilaments (up-down method or percentage response to 10 applications of innocuous or noxious von Frey monofilament). While the spinal nerve injured rats also develop cold allodynia and thermal hyperalgesia, they have a greater degree of mechanical allodynia and most pharmacological studies with these animals have involved mechanical allodynia endpoints.

Another widely used model of direct nerve injury is a partial nerve ligation model (PNL) ( ). The sciatic nerve is exposed unilaterally, just distal to the descendence of the posterior biceps semitendinosus nerve from the sciatic. The dorsal 1/3-1/2 of the nerve thickness is then tightly ligated with an 8-0 silk suture. Following injury, these animals develop guarding behavior of the injured hind limb suggesting the possibility of spontaneous pain. In addition, the animals develop mechanical allodynia as well as thermal hyperalgesia and bilateral mechanical hyperalgesia. Inflammation/neuritis/nerve compression

Neuropathic pain can also result from inflammation around peripheral nerves and peripheral nerve compression. Two preclinical models have been developed to attempt to mimic this phenomenon. The first model is the chronic constriction injury (CCI) (Bennett model) (Figure 4) of the sciatic nerve model.33 In this model, a 1.5 cm incision is made 0.5 cm below the pelvis. The biceps femoris and the gluteus superficialis are separated and the sciatic nerve exposed, isolated, and four loose ligatures (5-0 chromic catgut) with 1 mm spacing are placed around it. CCI animals develop mechanical allodynia, cold allodynia, and thermal hyperalgesia. When compared to the 'SNL injured animals,' CCI animals do develop thermal hyperalgesia and cold allodynia to a greater extent.

The second model, developed more recently, is the SIN model or zymosan-induced sciatic inflammatory neuritis.34 In this model, a chronic indwelling perisciatic catheter is used to inject zymosan around the sciatic nerve. After aseptic exposure of the sciatic nerve at midthigh level, the gelfoam is threaded around the nerve so as to minimize nerve displacement. Suturing and insertion of a sterile 'dummy' injection tube during implantation maintained catheter patency and ensured replicable drug delivery close to the nerve. After anchoring to the muscle, the external end is tunneled subcutaneously to exit 1 cm rostral to the tail base. After removal of the 'dummy' injector, the external end of the silastic tube is protected. Usually, catheter placement can be verified at sacrifice by visual inspection. The catheter

Figure 4 Animal models of neuropathic pain. This schematic illustrates the three main rodent models of neuropathic pain associated with direct nerve injury; the L5-L6 spinal nerve ligation (SNL) model (Chung model), the chronic constriction injury (CCI) of the sciatic nerve model (Bennett model), and the partial nerve ligation (PNL) model (Seltzer model).

Figure 4 Animal models of neuropathic pain. This schematic illustrates the three main rodent models of neuropathic pain associated with direct nerve injury; the L5-L6 spinal nerve ligation (SNL) model (Chung model), the chronic constriction injury (CCI) of the sciatic nerve model (Bennett model), and the partial nerve ligation (PNL) model (Seltzer model).


Tibial is used for a single injection 4-5 days after surgery conducted in freely moving rats. In this model, perisciatic zymosan injection induces unilateral mechanical allodynia at low dose and bilateral mechanical allodynia at high dose. Interestingly, the same high dose injected into gelfoam in neighboring muscles does not induce mechanical allodynia, suggesting that immune activation must occur in close proximity to peripheral nerves to create allodynia and that zymosan spread to systemic circulation cannot explain allodynia created by perisciatic zymosan. Interestingly, no thermal hyperalgesia is observed in this model. Diabetes

Another major cause of neuropathic pain in the clinic is neuropathic pain observed in diabetic patients. In rodents, this is mimicked by streptozotocin (STZ) injection to induce diabetes and subsequent neuropathic pain symptoms.33 Usually, diabetes is induced by a single injection of STZ (75 mgkg _ 1 intraperitoneal). Diabetes is confirmed by testing for blood glucose levels. Not all animals show signs of neuropathic pain immediately following STZ administration. Generally it takes usually between 4 and 8 weeks to observed neuropathic pain symptoms, mostly mechanical allodynia assessment with von Frey monofilaments, in a group of streptozotocin-treated rats. Chemotherapy-induced neuropathic pain (vincristine/paclitaxel/platine)

The last 'type' of neuropathic pain models are chemotherapy-induced neuropathic pain models. Cancer-related pain is a significant clinical problem that will likely increase in its extent as the average lifespan continues to rise and cancer therapies continue to improve. The two main sources of cancer-related pain are that from the malignancy itself and from the treatments utilized to alleviate the cancer (surgery, radiation, and chemotherapy). Peripheral neuropathy and subsequent neuropathic pain related to chemotherapeutic treatment can be dose limiting, and the pain is often resistant to standard analgesics. To date, no one drug or drug class is considered to be both a 'safe and effective analgesic' in the treatment of chemotherapy-induced pain, and three preclinical models of chemotherapy-induced neuropathic pain have been recently developed to further our understanding of the pathophysiology of such neuropathic pain states. Chemotherapy-induced neuropathic pain can be induced by the injection of either vincristine, platine, or paclitaxel.33 Depending on the experimental protocol, they can be injected as a bolus, for several days or weeks or as a continuous intravenous infusion using osmotic pump. Interestingly, as observed in the clinic, thermal hyperalgesia is not observed in these animals. However, both mechanical allodynia and cold allodynia are observed.

The differential efficacy of analgesic medications for different types of pain that is seen in the clinic is also observed in animal pain models. For example, while opioid analgesics like morphine (Figure 5) are potent and efficacious in all animal pain models, anti-inflammatory agents such as ibuprofen and celecoxib (Figure 5) are most potent and effective in animal models associated with inflammation, and anti-epileptics like lamotrigine and gabapentin (Figure 6) are most potent and efficacious in animal models of neuropathic pain (Table 1). As preclinical models of the various forms of pain appear to have selective and differential predictive validity for efficacy in the clinical setting, they should be useful in determining if new chemical entities (NCEs) with a novel molecular mechanism have the promise to be broad-spectrum analgesics.

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