All animal procedures and protocols were approved by the Minneapolis Veterans Affairs Health Care System (VAHCS) Institutional Animal Care and Use Committee and conformed to the "Guide for the Care and use of Laboratory Animals" (The National Academic Press, USA)12.
1. Choice of animals
NOTE: C57Bl6 mice are used for all three arthritis models.
2. Intra-articular injections into the knee
3. Production of arthritis
4. Measurement of evoked joint pain
5. Measurement of spontaneous joint pain.
NOTE: The Advanced Dynamic Weight Bearing (ADWB) device calculates the percent of total time the animal spends on each limb and the percent of total weight borne on each limb.
Acute and chronic inflammatory and chronic degenerative joint pain was produced by IA injection into the left knee of C57BL/6J male and female mice prior to pain assessment using the protocol outlined above. Joint pain was measured as evoked pain scores (EPS) and spontaneous pain behaviors measured with an advanced dynamic weight bearing (ADWB) device. For the purposes of this report, individual comparisons were made using Student's t tests. For experiments where analgesic doses are being compared, ANOVA may be a more appropriate statistical comparison.
EPS (the sum of tallied fights plus vocalizations) increased with all three types of joint pain (Figure 1). ADWB measures of percent weight bearing on the left hind limb compared to naïve mice were reduced in acute and chronic inflammatory joint pain. Females but not males with chronic degenerative joint pain (COL) showed similar reductions in ADWB measures (Figure 2A). Males with acute inflammatory joint pain transferred more weight to the forelimbs than naïve animals. Weight bearing on forelimbs increased in female mice compared to naïve but not in males with chronic degenerative joint pain (COL) (Figure 2B). ADWB measures of percent time on the affected left hind limb decreased compared to naïve in mice with acute and chronic inflammatory joint pain, but this was not statistically significant. These animals did spend significantly less time on the left hind limb than on the right hind limb. In both males and females with chronic degenerative joint pain, there was no difference in time spent on the left hind limbs compared to naïve. Females with chronic degenerative joint pain spent more time on the nonarthritic right hind limb compared to naive (Figure 3A). Mice with acute inflammatory joint pain and females with chronic degenerative joint pain spent significantly more time on the forelimbs than naïve animals (Figure 3B).
Figure 1. Evoked pain scores for left (blue column) and right (red column) hind limbs. Right hind limbs served as internal controls and did not undergo any IA injections. Measurements were taken in the following conditions: uninjected naïve (male and female), acute inflammatory carrageenan-induced arthritis (CAR), chronic inflammatory Complete Freund's Adjuvant-induced arthritis (CFA) and chronic noninflammatory Collagenase-induced arthritis (COL – male and female). In naïve animals the EPS of the left hind knee was not statistically different from the right in males and only minimally different in females (**P = 0.038) likely due to some increased pain response in the knee examined second (the left) due to crossover pain response. In all arthritic groups the arthritic left knee EPS was significantly greater than the nonarthritic contralateral right knee. When comparing the EPS of the left knee of arthritis groups to naïve animals, all arthritis groups had significantly greater EPS scores in the arthritic left knee compared to naïve left knees. This was true in both males and females. *P < 0.05 compared to male naïve, #P < 0.05 compared to female naïve. All error bars are SEM in all figures. Please click here to view a larger version of this figure.
Figure 2: Spontaneous pain behavior measured by percent body weight borne on limbs. (A) Spontaneous pain behavior measured by total percent body weight on the left (blue column) and right (red column) hind limbs. In both male and female naïve animals, there was no significant difference between percent weight borne on the left and right hind limbs. Male mice with CAR and CFA induced joint pain and females with COL induced joint pain bore significantly less weight on the left hind limb compared to the naive. *P < 0.05 compared to male naïve, #P < 0.05 compared to female naïve. (B) Spontaneous pain behavior measured by total percent body weight placed on the forelimbs. In males with CAR induced acute joint pain and females with COL induced chronic joint pain, there was a significant increase in the amount of weight borne on the forelimbs. This was not seen in males with CFA or COL induced joint pain. Naïve females tended to bear more weight on the forelimbs than naïve males (P = 0.02). Additional weight may be borne by other body parts such as the tail or rump. These were usually minimal and did not contribute significantly to the understanding of the pain response and are therefore not shown. *P < 0.05 compared to male naïve, #P < 0.05 compared to female naïve and to male 4 week COL in forelimb weightbearing. Please click here to view a larger version of this figure.
Figure 3: Spontaneous pain behavior measured by percent time spent on limbs. (A) Spontaneous pain behavior measured by total percent time spent on the left (blue column) and right (red column) hind limbs. There was no significant difference between right and left hind limbs in male and female naïve mice with respect to time spent on the limb. Male mice with CAR and CFA induced arthritis appeared to spend less time weightbearing with the arthritic left hind limb compared to naïve, but this was not statistically significant and likely due to increased variability in this measure in these mice. There was a significant difference in these animals comparing right to left as the right hind limb spent more total time weightbearing than the left. In females with COL induced arthritis, the right hind limb was weightbearing a greater proportion of time than in naïve females. *P < 0.05 comparing right to left, #P < 0.05 compared to female naïve and to male 4 week COL in time spent on forelimbs. (B) Spontaneous pain behavior measured by total amount of time spent on the forelimbs. In males with CAR induced arthritis and females with COL induced arthritis the mice spent significantly more time weightbearing on the fore limbs than did the naïve animals. Female naïve mice spent more time on the forelimbs than male naïve animals. *P < 0.05 compared to male naïve, #P < 0.05 compared to female naïve. Please click here to view a larger version of this figure.
Advanced Dynamic Weightbearing Device | Bioseb In Vivo Research Instruments, Vitrolles, France | Model: BIO-DWB-M – For mice | |
C57/Bl6 mice (male and female) | Jackson Labs, Bar Harbor ME | Stock No: 000664 | Black 6 | |
Carrageenan | Sigma-Aldrich, St. Louis, MO, USA | 22049-5G-F | |
Complete Freund's Adjuvant | Sigma-Aldrich, St. Louis, MO, USA | F5881 | |
Palpometer | Palpometer Systems, Inc. Victoria, B.C | Not available – no longer in business | |
Type IV Collagenase | Worthington Biomedical Corp, Lakeville, NJ | LS004210 |
Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.
Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.
Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.