Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats
Instructor Prep
concepts
Student Protocol
The animal protocols were approved by Zhejiang Chinese Medical University Animal Ethics Committee.
1. Animals
- Obtain male Sprague-Dawley (SD) rats (280–320 g, 8-10 weeks of age) from Shanghai Laboratory Animal Center. House the animals in Zhejiang Chinese Medical University Laboratory Animal Center. Note that the breeding conditions should include 12 h/ 2h light/dark cycles and keep temperature constant at 24 °C. Provide water and food ad libitum. Note that a total of 48 rats are used in this study. One note, in this model, we need to observe the pain response during the whole process. If pain medication is applied, then we cannot successfully establish the pain model. After the experiments, the rats are sacrificed.
2. CPIP model establishment
- Anesthetize all rats (including sham and CPIP model groups) with sodium phenobarbital (50 mg/kg, intraperitoneal injection [i.p.]). Maintain anesthesia with up to 20 mg/kg/h phenobarbital [i.p.], if necessary. Check the reflexes of each animal by pinching its hind paw or tail tip using forceps. Make sure that the rats are not responsive before model establishment. Place vet ointment on eyes to avoid dryness during the procedure. Place the anesthetized rats on a heated pad maintained at 37 °C for the following procedure.
- Ischemia and reperfusion of the hind paw
- Lubricate the right hind paw and ankle with glycerol once the rat is anesthetized.
- Slide a Nitrile 70 Durometer O-ring with a 7/32" (5.5 mm) internal diameter into the larger side of a 1.5 mL Eppendorf tube (with the snap-cap cut off before use). Carefully insert the hind paw into the hollow Eppendorf tube until reaching the bottom.
- Gradually slide the O-ring from the tube to the right hind limb near the ankle joint and place for 3 h. Apply the same treatment to a sham group of rats, except that a broken O-ring, which is cut off and should not induce ischemia, should be placed around the ankle.
- Cut off the O-ring 3 h after the ischemia step. Carefully watch the rat until it recovers enough consciousness to maintain sternal recumbency. Note that the rat that received anesthesia should not be placed back to the company of other rats until it fully recovered.
3. Nocifensive behavioral tests
- Place the rat in a transparent Plexiglas chamber that is sitting on a mesh floor. Habituate the rat for 0.5 h before any behavioral testing.
- Mechanical allodynia
- Use von Frey filaments (0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, 15.0, and 26.0 g filaments) for the test. Begin the test from the middle filament (4.0 g). Vertically apply the filaments to the middle plantar surface of the hind paw. Slightly apply suitable force to bend the filament for up to 5 s. A sudden retraction of the hind paw in response to the stimuli is considered a nocifensive behavior. Conduct the mechanical allodynia test on days -3, -2, -1, and every other day until day 13.
- Apply the up-down testing method to test the threshold. Apply the Dixon method for calculating 50% paw withdrawal threshold (PWT)13,14,15.
- Thermal hyperalgesia
- Use Hargreaves' method to examine thermal hyperalgesia. Directly aim the light beam emitted from a bulb (50 W) to the hind paw to measure the paw withdrawal latency (PWL). Set 20 s as the cut-off threshold to avoid excessive injury from the heating.
- Repeat each test 3x in 5 min intervals for each hind paw. Take the average of these three tests as the PWL of each rat16. Conduct the thermal hyperalgesia test on days -3, -2, -1, and every other day until day 13.
- Capsaicin-induced acute nocifensive behavior
- Prepare capsaicin stock solution (200 mM) using dimethyl sulfoxide (DMSO) and further dilute to 1:1000 in sterile phosphate-buffered saline (PBS) for hind paw injection. The final DMSO concentration in PBS is 0.1% (vehicle contains 0.1% DMSO in PBS only). Inject capsaicin or vehicle into the hind paw (intraplantar injection) at a volume of 50 μL using a 30 G needle attached to 1 mL syringe.
- Record the nocifensive behavior (i.e., licking, biting, or flinching of the injected paw) using a video camera for 10 min right after the injection and quantified thereafter as previously described17,18,19.
- Hind paw edema evaluation: Evaluate the hind paw edema by measuring the increase in paw diameter. Measure with a digital caliper and calculate the difference between the basal value and the test value observed at different time points. Assess the changes in paw thickness at 15 min, 24 h, 48 h, and 72 h after model establishment.
Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats
Learning Objectives
After placing the O-ring on the ankle, the ipsilateral hind paw skin showed cyanosis, an indication of tissue hypoxia (Figure 1A). After cutting the O-ring, the ipsilateral hind paw began to fill with blood and showed robust swelling, which demonstrated an intense sign of hyperemia (Figure 1A). The paw swelling gradually diminished and returned to normal 48 h after the ischemic/reperfusion procedure (two-way ANOVA with Sidak post-hoc test, Figure 1B). All of these signs are consistent with previous studies6,12.
Then, mechanical allodynia was measured using a von Frey hair test. The ipsilateral hind paw of the CPIP group exhibited obvious mechanical allodynia 1 day after model establishment compared to the sham group. The mechanical allodynia of the ipsilateral hind paw persisted until 13 days of the observation timeframe (two-way ANOVA with Sidak post-hoc test, Figure 1C). The contralateral hind paw of the CPIP group also displayed mechanical hyperalgesia similar to the ipsilateral hind paw, lasting for 13 days (two-way ANOVA with Sidak post-hoc test, Figure 1D).
Thermal hyperalgesia was then measured using a Hargreaves' test. Bilateral hind paws of CPIP rats exhibited significantly reduced withdrawal latency in response to noxious thermal stimuli, a sign of thermal hyperalgesia, compared to the sham group rats (two-way ANOVA with Sidak post hoc test, Figure 1E,F). This observation is consistent with previous studies11,12. The thermal hyperalgesia of the ipsilateral hind paw persisted until the end of the observation timeframe, whereas the thermal hyperalgesia of the contralateral hind paw lasted for 7 days (two-way ANOVA with Sidak post-hoc test, Figure 1E,F). The above results suggest that CPIP rats develop robust and persistent mechanical and thermal hypersensitivities, consistent with previous observations11,12.
CRPS patients exhibited an obvious increased response to capsaicin-induced pain in affected areas20. It was then examined whether the CPIP rat model can recapitulate this phenomenon. Nocifensive behavior in CPIP rats was observed in response to intraplantar capsaicin (a TRPV1 agonist) injection into the ipsilateral hind paw. First, the nocifensive responses of the rats when a vehicle was injected were tested. The sham group showed a slight nocifensive response to vehicle injection, whereas the CPIP group showed a significantly higher response compared to the sham group (one-way ANOVA with Sidak post-hoc test, Figure 2). Furthermore, capsaicin injection resulted in robust nocifensive response in the sham group (Figure 2). More importantly, CPIP rats showed significantly higher responses to capsaicin injection than the sham group (Figure 2). These data suggest that CPIP rats exhibited enhanced nocifensive responses to capsaicin, a phenomenon mimicking human patients with CRPS-I.
Figure 1: The CPIP rat model showed thermal and mechanical pain hypersensitivities in bilateral hind limbs. (A) Typical images taken during different time points (during ischemia, 10 min after reperfusion, and 7 days later). (B) Ipsilateral hind paw thickness measurements of both CPIP and sham groups. (C,D) 50% paw withdraw threshold (50% PWT, index of mechanical hyperalgesia) of ipsilateral (C) and contralateral (D) hind paws of rats. (E,F) Paw withdrawal latency (PWL, index of thermal hyperalgesia) of ipsilateral (E) and contralateral (F) hind paws of rats (n = 8 rats per group, **p < 0.01). Results are expressed as mean ± SEM (two-way ANOVA followed by Sidak post-hoc test). Please click here to view a larger version of this figure.
Figure 2: CPIP rats exhibited more nocifensive behaviors compared to sham rats in response to intraplantar capsaicin injection into ipsilateral hind paws. The cumulated time the rats spent licking, biting, or flinching the hind paws was calculated over 10 min after vehicle (0.1% DMSO in PBS, intraplantar) or capsaicin (10 nmol in 50 μL volume, intraplantar) injection (n = 8 rats per group, **p < 0.01). Results are expressed as mean ± SEM (one-way ANOVA followed by Sidak post-hoc test). Please click here to view a larger version of this figure.
List of Materials
| 1.5 ml Eppendorf tube | Eppendorf | 22431021 | |
| DMSO | Sigma-Aldrich | D1435 | |
| Capsaicin | APEXBIO | A3278 | |
| Digital caliper | Meinaite | NA | |
| O-ring | O-Rings West | Nitrile 70 Durometer | 7/32 in. internal diameter |
| Plantar Test Apparatus | UGO Basile, Italy | 37370 | |
| von Frey filaments | UGO Basile, Italy | NC12775 |
Lab Prep
Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.
Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.
Procedure
Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.