Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Summary

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Abstract

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.

Introduction

Complex regional pain syndrome (CRPS) reprents complex and chronic pain symptoms resulting from fractures, trauma, surgery, ischemia or nerve injury^1,2,3. CRPS is classified into 2 subcategories: CRPS type-I and type-II (CRPS-I and CRPS-II)⁴. Epidemiological studies revealed that the prevalence of CRPS was approximately 1:2000⁵. CRPS-I, which shows no obvious nerve damage, can result in chronic pain and dramatically affects the life quality of the patients. Current available treatments show inadequate therapeutic effects. Therefore, CRPS-I still remains an important and challenging clinical problem that needs to be addressed.

Establishing a preclinical animal model mimicking CRPS-I is crucial for exploring the mechanisms underlying CRPS-I. In order to address this issue, Coderre et al. designed a rat model by applying prolonged ischemia and reperfusion to the hind limb to recapitulate CRPS-I⁶. It is known that ischemia/reperfusion injury is among one of the major causes of CRPS-I⁷. The rat CPIP model exhibits many CRPS-I-like symptoms, which include hind limb edema and hyperemia in the early stage after model establishment, followed with persistent thermal and mechanical hypersensitivities⁶. With the aid from this model, it is proposed that central pain sensitization, peripheral TRPA1 channel activation and reactive oxygen species generation, etc. contribute to CRPS-I^8,9,10. We recently successfully established the CPIP rat model and performed RNA-sequencing of the dorsal root ganglia (DRGs) that innervate the affected hind paw¹¹. We discovered some potential mechanisms that are possibly involved in mediating the pain hypersensitivities of CRPS-I¹¹. We further identified transient receptor potential vanilloid 1 (TRPV1) channel in DRG neurons as an important contributor to the mechanical and thermal hypersensitivities of CRPS-I¹².

In this study, we described the detailed procedures involved in the establishment of the rat model of CPIP. We further evaluated the rat CPIP model by measuring the mechanical and thermal hypersensitivities as well as its responsiveness to acute capsaicin challenge. We propose that the rat CPIP model can be a reliable animal model for further investigation of the mechanisms involved in CRPS-I.

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Protocol

The animal protocols were approved by Zhejiang Chinese Medical University Animal Ethics Committee.

1. Animals

1. 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

1. 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.
2. Ischemia and reperfusion of the hind paw
   1. Lubricate the right hind paw and ankle with glycerol once the rat is anesthetized.
   2. 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.
   3. 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.
   4. 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

1. 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.
2. Mechanical allodynia
   1. 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.
   2. Apply the up-down testing method to test the threshold. Apply the Dixon method for calculating 50% paw withdrawal threshold (PWT)^13,14,15.
3. Thermal hyperalgesia
   1. 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.
   2. 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.
4. Capsaicin-induced acute nocifensive behavior
   1. 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.
   2. 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 described^17,18,19.
5. 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.

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Results

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 pos...

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Discussion

This protocol describes the detailed methods for establishing a rat CPIP model by applying ischemia/reperfusion to hind limbs of the rats. It involves the evaluation of hind limb appearance, edema, mechanical/thermal hypersensitivities, and acute nocifensive behaviors in response to capsaicin injection.

Limb ischemia/reperfusion is a common factor contributing to CRPS-I in human patients¹². This protocol describes how to establish the rat CPIP model, which is a commonly...

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Materials

Name	Company	Catalog Number	Comments
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