Relieving Effects Of Acupuncture On Complete Freund's Adjuvant-induced Peripheral Chronic Inflammatory Pain In Mice

Inflammatory pain is one of the most common pathologic pains in clinic. Inflammatory pain arises as a debilitating consequence of injury (including the wound, germ, virus infection and surgical operation) to the peripheral tissue, which is characterized by combination of spontaneous burning pain, hyperalgesia and allodynia. Complete Freund's adjuvant (CFA) is often been used to make various chronic inflammatory pain models for basic study. The researches about it's peripheral and central mechanism have been the important focus of the international pain research organization. Substantial evidences have indicated that a number of bioactive substances were involved in the formation and modulation of inflammatory pain. However, the etiological mechanism of inflammatory pain has been poorly understood.Acupuncture, a traditional therapeutic modality from Traditional Chinese Medicine, has been used for more than 2000 years to treat a variety of diseases and symptoms with few side effects. Numerous clinical and basic studies show that acupuncture can effectively relieve pain with acupoint specificity. However, the mechanism underlying the pain-relieving effect is far from clear. To investigate the mechanisms underlying acupuncture analgesia, many studies have been conducted using electro-acupuncture (EA) on various experimental animal models.Modern biomedical studies demonstrate that peripheral information (e.g. pain, analgesia) is first integrated in the spinal cord. This process mainly depends on two kinds of neurotransmitters (excitatory and inhibitory) released from the dorsal horn neurons. They influence the excitement of the related neurons, then interfere the integration and conduction of afferent information in the spinal cord, ultimately affect the particular sense in the brain. Gamma-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter and wildly distributed in the central nervous system (CNS) of the mammalian. It is highly concentrated in the superficial dorsal horn of the spinal cord where primary nociceptive afferents terminate. GABA acts through two receptor subtypes, GABA_a and GABA_b, contributing to inhibitory control at the spinal level. At present, the role of spinal GABA_b receptor in pain modulation has been definit. However, it is less clear for GABA_a receptor.In our studies, we employed the peripheral chronic inflammatory pain model induced by the complete Freund's adjuvant (CFA) in mice, to investigate the analgesic effects of electro-acupuncture (EA) treatment at different acupoints and observe the involvement of spinal GABA_a receptor in chronic pain and acupuncture analgesia.The results were following:1 CFA-induced peripheral inflammatory pain model1.1 Production of modelInflammatory pain was induced by an injection of 20μl complete Freund's adjuvant (CFA) into the right paw of the mouse. The inflammation, manifested as redness, edema, and algesia, was limited to the injected paw. Control group was injected normal saline (NS, same volume as used for the CFA injection). Paw withdrawal latency (PWL) was used to assess the inflammatory pain. Besides the PWL tests, the circumference of the paw around the NS- or CFA- injected point was measured to assess the severity of inflammation.1.2 Significant decreased PWL in the pain modelUnder controlled environmental conditions, PWL was measured by observing the animal's paw-withdrawal response after applying initial heat to the left and right paws. The results indicated that PWL was significantly shortened on the right side (injected side) 24 hours after injection of CFA. The shortened PWL could remain 14 days. No obvious changes were observed in the non-injected side or Saline-injected side. 1.3 Swelling in the pain modelBesides the PWL tests, the circumference of the paw around the saline- or CFA-injected point was measured to assess the severity of inflammation. The measurement was done by slightly winding a thread around the paw and reading the thread length. The results indicated that the circumference of the right paws was significantly increased 24 hours after injection of CFA and lasted during the 21-day observation period. In none of the tested groups could we observe significant changes in the circumference of the left paws.2 Effects of EA on CFA-induced peripheral inflammatory pain2.1 Effects of EA at bilateral acupoints 'Zu-san-li' and 'Kun-lun' on paw circumferencesExcept for the saline-injection group, the circumference of the right paws was significantly increased in all other CFA-injection groups and lasted during the 21-day observation period. In none of the tested groups could we observe significant changes in the circumference of the left paws.2.2 Immediate effects of EA at bilateral acupoints 'Zu-san-li' and 'Kun-lun' on PWL responsesThe mice were separated into 4 groups: (1) the saline group (sln) with saline injection; (2) the model group (mdl) with CFA injection; (3) the EA group (mdl EA) with CFA injection and treated with EA; (4) the sham EA group (shm EA) with CFA injection and treated by sham EA. We found that PWLs were significantly shortened on the right side (injected side) after injection (injct) of CFA in the mdl, mdl EA, and shm EA groups, but not in the sln group. The shortened PWL remained during the entire 80 min observation in the mdl and shm EA group. In the EA-treated mdl EA group, the shortened PWL did recover temporarily in the time period from 20 to 30 min after the EA treatment, but returned thereafter to the shortened PWL time seen for the mdl and shm EA groups.2.3 Cumulative effects of EA at bilateral acupoints 'Zu-san-li' and 'Kun-lun' on PWL responses Similar to the immediate observation, there was a significant decrease in PWL in the mdl, mdl EA, and shm EA groups after injection of CFA on the right side in the cumulative observation. The decreased PWL continued during the 14-day lasting observation in the mdl and shm EA groups. After repetitive (2 times) EA treatment the decreased PWL of the mdl EA group recovered at day 8 to the value seen in the sln group. There were no apparent changes in PWL on the left side in all 4 groups tested.2.4 Immediate effects of EA at bilateral acupoints 'Shou-san-li' and 'Nei-guan' on PWL responsesCompared with EA at acupoints 'Zu-san-li'and 'Kun-lun', there were no apparent increase in PWL in the EA at acupoints 'Shou-san-li' and 'Nei-guan'.3 Involvement of spinal GABA_a receptor in the CFA-induced peripheral inflammatory pain and acupuncture analgesia3.1 The effect of intrathecal injecting bicuculline on PWL responses in pain model miceWe observed bicuculline (Bic)'s effect, at seven different doses (1, 0.1, 0.05, 0.02, 0.01, 0.005, 0.0025μg/5μl) on the PWL of the CFA-injected paw. The results revealed that highest doses of bicuculline (1 and 0.1μg/5μl) produced spontaneous vocalization, allodynia and caudally directed biting and scratching behavior. Other doses had no effect on PWL during the 100 min.3.2 The effect of intrathecal injecting bicuculline on PWL responses in normal miceWe observed the effect of intrathecal injecting (i.t.) higher dose (0.05μg/5μl) of bicuculline (Bic), a GABA_a receptor antagonist on PWL in normal mice. Experiments were divided into two groups: Normal + i.t. Implantation group (normal mice only receiving intrathecal surgery) and Normal + i.t. 0.05μg/5μl group (normal mice injected Bic, at 0.05μg/5μl). The results showed that compared with the pre-drug value, dose of 0.05μg/5μl Bic significantly decreased the PWL in 20 minutes after injecting.3.3 The effect of intrathecal injecting bicuculline on EA analgesiaWe observed bicuculline (Bic), at higher dose (0.05μg/5μl) on EA relieving pain. Mice were divided into four groups: Model + i.t. NS group, Model +EA group, Model +sham EA group and Model +EA+ i.t. 0.05μg/5μl Bic. The results showed that compared with Md1+EA group, the increased PWL after EA treatment did decrease after i.t. injecting Bic.Conclusion of the study:1. CFA is a potent agent that can produce inflammation of the tissue, and it has been clinically used as a model of peripheral inflammatory pain in animals such as rats and mice representing a kind of peripheral inflammatory disease. In our study, we successfully made CFA-induced peripheral chronic inflammatory pain models.2. EA effectively relieves the peripheral chronic inflammatory pain by CFA, in mice, at acupoints within or near the same spinal segment of the pain location.3. Spinal GABA_A receptor might participate in CFA-induced peripheral inflammatory pain modulation and EA analgesia effect.