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Effect of electroacupuncture on hyperalgesia and Fos protein expression in rats with persistent inflammation – a new animal model
Effect of electroacupuncture on hyperalgesia and Fos protein expression in rats with persistent inflammation – a new animal model Lixing Lao, Grant Zhang, Feng Wei, Brian M. Berman, Ke Ren INTRODUCTION
L. Lao, G. Zhang, B. M. Berman, Complementary Medicine Program, School of Medicine; F.Wei, K. Ren, Dept. of OCBS, Dental School, University of Maryland, Baltimore, MD 21201, USA This is an abstract from the Society for Acupuncture Research (SAR) annual conference proceedings
Acupuncture, a traditional therapeutic modality, has been used in China and other Asian countries for thousands of years for treating a variety of diseases and symptoms, including inflammation and arthritic pain. To investigate the mechanism of action of acupuncture, animal behavioral models have been used to evaluate the analgesic effects of acupuncture (AA). Persistent inflammatory pain models have been recently developed in the rat using inflammatory agents such as yeast, carrageenan, and complete Freund’s adjuvant (CFA), which produce inflammation lasting for hours, days, or weeks (Kayser & Guilband 1987, Winter & Flataker 1965, Larson et al. 1986). CFA-mediated persistent inflammatory pain involves a pathogenic condition that mimics sub-acute rheumatoid arthritis and can be quantitatively. This animal model has advanced the understanding of the mechanisms of persistent pain and the actions of pharmaceutical drugs. Few studies have used this behavioral model to study the mechanism of electroacupuncture (EA). Fos, the protein encoded by the c-fos prooncogene, is a useful marker of neuronal activity in response to noxious stimulation, and can be used to map the functionally related neuronal pathways (Morgan et al. 1987, Hunt et al. 1987, Presley et al.
Clinical Acupuncture and Oriental Medicine (2000) 1, 112–114 © 2000 Harcourt Publishers Ltd
1990, Bullitt 1990). More recently, Fos-like immunoreactivity (Fos-LI) has been used to investigate the mechanism of AA by identifying neurons that are activated and/or suppressed by acupuncture in animals (Lee & Beitz 1993, Ji et al. 1993, Zhou et al. 1993, Guo et al. 1996).
OBJECTIVES This study was designed to 1) evaluate the usefulness of an animal model of inflammatory pain in the study of acupuncture analgesia (AA); 2) examine the effect of EA on persistent inflammation and hyperalgesia; and 3) investigate the mechanism of AA by examining the Fos protein expression in the spinal cord.
METHODS AND MATERIAL Male Sprague-Dawley rats weighing 300–400 g were used. The inflammation and hyperalgesia were induced by injecting complete Freund’s adjuvant (CFA, 0.05 ml, 0.025 mg Mycobacterium tuberculosis) into the plantar suface of left hindpaw of the rat. The inflammation, manifested as redness, edema, and hyper-responsiveness to noxious stimuli, was limited
Electroacupuncture on hyperalgesia and Fos protein expression
to the injected paw. Hyperalgesia was determined by a decrease in paw withdrawal latencies (PWL) to a thermal stimulus (Hargreaves et al. 1988, Hylden et al. 1989, Ren et al. 1992), and inflammation was determined by measuring the diameter of the hindpaw thickness with a caliper. The inflammation appeared shortly after the injection, peaked at 24 h, and lasted for about two weeks. Rats were divided into EA-treatment (n=6) and placebo-control (n = 6). For EA treatment, the equivalent human acupuncture point Huantiao (G30) was selected at the rat’s hindlimbs bilaterally (O’Connor & Bensky 1981). Two acupuncture needles (gauge # 32, 1 inch in length) were inserted into the two acupuncture points, and a pair of electrodes from an electrical stimulator (A300, WPI) was attached to the ends of the needles. Commonly used clinical EA parameters were chosen at 3 V, 10 Hz and 0.1 ms pulse width for 20 min. The EA was applied immediately after the injection of CFA, and 2 h later. For placebo control, the needles were taped on the surface of G30 and a pair of electrodes from the stimulator was attached to the ends of the needles but no electrical current was delivered. For Fos protein expression, the L4–L5 segments of the spinal cord from the CFA-inflamed rats with or without EA treatment (n = 3 for each group) were examined. The same EA treatment was given as in the behavioral study. In addition, non-inflamed rats (no CFA injected, n = 2) that received EA treatment were included as EA control. All rats were perfused at 2.5 h following the EA treatment, and the tissues were immediately processed for immunocytochemistry.
RESULTS The results showed that acupuncture-treated rats had a significantly longer PWL in the inflamed paw than that in control rats at 2.5 h and five days after the injection of CFA (p < 0.05). No significant differences were found between these two groups at 5 h and 24 h. The paw edema was also significantly reduced in the acupuncture-treated rats compared to the controls at 24 h after the injection (p < 0.01). The data suggest that EA treatment at an early stage of the inflammation may be effective in delaying the onset and facilitating the recovery of the inflammatory hyperalgesia, but may not be efficacious against severe hyperalgesia. The results from Fos protein experiment showed that, on the ipsilateral side on the inflamed hindpaw, Fos-expression in medial half of the laminae I–II in the EA-treated rats were significantly less than that in placebo controls (p < 0.01). An increased number
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of Fos-LI neurons were found in both EA treated rats and the placebo control rats in laminae III–VI (regions). On the contralateral side of the inflamed (hind) paw, there was no significant difference in laminae I–II between these two groups. However, in the III–VI region, the EA treated rats had significantly more Fos-LI cells than placebo control rats (p < 0.05). These data suggest that EA suppressed the inflammation induced Fos expression in neurons (laminae I–II) that are involved in receiving noxious stimulation but selectively activated Fos expression in neurons in other regions (laminae III–VI). The data also showed that Fos-LI neurons in EA-treated naive (non-inflamed) rats were increased in the selective region (laminae III–VI, but not laminae I–II) on both the ipisilateral and contralateral sides of the spinal cord, suggesting that low intensity EAinduced Fos protein expression is mainly in nonnociceptive neurons.
DISCUSSION The classically used animal models in AA studies are normal (intact) animal models with transient noxious stimuli. Although they have been proven valuable in establishing that AA does, in fact, exist, they are of limited clinical relevance. To evaluate EA in clinically relevant pathological conditions, a persistent inflammatory pain animal model would be more appropriate. In the present study, we used a rat model of persistent pain and inflammation to investigate the analgesic, anti-hyperalgesic and anti-inflammatory effects of acupuncture. This model allows us to address clinically relevant conditions such as rheumatoid arthritic pain and inflammation; to conduct the behavioral study with non-restrained animals to minimize possible stress induced analgesia (SIA) both during EA and subsequent behavioral tests; to assess the anti-hyperalgesic and anti-inflammatory effects of EA; and to have sufficient time (up to two weeks) to evaluate post-treatment effects of EA. The Fos protein immunocytochemistry studies using this animal model suggest that the effect of EA may be mediated by selectively activating non-nociceptive neurons, but inhibiting nociceptive neurons in the spinal cord. This study provides an ideal animal model for further exploring the mechanisms of action of acupuncture.
REFERENCES Bullit E. Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J Comp Neuro 1990; 296: 517–530.
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Electroacupuncture on hyperalgesia and Fos protein expression
Guo HF, Tian J, Wang X, Fang Y, Hou Y, Han J. Brain substrates activated by electroacupuncture of different frequencies (I): comparative study on the expression of oncogene c-fos and genes coding for three opioid peptides. Mol Brain Res 1996; 43: 157–166. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988; 32: 77–88. Hunt SP, Pini A, Evan G. Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature (Lond.) 1987; 328: 632–634. Hylden JLK, Nahin RL, Traub RJ, Dubner R. Expansion of receptive fields of spinal lamina I projection neurons in rats with unilateral adjuvant-induced inflammation: the contribution of dorsal horn mechanisms. Pain 1989; 37: 229–243. Ji RR, Zhang ZW, Zhou Y, Zhang Q, Han JS. Induction of c-fos expression in the rostral medulla of rats following electroacupuncture stimulation. Int J Neurosci 1993; 72: 183–91. Kayser V, Guilbaund G. Local and remote modifications of nociceptive sensitivity during carrageenin-induced inflammation in the rat. Pain 1987; 28: 99–107. Larson AA, Brown DR, EI-Atrash S, Walser MM. Pain threshold changes in adjuvant-induced inflammation: a
possible model of chronic pain in the mouse. Pharmacol Biochem Behav 1986; 24: 49–53. Lee JH, Beitz AJ. The distribution of brain-stem and spinal cord nuclei associated with different frequencies of electroacupuncture analgesia. Pain 1993; 52: 11–28. Morgan JI, Cohen DR, Hempstead JL, Curran T. Mapping patterns of c-fos expression in the central nervous system after seizures. Science 1987; 237: 192–197. O’Connor J, Bensky D (eds). Acupuncture-A comprehensive text. Chicago: Eastland Press; 1981. Presley RW, Menetrey D, Levine JD, Basbaum AI. Systemic morphine suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord. J Neurosci 1990; 10: 323–335. Ren K, Williams GM, Hylden JLK, Ruda MA, Dubner R. The intrathecal administration of excitatory amino acid receptor antagonists selectively attenuated carrageenaninduced behavioral hyperalgesia in rats. Eur J Pharmacol 1992; 219: 235–243. Winter, Flataker (1965) Reaction thresholds to pressure in edematous hidpaws of rats and responses to analgesic drugs. J Pharmacol Exp Ther 150: 165–171. Zhou Z, He L, Zjou J. Expression of C-fos protein in CNS following electroacupuncture of the “quanliao” and the electrostimulation of the rat tail. Chen Tzu Yen Chiu 1993; 18: 14–20.
L. Lao, G. Zhang, B. M. Berman, Complementary Medicine Program, School of Medicine; F.Wei, K. Ren, Dept. of OCBS, Dental School, University of Maryland, Baltimore, MD 21201, USA This is an abstract from the Society for Acupuncture Research (SAR) annual conference proceedings
Acupuncture, a traditional therapeutic modality, has been used in China and other Asian countries for thousands of years for treating a variety of diseases and symptoms, including inflammation and arthritic pain. To investigate the mechanism of action of acupuncture, animal behavioral models have been used to evaluate the analgesic effects of acupuncture (AA). Persistent inflammatory pain models have been recently developed in the rat using inflammatory agents such as yeast, carrageenan, and complete Freund’s adjuvant (CFA), which produce inflammation lasting for hours, days, or weeks (Kayser & Guilband 1987, Winter & Flataker 1965, Larson et al. 1986). CFA-mediated persistent inflammatory pain involves a pathogenic condition that mimics sub-acute rheumatoid arthritis and can be quantitatively. This animal model has advanced the understanding of the mechanisms of persistent pain and the actions of pharmaceutical drugs. Few studies have used this behavioral model to study the mechanism of electroacupuncture (EA). Fos, the protein encoded by the c-fos prooncogene, is a useful marker of neuronal activity in response to noxious stimulation, and can be used to map the functionally related neuronal pathways (Morgan et al. 1987, Hunt et al. 1987, Presley et al.
Clinical Acupuncture and Oriental Medicine (2000) 1, 112–114 © 2000 Harcourt Publishers Ltd
1990, Bullitt 1990). More recently, Fos-like immunoreactivity (Fos-LI) has been used to investigate the mechanism of AA by identifying neurons that are activated and/or suppressed by acupuncture in animals (Lee & Beitz 1993, Ji et al. 1993, Zhou et al. 1993, Guo et al. 1996).
OBJECTIVES This study was designed to 1) evaluate the usefulness of an animal model of inflammatory pain in the study of acupuncture analgesia (AA); 2) examine the effect of EA on persistent inflammation and hyperalgesia; and 3) investigate the mechanism of AA by examining the Fos protein expression in the spinal cord.
METHODS AND MATERIAL Male Sprague-Dawley rats weighing 300–400 g were used. The inflammation and hyperalgesia were induced by injecting complete Freund’s adjuvant (CFA, 0.05 ml, 0.025 mg Mycobacterium tuberculosis) into the plantar suface of left hindpaw of the rat. The inflammation, manifested as redness, edema, and hyper-responsiveness to noxious stimuli, was limited
Electroacupuncture on hyperalgesia and Fos protein expression
to the injected paw. Hyperalgesia was determined by a decrease in paw withdrawal latencies (PWL) to a thermal stimulus (Hargreaves et al. 1988, Hylden et al. 1989, Ren et al. 1992), and inflammation was determined by measuring the diameter of the hindpaw thickness with a caliper. The inflammation appeared shortly after the injection, peaked at 24 h, and lasted for about two weeks. Rats were divided into EA-treatment (n=6) and placebo-control (n = 6). For EA treatment, the equivalent human acupuncture point Huantiao (G30) was selected at the rat’s hindlimbs bilaterally (O’Connor & Bensky 1981). Two acupuncture needles (gauge # 32, 1 inch in length) were inserted into the two acupuncture points, and a pair of electrodes from an electrical stimulator (A300, WPI) was attached to the ends of the needles. Commonly used clinical EA parameters were chosen at 3 V, 10 Hz and 0.1 ms pulse width for 20 min. The EA was applied immediately after the injection of CFA, and 2 h later. For placebo control, the needles were taped on the surface of G30 and a pair of electrodes from the stimulator was attached to the ends of the needles but no electrical current was delivered. For Fos protein expression, the L4–L5 segments of the spinal cord from the CFA-inflamed rats with or without EA treatment (n = 3 for each group) were examined. The same EA treatment was given as in the behavioral study. In addition, non-inflamed rats (no CFA injected, n = 2) that received EA treatment were included as EA control. All rats were perfused at 2.5 h following the EA treatment, and the tissues were immediately processed for immunocytochemistry.
RESULTS The results showed that acupuncture-treated rats had a significantly longer PWL in the inflamed paw than that in control rats at 2.5 h and five days after the injection of CFA (p < 0.05). No significant differences were found between these two groups at 5 h and 24 h. The paw edema was also significantly reduced in the acupuncture-treated rats compared to the controls at 24 h after the injection (p < 0.01). The data suggest that EA treatment at an early stage of the inflammation may be effective in delaying the onset and facilitating the recovery of the inflammatory hyperalgesia, but may not be efficacious against severe hyperalgesia. The results from Fos protein experiment showed that, on the ipsilateral side on the inflamed hindpaw, Fos-expression in medial half of the laminae I–II in the EA-treated rats were significantly less than that in placebo controls (p < 0.01). An increased number
113
of Fos-LI neurons were found in both EA treated rats and the placebo control rats in laminae III–VI (regions). On the contralateral side of the inflamed (hind) paw, there was no significant difference in laminae I–II between these two groups. However, in the III–VI region, the EA treated rats had significantly more Fos-LI cells than placebo control rats (p < 0.05). These data suggest that EA suppressed the inflammation induced Fos expression in neurons (laminae I–II) that are involved in receiving noxious stimulation but selectively activated Fos expression in neurons in other regions (laminae III–VI). The data also showed that Fos-LI neurons in EA-treated naive (non-inflamed) rats were increased in the selective region (laminae III–VI, but not laminae I–II) on both the ipisilateral and contralateral sides of the spinal cord, suggesting that low intensity EAinduced Fos protein expression is mainly in nonnociceptive neurons.
DISCUSSION The classically used animal models in AA studies are normal (intact) animal models with transient noxious stimuli. Although they have been proven valuable in establishing that AA does, in fact, exist, they are of limited clinical relevance. To evaluate EA in clinically relevant pathological conditions, a persistent inflammatory pain animal model would be more appropriate. In the present study, we used a rat model of persistent pain and inflammation to investigate the analgesic, anti-hyperalgesic and anti-inflammatory effects of acupuncture. This model allows us to address clinically relevant conditions such as rheumatoid arthritic pain and inflammation; to conduct the behavioral study with non-restrained animals to minimize possible stress induced analgesia (SIA) both during EA and subsequent behavioral tests; to assess the anti-hyperalgesic and anti-inflammatory effects of EA; and to have sufficient time (up to two weeks) to evaluate post-treatment effects of EA. The Fos protein immunocytochemistry studies using this animal model suggest that the effect of EA may be mediated by selectively activating non-nociceptive neurons, but inhibiting nociceptive neurons in the spinal cord. This study provides an ideal animal model for further exploring the mechanisms of action of acupuncture.
REFERENCES Bullit E. Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J Comp Neuro 1990; 296: 517–530.
114
Electroacupuncture on hyperalgesia and Fos protein expression
Guo HF, Tian J, Wang X, Fang Y, Hou Y, Han J. Brain substrates activated by electroacupuncture of different frequencies (I): comparative study on the expression of oncogene c-fos and genes coding for three opioid peptides. Mol Brain Res 1996; 43: 157–166. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988; 32: 77–88. Hunt SP, Pini A, Evan G. Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature (Lond.) 1987; 328: 632–634. Hylden JLK, Nahin RL, Traub RJ, Dubner R. Expansion of receptive fields of spinal lamina I projection neurons in rats with unilateral adjuvant-induced inflammation: the contribution of dorsal horn mechanisms. Pain 1989; 37: 229–243. Ji RR, Zhang ZW, Zhou Y, Zhang Q, Han JS. Induction of c-fos expression in the rostral medulla of rats following electroacupuncture stimulation. Int J Neurosci 1993; 72: 183–91. Kayser V, Guilbaund G. Local and remote modifications of nociceptive sensitivity during carrageenin-induced inflammation in the rat. Pain 1987; 28: 99–107. Larson AA, Brown DR, EI-Atrash S, Walser MM. Pain threshold changes in adjuvant-induced inflammation: a
possible model of chronic pain in the mouse. Pharmacol Biochem Behav 1986; 24: 49–53. Lee JH, Beitz AJ. The distribution of brain-stem and spinal cord nuclei associated with different frequencies of electroacupuncture analgesia. Pain 1993; 52: 11–28. Morgan JI, Cohen DR, Hempstead JL, Curran T. Mapping patterns of c-fos expression in the central nervous system after seizures. Science 1987; 237: 192–197. O’Connor J, Bensky D (eds). Acupuncture-A comprehensive text. Chicago: Eastland Press; 1981. Presley RW, Menetrey D, Levine JD, Basbaum AI. Systemic morphine suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord. J Neurosci 1990; 10: 323–335. Ren K, Williams GM, Hylden JLK, Ruda MA, Dubner R. The intrathecal administration of excitatory amino acid receptor antagonists selectively attenuated carrageenaninduced behavioral hyperalgesia in rats. Eur J Pharmacol 1992; 219: 235–243. Winter, Flataker (1965) Reaction thresholds to pressure in edematous hidpaws of rats and responses to analgesic drugs. J Pharmacol Exp Ther 150: 165–171. Zhou Z, He L, Zjou J. Expression of C-fos protein in CNS following electroacupuncture of the “quanliao” and the electrostimulation of the rat tail. Chen Tzu Yen Chiu 1993; 18: 14–20.