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ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION IN THE SPINAL CORD INDUCED BY NOXIOUS STIMULATION OF THE RAT BLADDER
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Vol. 160,2274-2279, December 1998 Printed in U.S.A.
THE JOURNAL OF UROLOGY Copyright 8 1998 by AWRICAN UROIXICAL ASSOCUTION, INC.
ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION IN THE SPINAL CORD INDUCED BY NOXIOUS STIMULATION OF THE RAT BLADDER CARL J. CHANG, SHIH-TSUNG HUANG, KENNETH HSU, AUSTIN LIN, MARSHALL L. STOLLER AND TOM F. LUE" From the Department of Urology, School of Medicine, University of California at Sun Francisco, S u n Francisco, C A
ABSTRACT
Purpose: The present study investigated the effects of noxious stimulation of the lower urinary tract on neuronal fos protein expression in the spinal micturition center of rats and also examined the effects of electroacupuncture (EA)on fos expression induced by noxious stimulation of the lower urinary tract. Materials and Methods: Experiments were conducted on 21 female Sprague-Dawley rats divided into four groups. Group 1rats (n = 5) served as normal controls. Group 2 rats (n = 5) received EA at the Sanyinjiao acupoint. Group 3 animals (n = 6) were catheterized through the urethra and instilled with 1%acetic acid, and group 4 (n = 5) animals received EA 1hour before 1%acetic acid instillation. All animals underwent arterial perfusion, laminectomy, and spinal cord removal. Spinal cords were sectioned and processed for immunohistochemical staining for fos protein. Results: No fos protein was detected in any spinal neurons in normal control animals, and either none or few (0 to 4 cells/section) fos-immunoreactive (fos-IR) cells were seen in animals treated with EA. Noxious stimulation of the lower urinary tract with 1%acetic acid drastically increased the number of fos-IR neurons (30 to 127 cells&, section, mean 76.17 2 13.98; 28 to 77, cells/Sl section, mean 59 2 8.30; 7 to 35 cells/S2 section, mean 19.83 5 4.10). However, EA administered 1 hour before 1%acetic acid instillation significantly decreased the number of fos-IR neurons resulting from chemical irritation (0 to 50 cells&, section, mean 19.8 2 9.33; 0 to 47 cells/Sl section, mean 13.2 ? 9.12; 0 to 37 cells/S2 section, mean 13.6 t 7.31). Conclusions: Our study demonstrates that bladder instillation with 1%acetic acid induces fos protein expression in the spinal micturition center of the rat and that electroacupuncture can reduce this expression. These results suggest a link between electroacupuncture and reduction in spinal neuronal cell activity. KEY WORDS: c-fos, electroacupuncture, spinal cord, immunohistochemistry
Many studies have been conducted in recent years to exAcupuncture is considered a new alternative form of medicine in western society but has been widely used for over amine the effects and mechanism of acupuncture on pain 5000 years in China as a n integral part of traditional Chinese relief; however, most of these studies have only focused on medicine in the treatment and prevention of disease. Over the philosophical aspect of acupuncture and not on its physthe centuries, its use has spread to other Asian countries and iological mechanism. The precise effects of EA on the central eventually to Europe and America. Acupuncture involves the nervous system are still not completely known, primarily insertion of fine needles, often used together with electrical because of the difficulty of tracing neuronal pathways by stimuli, on the surface of the body to influence and modify its traditional methods. physiological functioning. Generally used for analgesia, elecHunt et a17 first suggested the relationship between the troacupuncture (EA) is a common form of acupuncture treat- c-fos proto-oncogene and nociception. The c-fos oncogene is ment that involves the use of very small electrical impulses one of the 50 to 100 immediate early genes that are encoded through acupuncture needles. Acupuncture has been used when cells are stimulated. Fos protein forms a component of primarily for pain relief and as treatment for a wide variety the mammalian transcription factor activator protein-1 (A€'of ailments and condition^.'-^ In urology, acupuncture has been used clinically to tempo- l),' which in turn regulates gene expression and long-term rarily relieve the symptoms of functional disorders of the developmental changes in cellular state.g Immunohistochemlower urinary tract, such as interstitial cystitis, incontinence, ical staining with anti-fos protein antibody can be used to urinary retention, nocturnal e n ~ r e s i s and , ~ frequency, ur- identify the activated neurons in the spinal cord. Previous gency, and dysuria in female patient^.^.^ Located 5 cm. di- studies have been conducted in which fos protein served as a rectly above the medial malleolus in humans, the Sanyinjiao metabolic marker for nociceptive stimulation in the central (spleen 6 (Sp-6))acupoint has traditionally been used to treat nervous system. Using fos as a marker, Birder and de Groat" demonstrated that fos protein is expressed in the these urinary problems. spinal cord after irritation of the lower urinary tract. Other Accepted for publication July 10, 1998. studies have investigated the effects of different types of * Requests for r g r i n t s : Department of Urology, University of Cal- stimulation on fos expression in the spinal cord. Some sugifornia, School of edicine, San Francisco, CA 94143-0738. Funded by the LITEON Cultural and Educational Foundation, gest that electrostimulation" or EA induces fos expresTaipei, Taiwan, sion,12-16 whereas others have demonstrated that EA sup2274
ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION
presses noxious stimuli-induced fos expression in the central nervous system.17 However, there have been no studies so far that investigate the effects of EA on fos expression induced by noxious stimulation of the urinary tract. MATERIALS AND METHODS
Experiments were performed on 21 female, 2 to 3-month old Sprague-Dawley rats weighing between 250 and 300 gm. To minimize unintended fos protein expression induced by the slightest environmental stimuli, all animals were carefully and gently handled and kept a t room temperature during all experimental procedures. Each animal was worked on during similar times of day to eliminate diurnal effects that might have altered the true degree of fos expression. Non-treated animals. Normal controls (group 1) (n = 5) were used to determine the degree of fos expression within the dorsal horn of the spinal cord in non-stimulated animals. After brief inhalation of methoxyflurane pre-anesthesia, all animals were anesthetized with sodium pentobarbital (35 mg./100 g i.p.). Each rat was cannulated through the carotid artery with PE50 tubing and perfused with 120 ml. normal saline followed by 60 ml. NOS fixative (25 ml. concentrated 37% formaldehyde, 1 ml. concentrated picric acid in 500 ml. 0.1 M phosphate buffer solution (PBS) pH 8.0). Posterior laminectomy was then carefully performed, and the spinal cord posterior to the cervical spinal level was removed for subsequent immunohistochemical analysis (described below). Treated animals. To examine the effects of different stimuli on fos expression, a total of 16 rats was divided into three groups (groups 2 through 4). Group 2 animals (n = 5) received electroacupuncture treatment only, and those in group 3 (n = 6) were subject to 1%acetic acid bladder instillation; group 4 animals (n = 5) received both electroacupuncture and 1% acetic acid instillation. All animals received methoxyflurane pre-anesthesia and sodium pentobarbital anesthesia as described above for control animals. Each animal was gently placed and secured in a supine position, and a stainless steel acupuncture needle was carefully inserted perpendicularly at a depth of 5 mm. into each hind leg a t the Sp6 acupoint. The needle in the left hind leg was stimulated using a custom-built pulse generator (Department of Urology, UCSF, San Francisco, CA) a t 20 pulses/s with a 0.2 ms monophasic charge-balanced rectangular pulse at an intensity of 1 mA for a total of 25 minutes. Animals were sacrificed two hours after the onset of electroacupuncture administration and subsequently underwent perfusion, laminectomy, and spinal cord removal as described above for control animals. Group 3 animals were catheterized through the urethra and instilled with 0.3 ml. 1%acetic acid for 15 minutes. Before catheterization, mineral oil was applied to the opening of the urethra to prevent chemical irritation of the perineal skin and vaginal mucosa. Instillation was performed with a 10 ml. syringe, and after 15 minutes the acetic acid was drawn out of the bladder, followed by 3 rinses with 0.3 ml. normal saline. Animals then underwent perfusion, laminectomy, and spinal cord removal 2 hours after the initiation of acetic acid instillation. Animals in group 4 underwent 25 minutes of electroacupuncture followed by instillation of 0.3 ml. 1%acetic acid 1 hour later. These rats underwent the same procedures and were euthanized 2 hours after initiation of acetic acid instillation as the rats in group 3. Zmmunohistochemistry. Spinal cords were postfixed for 4 hours in NOS fixative (25 ml. concentrated 37% formaldehyde/l ml. concentrated picric acid in 500 ml. 0.1 M PBS pH 8.0) and then cryoprotected in a solution of 30% sucrose in PBS pH 8.0 for 24 hours. The lumbral-sacral segments were isolated, embedded in O.C.T. compound (Tissue Tek@,Miles
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Laboratory, Sakura Finetek, Torrence, CA) and liquid nitrogen, and sectioned into 50 pm. serial transverse sections with a cryostat microtome. Every fifth section from L, to S, was processed for fos immunohistochemical staining using an avidin-biotin procedure." Spinal cord sections were rinsed for 30 minutes in a solution of 0.3% hydrogen peroxide in PBS pH 8.0 to remove excess red blood cells and to improve clarity. Sections were then incubated in 3% goat serum in PBS pH 7.4/Triton X for 2 hours. Spinal cord sections were incubated in antisera for fos (1:1000, Oncogene Research Products, Cambridge, MA, cat.# PC05, batch D02473-1) in PBS pH 8.0 at 4C for 48 hours. The primary antisera was raised in rabbits against the peptide corresponding to residues 4 to 17 of the fos protein in humans. Sections were subsequently incubated in anti IgG solution (1:600) for 2 hours. Sections were then incubated in an alpha-biotin complex solution (1:200, Vector Labs, Burlingame, CA) in PBS pH 7.Li/Triton X for 1hour. Spinal cord sections were placed on an agitator during each incubation and rinse step and were rinsed in three 10-minute washes of PBS pH 8.0 between each step. After staining in 0.04% diaminobenzidine and 0.01% hydrogen peroxide in PBS pH 8.0, sections were placed on glass slides and allowed to dry overnight. Slides were rinsed in a series of 95% ethanol, 100% ethanol, and xylene dips and then coverslipped. To determine the specificity of the immunohistochemical staining for fos protein, negative controls were used in which omission of fos antiserum from the staining protocol abolished all specific fos protein staining in the spinal cord sections. Cell counting and statistical analysis. Spinal cord sections representative of the central portion of each spinal cord segment from L, to S , were selected from each animal. Sections were analyzed under a light microscope for cells positively stained for fos protein. Positive cells were observed and counted under 40x and lOOx magnification and resolved under 200x magnification when necessary. Only positively stained cells located in the dorsal commissure (DCM), sacral parasympathetic nucleus (SPN), medial dorsal horn (MDH), and lateral dorsal horn (LDN) were counted. Any questionable fos-positive nuclei under the latter magnification were not counted. Data were analyzed statistically using one way analysis of variance (ANOVA) and the two sample unpaired Student's t test assuming unequal variances to compare the counts of fos-positive cells in the spinal cord sections among the different groups. Values of p 50.05 were considered significant. RESULTS
Neurons positively stained for fos protein were seen as highly localized, round or oval, dark brown nuclei against a pale brown background. The intensity of the staining of fosimmunoreactive (fos-IR) nuclei appeared lighter in color in animals that underwent electroacupuncture 1 hour before acetic acid instillation. Counts of fos-IR neurons are given in the table . Group 1 animals, which received neither electroacupuncture nor acetic acid instillation, showed no fos expression at the L,-S, levels of the spinal cord. Group 2 rats, which received electroacupuncture only, showed either none or very few (from 0 to 4) fos-IR cells. Many fos-IR cells were seen in group 3 rats, which received 1%acetic acid instillation only (fig. 1). Most of these positively stained cells were found at the L, level (from 30 to 127, mean 76.17 2 13.98), fewer were present in the S , level (from 28 to 77, mean 59 +- 8.30) and even fewer were seen in the S, level (from 7 to 35, mean 19.83 i 4.10). Group 4 rats, which received electroacupuncture 1 hour before 1% acetic acid instillation, showed fewer fos-IR cells (fig. 2) than those in the group 3 rats (L,: from 0 to 50, mean 19.8 2 9.33; s,: from 0 to 47, mean 13.2 Ifr 9.12; S,: from 0 to 37, mean 13.6 2 7.31). Similar to group 3 rats,
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ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION
nerve section and other pathological stimuli can also induce c-fos. The induction can occur via increases in i n t r a c e h l a r calcium that act through a Ca2'/&P element on its ProGroup 1 (no electroacupuncture or acetic acid) moter, or via trophic and other factors that act through a mean SD Serum response element (SRE) on its promoter. Several studL , 0 0 0 0 0 0 +O ies show that calcium entry via voltage sensitive calcium s , o o o o o 0 5 0 channels (VSCCs) is important for inducing c-fOs.lg s, 0 0 0 0 0 20 We used pentobarbital anesthesia throughout the entire Group 2 (electroacupuncture only) study. Birder and de Groat" raised concerns that sodium SD mean pentobarbital as an anesthetic would completely eliminate L , 2 0 0 2 0 0.8 2 0.49 0.6 + 0.6 s , 0 0 0 3 0 fos protein expression in the spinal cord; however, our pres , 0 0 0 4 0 0.8 + 0.8 liminary study in which pentobarbital was used as a n anesthetic clearly showed that it did not suppress fos expression Group 3 (1% acetic acid only) ean SD in the spinal cord. In this study as well, we noticed that sodium pentobarbital did not reduce the number of fos-IR 76.17 2 13.98* 70 79 52 99 30 127 L, 59 2 8.30** 53 77 77 28 45 74 S, neurons in the spinal cord. Since pentobarbital was the only 19.83 24.10 35 22 19 11 25 S, 7 anesthetic that we used, all our results were comparable. Furthermore, several other studies investigating fos protein Group 4 (electroacupuncture + 1% acetic acid) mean SD expression in the central nervous system have also used 19.8 2 9.33* 25 0 0 50 L, 24 sodium pentobarbital as a n a n e s t h e t i ~ . ' ~ * ~ ~ . ~ ~ 13.2 2 9.12** 18 0 S, 0 1 47 Electroacupuncture was administered 1 hour before 1% 13.6 2 7.31 0 24 S, 0 7 37 acetic acid instillation in group 4 rats because our prelimiL,: p = 0.010*. S,: p = 0.004**, S,: p = 0.485 nary data and a study by Lee and Beitz" showed that the Asterisks indicate significant difference ( p 50.05) in number of fos-IR cells greatest degree of fos suppression occurs when EA is applied between 1% acetic acid only treated (group 3) and EA + 1%acetic acid treated (group 4 ) groups when analyzed with t w o sample unpaired Student's t test 45 minutes prior to noxious stimulation and that the effect assuming unequal variances. persists for some time. This effect cannot be explained by the action of ordinary nociceptive spinal cord modulatory mechanisms or simple postsynaptic inhibitory mechanisms begroup 4 rats also showed a decrease in the number of fos-IR cause these two inhibitory mechanisms usually begin within cells in the caudal direction. In the one way ANOVA, p values milliseconds of the onset of stimulation and terminate within were less than 0.01 between groups at all three spinal levels. seconds of offset. Lee and Beitz17 therefore hypothesize that A comparison of groups 3 (1%acetic acid instillation only) the lasting effect of EA is due to its activation of multiple and 4 (EA + 1%acetic acid instillation) using the two sample descending antinociceptive circuits that participate in nociunpaired Student's t test assuming unequal variances re- ceptive modulations beyond the termination of EA. Because vealed significantly fewer visible fos-IR nuclei in segments L, fos protein participates in the control of genetic events which and S , in group 4 rats than in group 3 rats (p =0.010 and lead to the establishment of prolonged functional changes in 0.004, respectively). There was no significant difference neuron^,^ we further postulate that EA-induced fos suppreswhen the S, segments from the two groups were compared sion and its modulatory effect on the neurons may be the using Student's t test (p =0.485). mechanism of lasting clinical improvement from intermittent acupuncture treatment. DISCUSSION We used acetic acid for bladder instillation because it was Acupuncture has been used extensively in the field of an- the most effective irritant for eliciting fos expression in the algesia and anesthesia. In urology, its use has been primarily spinal cord. Birder and de Groat" determined that among limited in modifying bladder function, especially in cases of turpentine oil, xylene, and 1%acetic acid, the latter is the female urethral syndrome, nocturnal enuresis, and bladder most effective in inducing an inflammatory reaction in the instability of unknown origin. Temporary improvements bladder, since it is water soluble and turpentine and xylene such as decrease in frequency and a lessened urgency have are not. In our preliminary study, acupuncture was unable to been found shortly after acupuncture the rap^.^ Electro- suppress fos expression in the spinal cord after 3%acetic acid stimulation has been used to enhance the effects of acupunc- instillation, and therefore a 1%solution was used instead. ture; however, long term follow-ups with patients were still We believe that 3%acetic acid solution may cause structural disappointing because of recurrent urgency and frequency damage to the bladder and that its effect is too strong to be within 3 to 6 months. suppressible by EA. To better understand the mechanism of electroacupuncPrevious studies have shown that maximum fos protein ture in voiding dysfunction, we used fos protein expression in expression occurs within 2 hours after stimulation and then spinal neuronal cells as a metabolic marker to trace the disappears g r a d ~ a l l yTherefore, . ~ ~ ~ ~we ~ chose ~ ~ ~to sacrifice neuronal activities in the spinal micturition center (L,-S.J. the rats 2 hours after bladder instillation to achieve maxiThe c-fos proto-oncogene is one of the first 50 to 100 imme- mum expression. Our preliminary data and a study by Herddiate early genes' found in cells with rapid growth. The egen et alZ4demonstrated that most of the fos expression in protein encoded from the c-fos oncogene is called fos protein, the ventral horn can be found 8 t o 12 hours after stimulation, which can be found within the nucleus. Fos protein acts as a which explains why we had little expression in the ventral third messenger which modulates cell activity. Most cells, horn. especially neurons, express fos protein after external stimuWe instilled 0.3 ml. of 1%acetic acid into the bladder, lation. stimulating the urinary tract to induce fos protein expression The induction of some members of the Immediate Early in the spinal micturition center. The expression of fos protein Gene (IEG) family such as c-jun and c-fos is one of the first over the dorsal commissure (DCM) of the L,-S, segments molecular events following peripheral nerve damage. The fos may be indirect evidence of interneuron cell activity, which and jun proteins act as a transcription factor and may stim- transfers the visceral-sensory impulse from the pelvic and ulate the expression of a number of genes implicated in nerve pudendal nerves. Stimulation of the bladder mucOSa came regeneration. The c-fos immediate early gene can be induced from the 1% acetic acid, while urethral stimulation came by mild stimuli such as light, stress, hyperosmolar solutions, from catheterization. Hydrodilation was not considered a and hormones. Ischemia, hypoxia, seizures, cortical injury, factor in our study since 0.3 ml. was the maximum volume Counts of fos-immunoreactive neurons in spinal seEments L A ,
ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION
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FIG.1. Light microscopy of spinal cord sections from rat showing distribution of fos ;immunoreactive neurons after bladder instillation of 1%acetic acid.A, L, section (x40). B, L, section (X200). C, S, section (X40). D,S, section (X200). E, S, section (X40). F, S, section ( ~ 2 0 0 ) . Pointer indicates c-fos stain positive neuronal cells.
instilled in the bladder. The sacral parasympathetic nucleus up. Others have shown a considerable increase in fos expres(SPN) contained parasympathetic neurons innervating the sion with EA when higher current stimulation is used (1 to 3 bladder through the pelvic nerve. Expression of fos protein mA)14 or when stimulation is delivered to the skin via a over these two areas (DCM and SPN) may be indicative of an conducting pad." The decrease in fos over the spinal micturition center when activated micturition reflex due to lower urinary tract stimEA was administered to group 4 animals does not indicate ulationZ5 The vaned counts of fos-IR neurons (table) were most that the neurons were not activated. It is possible that afferlikely due to differences in response from rat to rat, since ent impulses have been modulated by EA-stimulated interindividuals may respond differently to the same stimuli. neurons. Chang and Harlanz6 hypothesized that EA may Group 2 rats had very little fos protein expression after EA in stimulate neurons in the midbrain which then generate inthe spinal micturition center, which indicated that 1 mA hibitory impulses to suppress the micturition center. In adstimulation delivered though an acupuncture needle was dition, enkephalin and morphine receptors have also been below the threshold of a nociceptive stimulation in our set- identified and localized over the spinal micturition center
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FIG. 2. Light microscopy of spinal cord sections from rat showing distribution of fos - immunoreactive neurons after EA and 1% acetic acid instillation. A, L, section (X40). B, L, section (X200). C, S, section (X40). D, S, section (X200). E, S, section (X40). F, S, section ( X 2 0 0 ) . Pointer indicates c-fos stain positive neuronal cells.
and the bladder. Since release of endorphin has been shown to be the mediator of acupuncture-induced analgesia, the circulating enkephalin may combine with the receptors in the micturition center and the bladder to modulate nociception and thus reduce fos expression. This hypothesis is supported by studies by Presley e t al,27 showing that systemic morphine suppresses noxious stimulus-evoked fos expression in the rat spinal cord, and by Lee and Beitz?' showing that naloxone eliminates the suppressive effect of EA on rat spinal fos protein expression induced by applying formalin to the hind limb. Future studies are needed t o further delineate the molecular basis of neuromodulation in the spinal neuron after fos expression.
CONCLUSIONS
Our study demonstrates that bladder instillation with 1% acetic acid induces fos protein expression in the spinal micturition center of the rat and that electroacupuncture can reduce this expression. These results suggest a link between electroacupunctrurer and reduction in spinal neuronal cell activity. REFERENCES
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ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION Med. Rehabil., 66.443, 1985. 3. Hoffmann, P., Skarphedinsson, J. O., Delle, M. and Thoren, P.: Electrical stimulation of the gastrocnemius muscle in the spontaneously hypertensive rat increases the pain threshold: role of different serotonergic receptors. Acta Physiol. Scand., 138 125, 1990. 4. Andersson, S. and Lundeberg, T.: Acupuncture -from empiricism to science: functional background to acupuncture effects in pain and disease. Med. Hypotheses, 4 5 271, 1995. 5. Chang, P. L.: Urodynamic studies in acupuncture for women with frequency, urgency, and dysuria. J. Urol., 140 563,1988. 6. Chang, P. L., Wu, C. J. and Huang, M. H.: Long-term outcome of acupuncture in women with frequency, urgency, and dysuria. Am. J. Chin. Med., 21: 231, 1993. 7. Hunt, S. P., Pini, A. and Evan, G.: Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature, 3 2 8 632, 1987. 8. Curran, T. and Morgan, J. I.: Fos: a n immediate-early transcription factor in neurons. J. Neurobiol., 2 6 403, 1994. 9. Goelet, P., Castellucci, V. F., Schacher, S. and Kandel, E. R.: The long and the short of long-term memory -a molecular framework. Nature, 3 2 2 419, 1986. 10. Birder, L. A. and de Groat, W. C.: Increased c-fos expression in spinal neurons after irritation of the lower urinary tract. J. Neurosci., 12: 4878, 1992. 11. Willcockson, H. H., Taylor-Blake, B. and Light, A. R.: Induction of fos-like immunoreactivity by electrocutaneous stimulation of the rat hindpaw. Somatosens. Mot. Res., 1 2 151, 1995. 12. Dai, J., Y. Zhu, X. Li, D. Huang and Xu, S.: c-fos expression during electroacupuncture analgesia in rats - an immunohistochemical study. Acupunct. Electrother. Res. Int. J., 17: 165, 1992. 13. Hu, Z., Chen, B. and Tong, J.: The change of c-fos expression in ovariectomized rats following electroacupuncture treatment -an immunochemical study. Acupunct. Electrother. Res. Int. J., 18: 117, 1993. 14. Ma, Q. P., Zhou, Y. and Han, J. S.: Electroacupuncture accelerated the expression of c-fos protooncogene in dopaminergic neurons in the ventral teemental area of the rat. Int. J. Neu" rosci., 7 0 217, 1993. 15. Pan. B., Castro-Lopes, J. M. and Coimbra, A,: C-fos expression in the hypothalami-pituitary system induced by electroacupuncture or noxious stimulation. Neuroreport, 5 1649, 1994. 16. Pan, B., Castro-Lopes, J. M. and Coimbra, A,: Activation of
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anterior lobe corticotrophs by electroacupuncture or noxious stimulation in the anaesthetized rat, as shown by colocalization of fos protein with ACTH and P-endorphin and increased hormone release. Brain Res. Bull., 4 0 175, 1996. 17. Lee, J. and Beitz. A. J.: Electroacupuncture modifies the expression of c-fos in the spinal cord induced by noxious stimulation. Brain Res., 557: 80, 1992. 18. Hsu, S., W e , L. and Fanger, H.: Use of avidin-biotinperoxidase complex (ABC)in immunoperoxidase techniques. A comparison between ABC and unlabelled antibody (PAP)procedure. J. Histochem. Cytochem., 2 9 1349, 1981. 19. Harp, F. R. and Sagar, S. M.: Alterations in gene expression as a n index of neuronal injury: heat shock and the immediate early gene response. Neurotoxicology, 1 5 51-9, 1994. 20. Lee, J. and Beitz, A. J.: The distribution of brain-stem and spinal cord nuclei associated with different frequencies of electroacupuncture analgesia. Pain, 5 2 11, 1993. 21. Romita, V. V., Suk, A. and Henry, J. L.: Parametric studies on electroacupuncture-like stimulation in a rat model: effects of intensity, frequency, and duration of stimulation on evoked antinociception. Brain Res., 42:289, 1997. 22. Menetrey, D., Gannon, A,, Levine, J. D. and Basbaum, A. I.: Expression of c-fos protein in interneurons and projection neurons of the rat spinal cord in response to noxious somatic, articular, and visceral stimulation. J. Comp. Neurol., 285: 177, 1989. 23. Morgan, J. I. and Curran, T.: Stimulus-transcription coupling the nervous system: involvement of the inducible protooncogenes fos and jun. Ann. Rev. Neurosci., 14: 421, 1991. 24. Herdegen, T., Kovary, K., Leah,J. and Bravo, R.: Specific temporal and spatial distribution of JUN, FOS, and KROX-24 proteins in spinal neurons following noxious transsynaptic stimulation. J. Comp. Neurol., 3 1 3 178, 1991. 25. Mallory, B., Steers, W. D. and de Groat, W. C.: Electrophysiological study of micturition reflexes in rats. Experientia, 4 2 109, 1989. 26. Chang, S. L. and Harlan, R. E.: The fos proto-oncogene protein: regulation by morphine in the rat hypothalamus. Life Sci., 4 6 1825, 1990. 27. Presley, R. W., Menetrey, D., Levine, J. D. and Basbaum, A. I.: Systemic morphine suppresses noxious stimulus-evoked fos protein-like immunoreactivity in the rat spinal cord. J. Neurosci., 1 0 323, 1990.
Vol. 160,2274-2279, December 1998 Printed in U.S.A.
THE JOURNAL OF UROLOGY Copyright 8 1998 by AWRICAN UROIXICAL ASSOCUTION, INC.
ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION IN THE SPINAL CORD INDUCED BY NOXIOUS STIMULATION OF THE RAT BLADDER CARL J. CHANG, SHIH-TSUNG HUANG, KENNETH HSU, AUSTIN LIN, MARSHALL L. STOLLER AND TOM F. LUE" From the Department of Urology, School of Medicine, University of California at Sun Francisco, S u n Francisco, C A
ABSTRACT
Purpose: The present study investigated the effects of noxious stimulation of the lower urinary tract on neuronal fos protein expression in the spinal micturition center of rats and also examined the effects of electroacupuncture (EA)on fos expression induced by noxious stimulation of the lower urinary tract. Materials and Methods: Experiments were conducted on 21 female Sprague-Dawley rats divided into four groups. Group 1rats (n = 5) served as normal controls. Group 2 rats (n = 5) received EA at the Sanyinjiao acupoint. Group 3 animals (n = 6) were catheterized through the urethra and instilled with 1%acetic acid, and group 4 (n = 5) animals received EA 1hour before 1%acetic acid instillation. All animals underwent arterial perfusion, laminectomy, and spinal cord removal. Spinal cords were sectioned and processed for immunohistochemical staining for fos protein. Results: No fos protein was detected in any spinal neurons in normal control animals, and either none or few (0 to 4 cells/section) fos-immunoreactive (fos-IR) cells were seen in animals treated with EA. Noxious stimulation of the lower urinary tract with 1%acetic acid drastically increased the number of fos-IR neurons (30 to 127 cells&, section, mean 76.17 2 13.98; 28 to 77, cells/Sl section, mean 59 2 8.30; 7 to 35 cells/S2 section, mean 19.83 5 4.10). However, EA administered 1 hour before 1%acetic acid instillation significantly decreased the number of fos-IR neurons resulting from chemical irritation (0 to 50 cells&, section, mean 19.8 2 9.33; 0 to 47 cells/Sl section, mean 13.2 ? 9.12; 0 to 37 cells/S2 section, mean 13.6 t 7.31). Conclusions: Our study demonstrates that bladder instillation with 1%acetic acid induces fos protein expression in the spinal micturition center of the rat and that electroacupuncture can reduce this expression. These results suggest a link between electroacupuncture and reduction in spinal neuronal cell activity. KEY WORDS: c-fos, electroacupuncture, spinal cord, immunohistochemistry
Many studies have been conducted in recent years to exAcupuncture is considered a new alternative form of medicine in western society but has been widely used for over amine the effects and mechanism of acupuncture on pain 5000 years in China as a n integral part of traditional Chinese relief; however, most of these studies have only focused on medicine in the treatment and prevention of disease. Over the philosophical aspect of acupuncture and not on its physthe centuries, its use has spread to other Asian countries and iological mechanism. The precise effects of EA on the central eventually to Europe and America. Acupuncture involves the nervous system are still not completely known, primarily insertion of fine needles, often used together with electrical because of the difficulty of tracing neuronal pathways by stimuli, on the surface of the body to influence and modify its traditional methods. physiological functioning. Generally used for analgesia, elecHunt et a17 first suggested the relationship between the troacupuncture (EA) is a common form of acupuncture treat- c-fos proto-oncogene and nociception. The c-fos oncogene is ment that involves the use of very small electrical impulses one of the 50 to 100 immediate early genes that are encoded through acupuncture needles. Acupuncture has been used when cells are stimulated. Fos protein forms a component of primarily for pain relief and as treatment for a wide variety the mammalian transcription factor activator protein-1 (A€'of ailments and condition^.'-^ In urology, acupuncture has been used clinically to tempo- l),' which in turn regulates gene expression and long-term rarily relieve the symptoms of functional disorders of the developmental changes in cellular state.g Immunohistochemlower urinary tract, such as interstitial cystitis, incontinence, ical staining with anti-fos protein antibody can be used to urinary retention, nocturnal e n ~ r e s i s and , ~ frequency, ur- identify the activated neurons in the spinal cord. Previous gency, and dysuria in female patient^.^.^ Located 5 cm. di- studies have been conducted in which fos protein served as a rectly above the medial malleolus in humans, the Sanyinjiao metabolic marker for nociceptive stimulation in the central (spleen 6 (Sp-6))acupoint has traditionally been used to treat nervous system. Using fos as a marker, Birder and de Groat" demonstrated that fos protein is expressed in the these urinary problems. spinal cord after irritation of the lower urinary tract. Other Accepted for publication July 10, 1998. studies have investigated the effects of different types of * Requests for r g r i n t s : Department of Urology, University of Cal- stimulation on fos expression in the spinal cord. Some sugifornia, School of edicine, San Francisco, CA 94143-0738. Funded by the LITEON Cultural and Educational Foundation, gest that electrostimulation" or EA induces fos expresTaipei, Taiwan, sion,12-16 whereas others have demonstrated that EA sup2274
ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION
presses noxious stimuli-induced fos expression in the central nervous system.17 However, there have been no studies so far that investigate the effects of EA on fos expression induced by noxious stimulation of the urinary tract. MATERIALS AND METHODS
Experiments were performed on 21 female, 2 to 3-month old Sprague-Dawley rats weighing between 250 and 300 gm. To minimize unintended fos protein expression induced by the slightest environmental stimuli, all animals were carefully and gently handled and kept a t room temperature during all experimental procedures. Each animal was worked on during similar times of day to eliminate diurnal effects that might have altered the true degree of fos expression. Non-treated animals. Normal controls (group 1) (n = 5) were used to determine the degree of fos expression within the dorsal horn of the spinal cord in non-stimulated animals. After brief inhalation of methoxyflurane pre-anesthesia, all animals were anesthetized with sodium pentobarbital (35 mg./100 g i.p.). Each rat was cannulated through the carotid artery with PE50 tubing and perfused with 120 ml. normal saline followed by 60 ml. NOS fixative (25 ml. concentrated 37% formaldehyde, 1 ml. concentrated picric acid in 500 ml. 0.1 M phosphate buffer solution (PBS) pH 8.0). Posterior laminectomy was then carefully performed, and the spinal cord posterior to the cervical spinal level was removed for subsequent immunohistochemical analysis (described below). Treated animals. To examine the effects of different stimuli on fos expression, a total of 16 rats was divided into three groups (groups 2 through 4). Group 2 animals (n = 5) received electroacupuncture treatment only, and those in group 3 (n = 6) were subject to 1%acetic acid bladder instillation; group 4 animals (n = 5) received both electroacupuncture and 1% acetic acid instillation. All animals received methoxyflurane pre-anesthesia and sodium pentobarbital anesthesia as described above for control animals. Each animal was gently placed and secured in a supine position, and a stainless steel acupuncture needle was carefully inserted perpendicularly at a depth of 5 mm. into each hind leg a t the Sp6 acupoint. The needle in the left hind leg was stimulated using a custom-built pulse generator (Department of Urology, UCSF, San Francisco, CA) a t 20 pulses/s with a 0.2 ms monophasic charge-balanced rectangular pulse at an intensity of 1 mA for a total of 25 minutes. Animals were sacrificed two hours after the onset of electroacupuncture administration and subsequently underwent perfusion, laminectomy, and spinal cord removal as described above for control animals. Group 3 animals were catheterized through the urethra and instilled with 0.3 ml. 1%acetic acid for 15 minutes. Before catheterization, mineral oil was applied to the opening of the urethra to prevent chemical irritation of the perineal skin and vaginal mucosa. Instillation was performed with a 10 ml. syringe, and after 15 minutes the acetic acid was drawn out of the bladder, followed by 3 rinses with 0.3 ml. normal saline. Animals then underwent perfusion, laminectomy, and spinal cord removal 2 hours after the initiation of acetic acid instillation. Animals in group 4 underwent 25 minutes of electroacupuncture followed by instillation of 0.3 ml. 1%acetic acid 1 hour later. These rats underwent the same procedures and were euthanized 2 hours after initiation of acetic acid instillation as the rats in group 3. Zmmunohistochemistry. Spinal cords were postfixed for 4 hours in NOS fixative (25 ml. concentrated 37% formaldehyde/l ml. concentrated picric acid in 500 ml. 0.1 M PBS pH 8.0) and then cryoprotected in a solution of 30% sucrose in PBS pH 8.0 for 24 hours. The lumbral-sacral segments were isolated, embedded in O.C.T. compound (Tissue Tek@,Miles
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Laboratory, Sakura Finetek, Torrence, CA) and liquid nitrogen, and sectioned into 50 pm. serial transverse sections with a cryostat microtome. Every fifth section from L, to S, was processed for fos immunohistochemical staining using an avidin-biotin procedure." Spinal cord sections were rinsed for 30 minutes in a solution of 0.3% hydrogen peroxide in PBS pH 8.0 to remove excess red blood cells and to improve clarity. Sections were then incubated in 3% goat serum in PBS pH 7.4/Triton X for 2 hours. Spinal cord sections were incubated in antisera for fos (1:1000, Oncogene Research Products, Cambridge, MA, cat.# PC05, batch D02473-1) in PBS pH 8.0 at 4C for 48 hours. The primary antisera was raised in rabbits against the peptide corresponding to residues 4 to 17 of the fos protein in humans. Sections were subsequently incubated in anti IgG solution (1:600) for 2 hours. Sections were then incubated in an alpha-biotin complex solution (1:200, Vector Labs, Burlingame, CA) in PBS pH 7.Li/Triton X for 1hour. Spinal cord sections were placed on an agitator during each incubation and rinse step and were rinsed in three 10-minute washes of PBS pH 8.0 between each step. After staining in 0.04% diaminobenzidine and 0.01% hydrogen peroxide in PBS pH 8.0, sections were placed on glass slides and allowed to dry overnight. Slides were rinsed in a series of 95% ethanol, 100% ethanol, and xylene dips and then coverslipped. To determine the specificity of the immunohistochemical staining for fos protein, negative controls were used in which omission of fos antiserum from the staining protocol abolished all specific fos protein staining in the spinal cord sections. Cell counting and statistical analysis. Spinal cord sections representative of the central portion of each spinal cord segment from L, to S , were selected from each animal. Sections were analyzed under a light microscope for cells positively stained for fos protein. Positive cells were observed and counted under 40x and lOOx magnification and resolved under 200x magnification when necessary. Only positively stained cells located in the dorsal commissure (DCM), sacral parasympathetic nucleus (SPN), medial dorsal horn (MDH), and lateral dorsal horn (LDN) were counted. Any questionable fos-positive nuclei under the latter magnification were not counted. Data were analyzed statistically using one way analysis of variance (ANOVA) and the two sample unpaired Student's t test assuming unequal variances to compare the counts of fos-positive cells in the spinal cord sections among the different groups. Values of p 50.05 were considered significant. RESULTS
Neurons positively stained for fos protein were seen as highly localized, round or oval, dark brown nuclei against a pale brown background. The intensity of the staining of fosimmunoreactive (fos-IR) nuclei appeared lighter in color in animals that underwent electroacupuncture 1 hour before acetic acid instillation. Counts of fos-IR neurons are given in the table . Group 1 animals, which received neither electroacupuncture nor acetic acid instillation, showed no fos expression at the L,-S, levels of the spinal cord. Group 2 rats, which received electroacupuncture only, showed either none or very few (from 0 to 4) fos-IR cells. Many fos-IR cells were seen in group 3 rats, which received 1%acetic acid instillation only (fig. 1). Most of these positively stained cells were found at the L, level (from 30 to 127, mean 76.17 2 13.98), fewer were present in the S , level (from 28 to 77, mean 59 +- 8.30) and even fewer were seen in the S, level (from 7 to 35, mean 19.83 i 4.10). Group 4 rats, which received electroacupuncture 1 hour before 1% acetic acid instillation, showed fewer fos-IR cells (fig. 2) than those in the group 3 rats (L,: from 0 to 50, mean 19.8 2 9.33; s,: from 0 to 47, mean 13.2 Ifr 9.12; S,: from 0 to 37, mean 13.6 2 7.31). Similar to group 3 rats,
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ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION
nerve section and other pathological stimuli can also induce c-fos. The induction can occur via increases in i n t r a c e h l a r calcium that act through a Ca2'/&P element on its ProGroup 1 (no electroacupuncture or acetic acid) moter, or via trophic and other factors that act through a mean SD Serum response element (SRE) on its promoter. Several studL , 0 0 0 0 0 0 +O ies show that calcium entry via voltage sensitive calcium s , o o o o o 0 5 0 channels (VSCCs) is important for inducing c-fOs.lg s, 0 0 0 0 0 20 We used pentobarbital anesthesia throughout the entire Group 2 (electroacupuncture only) study. Birder and de Groat" raised concerns that sodium SD mean pentobarbital as an anesthetic would completely eliminate L , 2 0 0 2 0 0.8 2 0.49 0.6 + 0.6 s , 0 0 0 3 0 fos protein expression in the spinal cord; however, our pres , 0 0 0 4 0 0.8 + 0.8 liminary study in which pentobarbital was used as a n anesthetic clearly showed that it did not suppress fos expression Group 3 (1% acetic acid only) ean SD in the spinal cord. In this study as well, we noticed that sodium pentobarbital did not reduce the number of fos-IR 76.17 2 13.98* 70 79 52 99 30 127 L, 59 2 8.30** 53 77 77 28 45 74 S, neurons in the spinal cord. Since pentobarbital was the only 19.83 24.10 35 22 19 11 25 S, 7 anesthetic that we used, all our results were comparable. Furthermore, several other studies investigating fos protein Group 4 (electroacupuncture + 1% acetic acid) mean SD expression in the central nervous system have also used 19.8 2 9.33* 25 0 0 50 L, 24 sodium pentobarbital as a n a n e s t h e t i ~ . ' ~ * ~ ~ . ~ ~ 13.2 2 9.12** 18 0 S, 0 1 47 Electroacupuncture was administered 1 hour before 1% 13.6 2 7.31 0 24 S, 0 7 37 acetic acid instillation in group 4 rats because our prelimiL,: p = 0.010*. S,: p = 0.004**, S,: p = 0.485 nary data and a study by Lee and Beitz" showed that the Asterisks indicate significant difference ( p 50.05) in number of fos-IR cells greatest degree of fos suppression occurs when EA is applied between 1% acetic acid only treated (group 3) and EA + 1%acetic acid treated (group 4 ) groups when analyzed with t w o sample unpaired Student's t test 45 minutes prior to noxious stimulation and that the effect assuming unequal variances. persists for some time. This effect cannot be explained by the action of ordinary nociceptive spinal cord modulatory mechanisms or simple postsynaptic inhibitory mechanisms begroup 4 rats also showed a decrease in the number of fos-IR cause these two inhibitory mechanisms usually begin within cells in the caudal direction. In the one way ANOVA, p values milliseconds of the onset of stimulation and terminate within were less than 0.01 between groups at all three spinal levels. seconds of offset. Lee and Beitz17 therefore hypothesize that A comparison of groups 3 (1%acetic acid instillation only) the lasting effect of EA is due to its activation of multiple and 4 (EA + 1%acetic acid instillation) using the two sample descending antinociceptive circuits that participate in nociunpaired Student's t test assuming unequal variances re- ceptive modulations beyond the termination of EA. Because vealed significantly fewer visible fos-IR nuclei in segments L, fos protein participates in the control of genetic events which and S , in group 4 rats than in group 3 rats (p =0.010 and lead to the establishment of prolonged functional changes in 0.004, respectively). There was no significant difference neuron^,^ we further postulate that EA-induced fos suppreswhen the S, segments from the two groups were compared sion and its modulatory effect on the neurons may be the using Student's t test (p =0.485). mechanism of lasting clinical improvement from intermittent acupuncture treatment. DISCUSSION We used acetic acid for bladder instillation because it was Acupuncture has been used extensively in the field of an- the most effective irritant for eliciting fos expression in the algesia and anesthesia. In urology, its use has been primarily spinal cord. Birder and de Groat" determined that among limited in modifying bladder function, especially in cases of turpentine oil, xylene, and 1%acetic acid, the latter is the female urethral syndrome, nocturnal enuresis, and bladder most effective in inducing an inflammatory reaction in the instability of unknown origin. Temporary improvements bladder, since it is water soluble and turpentine and xylene such as decrease in frequency and a lessened urgency have are not. In our preliminary study, acupuncture was unable to been found shortly after acupuncture the rap^.^ Electro- suppress fos expression in the spinal cord after 3%acetic acid stimulation has been used to enhance the effects of acupunc- instillation, and therefore a 1%solution was used instead. ture; however, long term follow-ups with patients were still We believe that 3%acetic acid solution may cause structural disappointing because of recurrent urgency and frequency damage to the bladder and that its effect is too strong to be within 3 to 6 months. suppressible by EA. To better understand the mechanism of electroacupuncPrevious studies have shown that maximum fos protein ture in voiding dysfunction, we used fos protein expression in expression occurs within 2 hours after stimulation and then spinal neuronal cells as a metabolic marker to trace the disappears g r a d ~ a l l yTherefore, . ~ ~ ~ ~we ~ chose ~ ~ ~to sacrifice neuronal activities in the spinal micturition center (L,-S.J. the rats 2 hours after bladder instillation to achieve maxiThe c-fos proto-oncogene is one of the first 50 to 100 imme- mum expression. Our preliminary data and a study by Herddiate early genes' found in cells with rapid growth. The egen et alZ4demonstrated that most of the fos expression in protein encoded from the c-fos oncogene is called fos protein, the ventral horn can be found 8 t o 12 hours after stimulation, which can be found within the nucleus. Fos protein acts as a which explains why we had little expression in the ventral third messenger which modulates cell activity. Most cells, horn. especially neurons, express fos protein after external stimuWe instilled 0.3 ml. of 1%acetic acid into the bladder, lation. stimulating the urinary tract to induce fos protein expression The induction of some members of the Immediate Early in the spinal micturition center. The expression of fos protein Gene (IEG) family such as c-jun and c-fos is one of the first over the dorsal commissure (DCM) of the L,-S, segments molecular events following peripheral nerve damage. The fos may be indirect evidence of interneuron cell activity, which and jun proteins act as a transcription factor and may stim- transfers the visceral-sensory impulse from the pelvic and ulate the expression of a number of genes implicated in nerve pudendal nerves. Stimulation of the bladder mucOSa came regeneration. The c-fos immediate early gene can be induced from the 1% acetic acid, while urethral stimulation came by mild stimuli such as light, stress, hyperosmolar solutions, from catheterization. Hydrodilation was not considered a and hormones. Ischemia, hypoxia, seizures, cortical injury, factor in our study since 0.3 ml. was the maximum volume Counts of fos-immunoreactive neurons in spinal seEments L A ,
ELECTROACUPUNCTURE DECREASES C-FOS EXPRESSION
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FIG.1. Light microscopy of spinal cord sections from rat showing distribution of fos ;immunoreactive neurons after bladder instillation of 1%acetic acid.A, L, section (x40). B, L, section (X200). C, S, section (X40). D,S, section (X200). E, S, section (X40). F, S, section ( ~ 2 0 0 ) . Pointer indicates c-fos stain positive neuronal cells.
instilled in the bladder. The sacral parasympathetic nucleus up. Others have shown a considerable increase in fos expres(SPN) contained parasympathetic neurons innervating the sion with EA when higher current stimulation is used (1 to 3 bladder through the pelvic nerve. Expression of fos protein mA)14 or when stimulation is delivered to the skin via a over these two areas (DCM and SPN) may be indicative of an conducting pad." The decrease in fos over the spinal micturition center when activated micturition reflex due to lower urinary tract stimEA was administered to group 4 animals does not indicate ulationZ5 The vaned counts of fos-IR neurons (table) were most that the neurons were not activated. It is possible that afferlikely due to differences in response from rat to rat, since ent impulses have been modulated by EA-stimulated interindividuals may respond differently to the same stimuli. neurons. Chang and Harlanz6 hypothesized that EA may Group 2 rats had very little fos protein expression after EA in stimulate neurons in the midbrain which then generate inthe spinal micturition center, which indicated that 1 mA hibitory impulses to suppress the micturition center. In adstimulation delivered though an acupuncture needle was dition, enkephalin and morphine receptors have also been below the threshold of a nociceptive stimulation in our set- identified and localized over the spinal micturition center
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FIG. 2. Light microscopy of spinal cord sections from rat showing distribution of fos - immunoreactive neurons after EA and 1% acetic acid instillation. A, L, section (X40). B, L, section (X200). C, S, section (X40). D, S, section (X200). E, S, section (X40). F, S, section ( X 2 0 0 ) . Pointer indicates c-fos stain positive neuronal cells.
and the bladder. Since release of endorphin has been shown to be the mediator of acupuncture-induced analgesia, the circulating enkephalin may combine with the receptors in the micturition center and the bladder to modulate nociception and thus reduce fos expression. This hypothesis is supported by studies by Presley e t al,27 showing that systemic morphine suppresses noxious stimulus-evoked fos expression in the rat spinal cord, and by Lee and Beitz?' showing that naloxone eliminates the suppressive effect of EA on rat spinal fos protein expression induced by applying formalin to the hind limb. Future studies are needed t o further delineate the molecular basis of neuromodulation in the spinal neuron after fos expression.
CONCLUSIONS
Our study demonstrates that bladder instillation with 1% acetic acid induces fos protein expression in the spinal micturition center of the rat and that electroacupuncture can reduce this expression. These results suggest a link between electroacupunctrurer and reduction in spinal neuronal cell activity. REFERENCES
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