EFFECTS OF Pueraria mirifica ON NEUROPATHIC PAIN IN RATS

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1 EFFECTS OF Pueraria mirifica ON NEUROPATHIC PAIN IN RATS Kamolchanok Tanchotikul 1, Quankamon Dejativongse Na Ayudhya 1*, Orawan Piyaboon 1 and Supin Chompoopong 2 1 Department of Biology, Mahidol Wittayanusorn School, Buddhamonthon, Nakhon Pathom, Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Rajthevi, Bangkok, jam26112@hotmail.com 35 th Congress on Science and Technology of Thailand Chonburi, Thailand, October, 2009 Abstract: Pueraria mirifica is a plant in the Leguminosae family, locally known in Thai as Khao-kruea-khao. This plant s tuberous root contains phytoestrogen compounds which are similar to mammalian estrogen. Phytoestrogen comprises chemicals which inhibit inflammatory mediator. Moreover, it may help in nerve regeneration and prevent cell death as mammalian estrogen does. Therefore, this study was aimed to investigate the effects of Pueraria mirifica on neuropathic pain induced rats. The rats were divided into four groups, two control groups which are the general control (CT) and the surgical control (Sham) and two subjective groups which are the neuropathic pain induced group treated with 0.9% normal saline (SNI N) and the neuropathic pain induced group treated with Pueraria mirifica suspension (SNI PM). The neuropathic pain was induced by Spared Nerve Injury surgery where the two branches of Sciatic nerve were tight while the other branch was left freely. During surgical procedure, the spared nerve injury rats nerves were wrapped once with immersed gel foam in 1 ml of Pueraria mirifica suspension after injury (The suspension was prepared from the 700C dehydrated P. mirifica powder). The rats behavior was investigated by using von Frey filament for the mechanical pain test. The results indicated that the neuropathic pain induced group which received Pueraria mirifica suspension (SNI PM) showed the minimum of pain withdrawal threshold at day 5 after nerve surgery and increased to the maximum threshold closely to the baseline at g (normal state) in the sixth week. Whereas the withdrawal threshold of spared nerve injury group which received normal saline (SNI N) gradually responded to the minimum of pain withdrawal threshold between g at day 10 after nerve surgery and maintained this steady response throughout 10 weeks. Pueraria mirifica may decrease neuropathic pain after nerve injury. Less pain was found during the regeneration process of damaged nerves, and more movement could be enhanced, therefore, it may also promote the recovery of nerve function in rats.

2 Introduction: Neuropathic pain is initiated or caused by a primary lesion of dysfunction in the nervous system (IASP definition). The causes of neuropathic pain are unclear, but the main characteristics of it which are spontaneous pain, hyperalgesia (exaggerated respond to noxious or painful stimuli) and allodynia (mechanical pain) can be explained by the nervous sensitization process. The nervous sensitization occurs in both peripheral and central nervous system. This incidence occurs when there is an extra secretion of inflammatory mediators both from the injured common tissue and injured nervous tissue. (Ponparadi, 2008) Another explanation for neuropathic pain characteristics is the rewiring of synaptic connections. After peripheral nerve injury, A fiber (a type of nerve fiber) s phenotype switches and the fiber itself sprout in the spinal cord including the area that it has never innervated before, thus it can receive other signals such as pain signal which is supposed to be received by another nerve fiber. (Woolf and Mannion, 1999) Estrogen (endogenous gonadal steroid 17 β-estradiol; E2) influences growth, differentiation, maturation, and function of many different target tissues including the cells of the central and peripheral nervous system. Its actions play important roles in non-genomic effect-modulate intracellular signal transduction cascades to stimulate cell survival by means of preventing programmed cell death and functional recovery-and genomic effect mediated in gene expression which is similar results as neurotrophins, a family of proteins that induce the survival, development, proliferation and function of neurons. By Thai local intellect, Pueraria mirifica, a plant in the Legumninosae family, locally known in Thai as Khao-kruea-khao. This plant s tuberous root contains phytoestrogens, a group of natural compounds which diverse effects physiologically. Some components which is similar to mammalian estrogen (E2) can also act as the normal actions of E2 from their high binding affinity and transactivation to E2 receptors and some anti-inflammatory mediators, at least flavonoids were found (Scott M. Belcher et a1, 2001). Recently, Phytoestrogens are partially claimed to stimulate neurite growth and synaptogenesis, potentiate of regeneration (Islamov et a1, 2002), relieve neuropathic pain in rats (Shir et a1, 2002). This study is aimed to investigate the effect of Pueraria mirifica on neuropathic pain in rats. The rats behaviors were investigated by using mechanical pain test. So, if the results apparently show the relievable neuropathic pain, effect of Pueraria mirifica on nervous tissue will be study. Methods: Animal model This experiment used a total 12 male adult Sprague Dawley rats which are originally weighed gram advocated by the National Laboratory Animal Centre of Thailand. The rats were housed in standard cages (3 rats per cage) with free access to food and water, exposed to 12 hr light and dark cycle for 2 weeks before pre-operation behavior test.

3 Experimental designs The rats are randomly separated into 3 group. Group 1: control group (CT) no treat and operation on three rats Group 2: Sham operate group (Sham) Three rats were operated (only incised their muscles) to expose sciatic nerve, and got normal saline 1 milliliter. Group 3: Spared Nerve Injury (SNI) Six rats were got Spared Nerve Injury operation which incised muscles to expose sciatic plexus, subsequently indicated 3 sciatic branches, tied 2 branches (common peroneal and tibial) with strong ligation and spared the rest one, sural. Then separated the rats into 2 groups Group 3.1: untreated SNI (SNI-N) Three SNI rats were received 1 milliliter normal saline Group 3.2: treated SNI (SNI-PM) The other three SNI rats were received 1 milliliter suspension of Pueraria mirifica (0.02 gram per milliliter). The suspension of this powder was freshly prepared by dissolving dried powder of Pueraria mirifica into 0.9% normal saline solution. During surgical procedure, the spared nerve injury rats nerves were wrapped once with immersed gel foam in 1 milliliter of the suspension after injury. Remark: All rats were labeled the indicated number with 2 numbers. The front number means the group that the rat was from and the following means the number of rat in each group (e. g. the rat with label 3-1 indicated this rat was the first rat of group SNI-N)

4 Diagram 1 experimental designs Surgical Procedure All experimental procedures were done on rats that were deeply anesthetized with ketaminexylaxine 0.15ml/ 100g. Additional doses of the aenesthetics were shaved and sterilized with betadine and 70% ethanol alcohol. Sterile operating instruments were used. An approximately cm. skin incision was made between the gluteus maximus and the biceps femoris muscle. The muscles were teased with knife. The branches of right sciatic nerve, common peroneal and tibial branches, were tight with silk thread; only sural branch was not damaged. In some rats, the damaged nerves were wrapped by Pueraria mirifica-soaked gel foam (SNI-PM) and Sodium-Chloride soaked gel foam (SNI-N) once after ligation. Then, the cut was sew up and after recovery from anesthesia the rats were allowed to move freely in cages. Behavioral Pain Test (Mechanical Pain Test/ Von Frey filament Test) Animals were placed on an elevated wire grid and the plantar surface of the paw stimulated with a series of ascending force von Frey monofilaments. The threshold was taken as the lowest force that evoked a brisk withdrawal response to one of five repetitive stimuli (Isabelle Decosterd, Clifford J. Woolf, 2000). The lateral and medial plantar surface of the paw as well as its dorsal surface was tested. Result: Physical Appearance In term of physical appearance, all SNI rats operated (right) legs paws were curved downward compared to control and sham-operated rats.

5 Mechanical Pain Test As the results of mechanical pain test, the control and sham-operated rats responded consistently to the von Frey filament at the level of g., which is the withdrawal threshold of normal rats and is said to be the base line of this experiment. The behavior was found significantly different in SNI groups; SNI-N and SNI-PM. The SNI rats right (operated) paw responded to the von Frey filament, at the low level compared to the base line, by withdrawing their paws severely and abruptly. Three days after surgery, the withdrawal threshold of SNI rats obviously decreased and reached the lowest level below g. After day 3, the withdrawal threshold began to rise in both SNI groups. The differences between SNI-N and SNI-PM were clearly shown after week 3 of the surgery. The withdrawal threshold of SNI-PM rats further increased until reached the base line at week 6, showing a sign of recovery, whereas the SNI-N rats showed the extended pain response and their withdrawal threshold remained low at the level of g. throughout the experiment period, 10 weeks. In controversy, the left paws of the SNI rats expressed the same result as the control and shamoperated rats responding to the von Frey filament at the level of g. throughout the study. Mechanical Pain Test Rt. Figure 1 Withdrawal Threshold (g) of right paws

6 Mechanical Pain Test Lt. Figure 2 Withdrawal Threshold (g) of left paw Discussion: The results show that Pueraria mirifica suspension produces inhibition on mechanical pain in rats with neuropathic pain after spared nerve injury in Sciatic nerve. The fall of withdrawal threshold from the pre-operation day to post operation day (day 0-3) confirms that the neuropathic pain occurred in rats. Moreover, finding that the SNI-operated rats paws were curved downward was also the trace of neuropathic pain. Since the nerve damage occurred, the muscle fiber would subsequently lose control and bend downward as evidently shown in SNI-operated rats (Chompoopong et a1, 2007). After day 3 both P.mirifica treated SNI-operated group (SNI-PM) and normal saline treated SNIoperated group (SNI-N) responded to the von Frey filament at a gradually higher level at virtually same rate signified self-recovery. However, after week 3, there is difference between SNI-N and SNI-PM group. Therefore, P. mirifica may account for this incidence. The mechanisms underlying P.mirifica-induced inhibition of mechanical pain following nerve injury are unknown. However, some studies have provided evidence for possible explanations. (1) Phytoestrogens have immunomodulatory and anti-inflammatory activity as it reduced peripheral and central nuclear factor-kappab, nitric oxide system and pro-inflammatory cytokine over-activation which are ones of the causes of nervous sensitization (Pongparadi, 2008) and lead to neuropathic pain. (2) Some phytoestrogens such as Genistein bind estrogen receptorβ(erβ) with 87% affinity particularly expressed in neurons (Valsecchi AE, 2008) as similar as mammalian estrogen (17β-estradiol). Estrogen has 100% affinity to ERβ and a direct interaction between components of the neurotrophin-activated and E2-mediated MAPK signaling pathways, therefore its

7 signaling process enhances cell survival by preventing cell death and regulates the normal transcription (Scott M. Belcher et a1, 2001). (3) Phytoestrogens have an influence on stimulation of neurite growth and synaptogenesis, potentiate of nerve regeneration (Islamov et a1, 2002). Peripheral nerve injury induces sprouting of A fiber central terminals into lamina II. This sprouting is probably dependent on injury to the peripheral axons of C fibers. Thus, intrathecally supplied neurotrophic factors, which may act as a C fiber therapy, can prevent A fiber sprouting (Woolf and Mannion, 2008). Since phytoestrogens can prevent cell death and have an influence on nerve regeneration, the injured nerve fibers, especially C fiber, received P.mirifica which contains phytroestrogens may have regenerated. When C-fiber death decreased, A fiber would stop sprouting; then, the rewiring of synaptic terminal would cease and the nerve fiber may rearrange itself and function normally. Conclusion: Peuraria mirifica has some effects on the behavior of the spared nerve injury rats in term of decreasing neuropathic pain in neuropathy and may support the regeneration of damaged nervous tissue which leads to the recovery of neuropathic pain. References: Belcher SM, Zsarnovszky A (2001). Estrogenic Actions in the Brain: Estrogen, Phytoestrogens, and Rapid Intracellular Signaling Mechanisms, J Pharmacol Exp Ther. 299(2): Chompoopong S, Siriphorn A,Chongthumakhun S, Phasukdee N (2007). Comparison of sciatic functional recovery index during treatment with estrogen, genistein and Pueraria mirifica in rats. Siriraj Medical Journal, 59: Isabelle Decosterd, Clifford J. Woolf (2000). Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain. 87: Islamov RR, Hendricks WA, Jones RJ, Layll GJ, Spanier NS, Murashov AK (2002). 17-[3 estradiol stimulates regeneration of sciatic nerve in female mice. Brain Res. 943(2): Shir Y, Campbell JN, Raja SN, Seltzer Ze (2002). The correlation between dietary soy phytoestrogens and neuropathic pain behavior in rats after partial denervation. Anesth Analg. 94(2): Valsecchi AE, Franchi S, Panerai AE, Sacerdote P, Trovato AE, Colleoni M. Genistein (2008), a natural phytoestrogen from soy, relieves neuropathic pain following chronic constriction sciatic nerve injury in mice: anti-inflammatory and antioxidant activity. 107(1): Woolf, C. J., and Richard J.M. (1999). Neuropathic pain: aethiology, symptoms, mechanism, and management. The Lancet. 353:

8 Basic concepts in Acute & Chronic Pain. (2008). [Online] Available: Keywords: Pueraria mirifica, Neuropathic pain, Behavioral Test Acknowledgements: This research was supported by Young Scientist Competition (YSC 2009). Department of Anatomy, Faculty of Medicine Siriraj hospital, Mahidol University and the Biology Department of Mahidol Wittayanusorn School.