Original Article

Effect of Odorata Leaves Extract on Symptomatic Alterations in Diabetic Neuropathic Rats Induced by Streptozotocin and Chronic Constriction Injury Nataya Sritawan1,3, Sittichai Iamsaard1,3, Kowit Chaiciwamongkol1,3, Jintanaporn Wattanathorn2,3, Nongnut Uabundit1,3* 1Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen, 2Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand 3Integrative Complementary Alternative Medicine (ICAM) Research and Development Group, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand *Corresponding author, e-mail: [email protected]

Abstract 1) normal control, 2) DM + Vehicle, 3) DM + Diabetic neuropathy is a serious CCI + Vehicle, 4) DM + Sham + Vehicle, complication of diabetes. It caused by 5) DM + CCI + Ascorbic acid, 6) DM + CCI increase of reactive oxygen species + Gabapentin, 7), 8), and 9) DM + CCI + PO resulting in damaging of nerve fiber 3, 30, or 120 mg/kg BW groups, respectively. functions. This condition affects the life The neurobehavioral examinations were quality of diabetic patients. P. odorata (PO) measured on day 1, 7, 14 and 21 whereas is a Thai-local vegetable and proven to have nerve conduction velocity (NCV) was high antioxidant activity. Therefore, this measured on day 21 only. The results showed study aimed to investigate the effects of PO that PO leaves extract could recover sensory leaves extract on neuropathic symptoms in and motor functions and increased NCV rat model induced by streptozotocin (STZ) value. In conclusion, P. odorata leaves extract and chronic constriction injury (CCI). In is able to attenuate the neuropathic symp- methodology, the experiment male rats toms of sciatic nerve in diabetic rats. were induced to be DM by STZ before CCI Keywords: Diabetic neuropathy, of sciatic nerve. Then the animals were Persicaria odorata, Neuropathic symptoms, divided into 9 groups (n=6). There were Chronic constriction injury, Streptozotocin 76 Vol.11 No.4

1. Introduction oxygen species, especially natural Diabetic neuropathy is a common that have antioxidant capacity, has gained complication of diabetes mellitus, affecting extensive attention.7-12 as many as 50% of diabetic patients.1, 2 It is Persicaria odorata (or Polyganum characterized by a progressive loss of pe- odoratum) is a local of southeastern ripheral nerve fibers involved in sensory, Asia. P. odorata is classified in family of motor, autonomic or combined neuropa- and genus of Persicaria. This thies.3 Neuropathic symptoms were also plant can grow up15 to 30 cm. Its leaf is dark described as an abnormality of sensations green and the stem jointed off each leaf. such as dysesthesia (unpleasant abnormal The flavor of P. odorata is described as be- sensation), hyperalgesia (an increased ing pungent and spicy and is commonly response to painful stimuli), and allodynia used in cuisines.13 The previous studies (pain in response to a stimulus that does identified compounds in P. odorata includ- not normally provoke pain).4,5 The ing quercitol, flavonoids, azetidine 2-car- pathophysiology of the neuropathic boxylic acid, mucous polysaccharides and condition remains unclear, although it has steroidal compounds. 14-16 Moreover, P. odo- been associated with the degeneration of rata leaves have been shown to possess myelinated and unmyelinated sensory strong antioxidant activity and there are fibers, and reducing the peripheral nerve many beneficial effects such as antibacte- conduction.2 rial, antifungal and hepatoprotective Previous studies have described that effect.13,15,17,18 However, there is no report hyperglycemia leads to excess formation of about the effect of P. odorata on diabetic reactive oxygen species, induces oxidative neuropathy. Therefore, the purpose of this stress in neurons and activates multiple study was to assess the effect of P. odorata biochemical pathways which these factors leaves extract on diabetic neuropathic play an important role in the development symptoms in rat model induced by strepto- of diabetic neuropathy.6 Therefore, search- zotocin (STZ) and chronic constriction in- ing for effective drugs for treatment dia- jury (CCI) model. betic neuropathy by eliminating reactive

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2. Materials and methods induce diabetes mellitus in experimental 2.1 Plant material and extraction animals. 2,19-21 Fresh leaves of P. odorata (PO) were Diabetes mellitus (DM) was induced collected from Khon Kaen province of by a single dose of 55 mg/kg intraperitoneal Thailand. The fresh leaves of PO were dried (i.p.) streptozotocin (STZ) dissolved in 0.1 at 40 oC for 24 hours. The dried plants M sodium citrate buffer, pH 4.5. Then, blood materials were crushed and boiled in glucose levels (BGL) were obtained via tail distilled water for 30 minutes, filtered clip under anesthesia 3 days after STZ in- through nylon cloth and made to crude jection by using Accu-Check Performa test powder by using a lyophilizer. strips and meter. The BGL that higher than 2.2 Animals 250 mg/dl were considered as diabetic rats Male Wistar albino rats weighing and used for this study.1 180-220 grams were employed in this study. 2.4 Induction of peripheral neuropathy They were housed at the Animal Care Unit Peripheral neuropathy was induced of Faculty of Medicine, Khon Kaen by chronic constriction injury (CCI) after University, exposed to 12 hours light and induced diabetes.22 Each rat was anesthe- dark cycles, maintained in the animal tized with thiopental sodium (i.p.) 60 mg/ cages with free access to water and kg BW. At first, the hair of the rat’s lower standard laboratory diet (Charoen Pokphand back in the thigh region of right paw was Foods Public Company, Thailand). The shaved, and the skin was sterilized with experimental protocol was approved by the povidone-iodine. The skin of lateral surface Institutional Animal Ethics Committee of of right thigh was incised and sciatic nerve Khon Kaen University (AEKKU 16/2557). was exposed. Four ligations of 4-0 silk su- ture were ligated at proximal to the sciatic 2.3 Induction of diabetes mellitus trifurcation with about 1-mm spacing. Fi- STZ is a broad-spectrum antibiotic nally, skin was immediately sutured with extracted from Streptomyces acromogenes. 4-0 silk suture. The diabetogenic action of STZ was found 2.5 Experimental design to be a selective destruction of pancreatic The rats were divided into 9 groups beta cells and has been widely used to of 6 animals in each group as follow; 78 Vol.11 No.4

Group 1 (Normal control): Rats were Group 9 (DM + CCI + PO 120 mg/kg BW): not subjected to any surgical procedure. Diabetic rats with CCI model were adminis- Group 2 (DM + Vehicle): STZ-induced tered PO leaves extract dose 120 mg/kg BW. diabetic control rats were subjected and All rats were administered per oral distilled water was administered. (p.o.) for 21 consecutive days that starting Group 3 (DM + CCI + Vehicle): Dia- from the 1st day. During experiment, the betic rats were subjected to surgical proce- behavioral examinations including hot dure, ligated the sciatic nerve by CCI model plate, Von Frey hair, and sciatic functional and distilled water was administered. index tests were measured on day 1, 7, 14 Group 4 (DM + Sham + Vehicle): Dia- and 21 whereas nerve conduction velocity betic rats were subjected to surgical proce- test was measured on day 21 after CCI dure to expose the sciatic nerve without any procedure. ligation and distilled water was administered. 2.6 Behavioral examinations Group 5 (DM + CCI + Ascorbic acid): 2.6.1 Hot plate test Diabetic rats were subjected to a surgical Hot plate test was used to inves- procedure to ligate the sciatic nerve by CCI tigate sensory functional recovery. The rats model and ascorbic acid 100 mg/kg BW was were placed on a hot-plate surface with the administered. temperature adjust to 55 + 1 oC. The latency Group 6 (DM + CCI + Gabapentin): of the first sign of paw licking or jumping Diabetic rats were subjected to surgical (withdrawal response) to avoid the heat was procedure to ligate the sciatic nerve by CCI taken as an index of the pain threshold. The model and gabapentin 50 mg/kg BW was cut-off time of 10 seconds was maintained administered. to avoid damage to the paw.10 Group 7 (DM + CCI + PO 3 mg/kg BW): 2.6.2 Von Frey hair test Diabetic rats with CCI model were adminis- Von Frey hair test used for assess tered PO leaves extract dose 3 mg/kg BW. mechanical withdrawal threshold. Each rat Group 8 (DM + CCI + PO 30 mg/kg BW): was placed in a chamber with a wire grid Diabetic rats with CCI model were adminis- and the central region of the plantar surface tered PO leaves extract dose 30 mg/kg BW. of the hind paw was stimulated with a

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series of ascending force von Frey mono- (ii) toe spread factor (TSF) = (ETS- filaments. A trial of each monofilament was NTS)/NTS; applied 10 times that in increasing force (iii) intermediary toe spread factor until the rat withdraw the paw. The with- (ITF) = (EIT-NIT)/NIT. drawal threshold was taken as the lowest After that, these factors were then in- force that cause at least 5 times out of the corporated into the Bain-Mackinnon-Hunter 10 stimuli.21,23 (BMH) sciatic function index-formula26: 2.6.3 Sciatic functional index SFI = - 38.3 x PLF + 109.5 x TSF + 13.3 The sciatic functional index was x ITF - 8. used to assess the motor functional recovery 2.7 Nerve conduction velocity (NCV) following experimentally-induced injury in The NCV test is a measurement of the the sciatic nerve.24,25 The trials were done in speed of conduction of an electrical impulse a wooden walking track darkened at one end through a nerve that can determine nerve and covered with a sheet of white paper. The damage.27 All rats were anesthetized by rat hind paws were dipped in black India-ink intraperitoneal injection of thiopental and then each animal was allowed to walk sodium (60 mg/kg) for electrophysiological freely in above mentioned walking track. recording. The sciatic nerve in the right From the analysis of the footprints hind leg was carefully exposed. A bipolar of the feet, the following lengths were ob- stimulating electrode was placed under the tained: (i) distance from the heel to the third sciatic nerve between the constricted sites toe, the print length (PL); (ii) distance from that set to be approximately 5 mm whereas the first to the fifth toe, the toe spread (TS); a recording electrode was placed in the gas- and (iii) distance from the second to the trocnemius muscle. The NCV was deter- fourth toe, the intermediary toe spread (ITS). mined from the difference in latencies of the All three measurements were action potentials and the nerve length sepa- taken from the experimental (E) and normal rating the two stimulation points as follow: (N) sides. The factors were calculated as = proximal distance - distal distance/ follows: proximal latency - distal latency and ex- (i) print length factor (PLF) = (EPL- pressed in meters per second (m/s). (27) NPL)/NPL; 80 Vol.11 No.4

3. Results PO 30 and PO 120 mg/kg BW = 1.20 + 0.20, 3.1 Hot plate test 1.83 + 0.31 and 1.50 + 0.22 sec, respectively) All rats of the DM + CCI + Vehicle were significant lower than the DM + CCI + group significantly increased in hind paw Vehicle group on day 21 (3.20 + 0.37 sec) as withdrawal latency time as compared the shown in Figure 1. This result indicated that normal group on day 1 until day 21 after sur- the treatment with PO leaves extract gery. The hind paw withdrawal latency times achieved better sensory functional recovery in PO treated groups (the DM + CCI + PO 3, than vehicle after constriction injury.

Figure 1 Statistical analysis of the thermal withdrawal latency time in hot plate test. All data are represented as means + SEM (a p < 0.05 compared with Normal group, b p < 0.05 compared with the DM + Sham + Vehicle group, c p < 0.05 compared with the DM + CCI + Vehicle group).

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3.2 Von Frey hair test + 1.09 and 3.80 + 0.09 g, respectively) was The right hind paw withdrawal significantly decreased as compared with threshold in the DM + CCI + Vehicle group the DM + CCI + Vehicle group (50.20 + 9.06 significantly increased after constriction g) as shown in Figure 2. This result indi- injury until the end of experiment as com- cated that the treatment groups with PO pared the normal group. At day 21 after leaves extract achieved better sensory treatment, the withdrawal threshold in PO functional recovery than vehicle after con- treated groups (the DM + CCI + PO 3, PO striction injury. 30 and PO 120 mg/kg BW = 5.12 + 1.22, 5.67

Figure 2 Statistical analysis of the mechanical withdrawal threshold in Von Frey hair test. All data are represented as means + SEM (a p < 0.05 compared with Normal group, b p < 0.05 compared with the DM + Sham + Vehicle group, c p < 0.05 compared with the DM + CCI + Vehicle group).

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3.3 Sciatic functional index creased as compared with the DM + CCI + In the results at day 21 of experi- Vehicle group (-96.55 + 13.14), indicating ment (Table 1), the sciatic functional index that the treated groups with PO leaves (SFI) values in PO treated groups (the DM extract achieved better motor functional + CCI + PO 3, PO 30 and PO 120 mg/kg BW recovery than vehicle after constriction = -61.73 + 4.97, -47.51 + 9.15 and -40.91 + injury in rats. 10.20, respectively) were significantly in- Table 1 The sciatic functional index (SFI) values in all groups. All data are represented as means + SEM (a p < 0.05 compared with Normal group, b p < 0.05 compared with the DM + Sham + Vehicle group, c p < 0.05 compared with the DM + CCI + Vehicle group). Group Sciatic functional index values Day 0 Day 1 Day 7 Day 14 Day 21 Normal -13.97 + 1.85 -12.24 + 2.49 -5.42 + 4.34 -9.28 + 3.05 -6.71 + 2.72

DM + Vehicle -9.78 + 2.81 10.44 + 13.12c -3.97 + 6.46c -6.65 + 4.78c -1.73 + 7.73c

DM + CCI + Vehicle -11.60 + 5.08 -101.22 + 14.04a,b -110.06 + 13.45a,b -115.96 + 6.26a,b -96.55 + 13.14a,b

DM + Sham + Vehicle -11.79 + 3.69 12.99 + 15.45 -17.94 + 7.50 -13.93 + 6.03 -22.36 + 6.18

DM + CCI + Ascorbic acid -9.04 + 2.52 -93.98 + 17.40b -92.28 + 22.37b -58.11 + 11.95c -38.29 + 13.78c

DM + CCI + Gabapentin -8.01 + 6.33 -99.20 + 13.80b -91.46 + 15.11b -87.11 + 15.95b -64.30 + 10.07b,c

DM + CCI + PO 3 mg/kg BW -10.35 + 3.86 -126.51 + 0.55b -81.43 + 11.30b -69.22 + 4.64b,c -61.73 + 4.97b,c

DM + CCI + PO 30 mg/kg BW -11.96 + 5.29 -128.77 + 0.68b -91.06 + 21.55b -58.66 + 8.52b,c -47.51 + 9.15c

DM + CCI + PO 120 mg/kg BW -9.24 + 3.39 -120.88 + 5.70b -97.80 + 10.68b -84.97 + 13.37b -40.91 + 10.20c

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3.4 NCV test values in the PO 30 (0.86 + 0.04 m/s) and The sciatic nerve conduction velocity 120 mg/kg BW (0.96 + 0.08 m/s) treated of the DM + CCI + Vehicle group was sig- groups were significantly higher than that nificantly reduced to 0.61 + 0.04 m/s as in the DM + CCI + Vehicle group. These compared with normal (1.46 + 0.04 m/s) and results implied that PO leaves extract (30 the DM + Sham + Vehicle groups (1.13 + and 120 mg/kg BW) were effective at pre- 0.10 m/s) as shown in Table 2. The NCV venting the deficit in the NCV value.

Table 2 The results of electrophysiological analysis in all groups. All data are repre- sented as means + SEM (a p < 0.05 compared with Normal group, b p < 0.05 compared with the DM + Sham + Vehicle group, c p < 0.05 compared with the DM + CCI + Vehicle group). Nerve Conduction Velocity Group (m/s) Normal 1.46 + 0.04 DM + Vehicle 1.15 + 0.07c DM + CCI + Vehicle 0.61 + 0.04a,b DM + Sham + Vehicle 1.13 + 0.10 DM + CCI + Ascorbic acid 0.85 + 0.09 DM + CCI + Gabapentin 0.93 + 0.08c DM + CCI + PO 3 mg/kg BW 0.86 + 0.04 DM + CCI + PO 30 mg/kg BW 0.96 + 0.04c DM + CCI + PO 120 mg/kg BW 0.96 + 0.08c

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4. Discussion and Conclusion nerve damage and to provide information Diabetic neuropathy causes a pro- on the functional integrity of the myelin gressive degeneration of sensory and motor sheath.27 In the present study, NCV of sci- nerves fibers, leading to abnormal nerve atic nerve rats in the DM + CCI + Vehicle functions.6,28,29 CCI model of sciatic nerve group was significantly decreased as com- induced loss of sensibility in the lateral and pared with normal control group. Previ- central areas of the injured paw and it could ously, the morphological changes such as develop to peripheral neuropathic pain in demyelination occur in experimental dia- long-term.30,31 In this study, the CCI of sci- betic neuropathy that associated with the atic nerve significantly induced loss of NCV deficit.32 After treatment with P. odo- sensibility and motor function without rata leaves extract, the NCV was signifi- neuropathic pain symptoms as compared cantly increased as compared with the DM with normal control group. It is possible that + CCI + Vehicle group. This result sug- the duration of CCI experiment not enough gested that P. odorata leave can increase induced to be neuropathic pain. As the the electrical signal conduction through a same time, P. odorata leaves extract sig- sciatic nerve in diabetic neuropathic rats. nificantly decreased thermal withdrawal Moreover, P. odorata also has the latency time and mechanical withdrawal advantages including simple planting, low threshold as compared with the DM + CCI cost, and low toxicity. In the future, results + Vehicle group. Moreover, P. odorata from this study may be the basic informa- leaves extract significantly increased index tion for developing of P. odorata leaves as a of SFI test as compared with the DM + CCI product in treatment of diabetic neuropa- + Vehicle group. These results suggest that thy. P. odorata leaves extract could recover sensory and motor functions on diabetic 5. Acknowledgement neuropathy of sciatic nerve in rats. The authors would like to thank The NCV test was performed to as- teaching staffs and all members in Depart- sess the speed of conduction of an electrical ment of Anatomy and Integrative Comple- impulse through a nerve. It could determine mentary Alternative Medicine (ICAM) Re-

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search and Development Group, Faculty of search Association 2011;49:2557-63. Medicine, Khon Kaen University, Thailand 5. Woolf CJ, Mannion RJ. Neuropathic for their suggestions and supporting. pain: aetiology, symptoms, mecha- This study was granted by Faculty of nisms, and management. Lancet Medicine, Khon Kaen University, Thailand 1999;353:1959-64. (Grant Number IN58150). 6. Edwards JL, Vincent AM, Cheng HT, Feldman EL. Diabetic neuropathy: Reference mechanisms to management. Pharma- 1. Boulton AJ. The diabetic foot: from art cology & Therapeutics 2008;120:1-34. to science. The 18th Camillo Golgi 7. Morani AS, Bodhankar SL, Mohan V, lecture. Diabetologia 2004;47:1343-53. Thakurdesai PA. Ameliorative effects 2. Fox A, Eastwood C, Gentry C, Manning of standardized extract from Trigonella D, Urban L. Critical evaluation of the foenum-graecum L. seeds on painful streptozotocin model of painful peripheral neuropathy in rats. Asian diabetic neuropathy in the rat. Pain Pacific Journal of Tropical Medicine 1999;81:307-16. 2012;5:385-90. 3. Rathur HM, Boulton AJ. Recent ad- 8. Muthuraman A, Diwan V, Jaggi AS, vances in the diagnosis and manage- Singh N, Singh D. Ameliorative effects ment of diabetic neuropathy. The of Ocimum sanctum in sciatic nerve Journal of Bone and Joint Surgery transection-induced neuropathy in British 2005;87:1605-10. rats. Journal of Ethnopharmacology 4. Muthuraman A, Singh N, Jaggi AS. 2008;120:56-62. Protective effect of Acorus calamus L. 9. Vats V, Yadav SP, Grover JK. Ethanolic in rat model of vincristine induced extract of Ocimum sanctum leaves painful neuropathy: an evidence of partially attenuates streptozotocin-in- anti-inflammatory and anti-oxidative duced alterations in glycogen content activity. Food and Chemical Toxicolo- and carbohydrate metabolism in rats. gy: An International Journal Published Journal of Ethnopharmacology 2004; for the British Industrial Biological Re- 90:155-60.

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10. Sharma S, Kulkarni SK, Agrewala JN, ing homoisoflavone molecule from Vi- Chopra K. Curcumin attenuates ther- etnamese (Polygonatum odo- mal hyperalgesia in a diabetic mouse ratum) that induces apoptosis and model of neuropathic pain. European G2/M cell cycle arrest in breast cancer Journal of Pharmacology 2006;536: cell lines. Food Chemistry 2007;104:332- 256-61. 40. 11. Yang S, Gao Q, Xing S, Feng X, Peng 15. Somparn N, Jitvaropas R, Saenthawee- L, Dong H, et al. Neuroprotective ef- sak S, Thuppia A. Hepatoprotective fects of Buyang Huanwu decoction and antioxidant effects of against hydrogen peroxide induced odoratum L. extract against acetami- oxidative injury in Schwann cells. Jour- nophen-induced hepatotoxicity in rats. nal of Ethnopharmacology 2011;137: Thammasat Medical Journal 2013; 1095-101. 13:456-64. 12. Kandhare A, Raygude K, Shiva Kumar 16. Woraratphoka J, Intarapichet K-O, In- V, Rajmane A, Visnagri A, Ghule A, et drapichate K. Antioxidant activity and al. Ameliorative effects quercetin cytotoxicity of six selected, regional, against impaired motor nerve function, Thai vegetables. American-Eurasian inflammatory mediators and apoptosis Journal of Toxicological Sciences in neonatal streptozotocin-induced 2012;4:108-17. diabetic neuropathy in rats. Biomedi- 17. Nanasombat S, Teckchuen N. Antimi- cine & Aging Pathology 2012;2:173-86. crobial, antioxidant and anticancer 13. Ridzuan P, Aini H, Shah M, Roesnita., activities of Thai local vegetables. Aminah K. Antibacterial and antifungal Journal of Medicinal Plants Research properties of Persicaria odorata leaf 2009;3:443-9. against pathogenic bacteria and fungi. 18. Sasongko P, Laohankunjit N, Kerdch- The Open Conference Proceedings oechuen O. Antibacterial activity of the Journal 2013;4:71-4. from Persicaria odorata 14. Rafi M, Vastano B. Identification of a leaves. Agricultural Science Journal structure specific Bcl-2 phosphorylat- 2011;42:105-8.

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19. Akbarzadeh A, Norouzian D, Mehrabi machine for video recording of the rat’s MR, Jamshidi S, Farhangi A, Verdi AA, gait and sciatic functional index meas- et al. Induction of diabetes by Strepto- urement. A comparative study with zotocin in rats. Indian Journal of Clini- other methods. Journal of Neurosci- cal Biochemistry : IJCB 2007;22:60-4. ence Methods 2010;189:23-9. 20. Jacobs RL, House JD, Brosnan ME, 25. Varejao AS, Meek MF, Ferreira AJ, Brosnan JT. Effects of streptozotocin- Patricio JA, Cabrita AM. Functional induced diabetes and of insulin treat- evaluation of peripheral nerve regen- ment on homocysteine metabolism in eration in the rat: walking track analy- the rat. Diabetes 1998;47:1967-70. sis. Journal of Neuroscience Methods 21. Rajasekaran S, Sivagnanam K, Subra- 2001;108:1-9. manian S. Antioxidant effect of Aloe 26. Bain JR, Mackinnon SE, Hunter DA. vera gel extract in streptozotocin-in- Functional evaluation of complete sci- duced diabetes in rats. Pharmacologi- atic, peroneal, and posterior tibial nerve cal Reports : PR 2005;57:90-6. lesions in the rat. Plastic and Recon- 22. Muthuraman A, Singh N. Neuroprotec- structive Surgery 1989;83:129-38. tive effect of saponin rich extract of 27. Burnard SL, McMurchie EJ, Leifert WR, Acorus calamus L. in rat model of Patten GS, Muggli R, Raederstorff D, et chronic constriction injury (CCI) of al. Cilazapril and dietary gamma-lino- sciatic nerve-induced neuropathic lenic acid prevent the deficit in sciatic pain. Journal of Ethnopharmacology nerve conduction velocity in the strep- 2012;142:723-31. tozotocin diabetic rat. Journal of Diabe- 23. Erichsen HK, Blackburn-Munro G. tes and Its Complications 1998;12:65-73. Pharmacological characterisation of the 28. Aiyepola O, Brooks N, Oguntibeju O. spared nerve injury model of neuro- Oxidative Stress and Diabetic Compli- pathic pain. Pain 2002;98:151-61. cations: The Role of Antioxidant Vita- 24. Monte-Raso VV, Barbieri G, Mazzer N, mins and Flavonoids 2014. Fonseca Mde C, Barbieri CH. A new 29. Vincent AM, Russell JW, Low P, Feld- treadmill-type motorized walking belt man EL. Oxidative stress in the patho-

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genesis of diabetic neuropathy. Endo- 31. Kingery WS, Lu JD, Roffers JA, Kell DR. crine Reviews 2004;25:612-28. The resolution of neuropathic hyperal- 30. Cobianchi S, de Cruz J, Navarro X. As- gesia following motor and sensory sessment of sensory thresholds and functional recovery in sciatic axonot- nociceptive fiber growth after sciatic metic mononeuropathies. Pain 1994; nerve injury reveals the differential 58:157-68. contribution of collateral reinnervation 32. Asensio-Pinilla E, Udina E, Jaramillo J, and nerve regeneration to neuropathic Navarro X. Electrical stimulation com- pain. Experimental Neurology 2014; bined with exercise increase axonal 255:1-11. regeneration after peripheral nerve injury. Experimental Neurology 2009; 219:258-65.

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