Investigation of the Role of Asiatic Acid in Experimentally Created Peripheral Nerve Injuries in Rats

Abstract

Objective: The neuroprotective and regenerative effects of Asiatic Acid (AA) were examined histopathologically, electrophysiologically and clinically in axonotmesis and neurotmesis type injury models experimentally. Material and Method: 56 male Spraque-Dawley male rats were divided into seven subgroups. Group1 (control), Group2A (the group that received oral saline for 28 days after axonotmesis), Group2B (the group that received 10 / mg / kg / day x 28 days of oral AA treatment after axonotmesis), Group2C (the group that received 20 / mg / kg / day x 28 days of oral AA treatment after axonotmesis), Group3A (the group that received oral saline for 28 days after neurotmesis), Group3B (the group that received 10 / mg / kg / day x 28 days of oral AA treatment after (neurotmesis) and Group3C (the group that received 20 / mg / kg / day x 28 days of oral AA treatment after neurotmesis). For functional evaluation; walking test, sciatic function index, extensor postural thrust and gastrocnemius muscle weight index parameters were measured. Electromyography (EMG) was performed for electrophysiological evaluation. All groups were sacrificed at the end of the 28th day. Sciatic nerves and gastrocnemius muscles were excised. Sciatic nerves were histopathologically stained with hemotoxylin-eosin and toloudin blue, and gastrocnemius muscle with masson trichrome. Sciatic nerves; It was also stained immunohistochemically with anti-TNF-alpha, anti-TGF-beta, antiNGF and anti-S100. After staining, the preparations were examined by semi-quantitative and stereological methods. Results were analyzed statistically. Results: Clinical, electrophysiological and histopathological pathological findings were obtained in parallel with the current literature in the groups that underwent axonotmesis and neurotmesis. It was observed that all groups given AA were statistically significant in histopathological, clinical and electrophysiological evaluation compared to the trauma groups that were not given medication. In addition, it was observed that the dose differences within the treatment groups did not make a statistically significant difference in histopathological improvement. Conclusion: As a result of the analysis, in groups given oral asiatic acid; Significant positive images in the restoration of regenerated axon count, myelin formation and denervated muscle atrophy were detected and confirmed by clinical tests. Therefore, we believe that asiatic acid will contribute to the treatment of peripheral nerve injuries with its anti-inflammatory, antioxidant and neuroprotective effects and will shed light on other studies for the treatment of peripheral nerve injuries.

Keywords

• Asiatic acid
• peripheral nerve injuries
• trauma
• rat model

Deneysel Olarak Ratlarda Oluşturulan Periferik Sinir Yaralanmalarında Asiatik Asit’in Rolünün İncelenmesi

Öz

Amaç: Deneysel olarak aksonotmezis ve nörotmezis tipi yaralanma modellerinde Asiatik Asit’in (AA) nöroprotektif ve rejeneratif etkileri histopatolojik, elektrofizyolojik ve klinik olarak incelendi. Gereç ve Yöntem: 56 adet erkek Spraque-Dawley cinsi erkek sıçan, yedi alt gruba ayrıldı. Grup1 (kontrol), Grup2A (aksonotmezis sonrası 28 gün oral serum fizyolojik verilen grup), Grup2B (aksonotmezis sonrası 10/mg/kg/gün x 28 gün oral AA tedavisi verilen grup), Grup2C (aksonotmezis sonrası 20/mg/kg/gün x 28 gün oral AA tedavisi verilen grup), Grup3A (nörotmezis sonrası oral serum fizyolojik verilen grup), Grup3B (nörotmezis sonrası 10/mg/kg/gün x 28 gün oral AA tedavisi verilen grup) ve Grup3C (nörotmezis sonrası 20/mg/kg/gün x 28 gün oral AA tedavisi verilen grup) olarak tanzim edildi. Fonksiyonel değerlendirme için yürüme testi, siyatik fonksiyon indeksi, ektansör postural thrust ve gastroknemius kası ağırlık indeksi parametrelerine bakıldı. Elektrofizyolojik değerlendirme için elektromyografi (EMG) yapıldı. Tüm gruplar 28.gün sonunda sakrifiye edildi. Siyatik sinirler ve gastroknemius kasları eksize edildi. Siyatik sinirler hemotoksilen-eozin ve toloudin mavisiyle, gastroknemius kası ise masson trikrom ile histopatolojik olarak boyandı. Siyatik sinirler; anti-TNF-alfa, anti-TGF-beta, anti-NGF ve anti-S100 ile immünohistokimyasal olarak da boyandı. Boyamalar sonrası preperatlar semikantitatif ve stereolojik yöntemlerle incelendi. Sonuçlar istatiksel olarak analiz edildi. Bulgular: Aksonotmezis ve nörotmezis yapılan gruplarda klinik, elektrofizyolojik ve histopatolojik olarak güncel literatür ile paralel patolojik bulgular elde edildi. AA verilen tüm grupların, ilaç verilmeyen travma gruplarına göre histopatolojik, klinik ve elektrofizyolojik değerlendirmede istatiksel olarak anlamlı olduğu gözlendi. Ayrıca tedavi grupları içerisindeki doz farklılıklarının histopatolojik iyileşmede istatiksel olarak anlamlı bir fark oluşturmadığı görüldü. Sonuç: Analizler sonucunda oral asiatik asit verilen gruplarda; rejenere akson sayısı, miyelin formasyonu ve denerve kas atrofisinin restorasyonunda pozitif yönde anlamlı görüntüler tespit ve klinik testlerle teyit edildi. Bu nedenle asiatik asitin antiinflamatuar, antioksidan ve nöroprotektif etkileriyle periferik sinir yararlanmalarının tedavisine katkılar sağlayacağı ve periferik sinir yaralanmalarının tedavisi için yapılacak diğer çalışmalara ışık tutacağı kanaatindeyiz.

Anahtar Kelimeler

• Asiatik asit
• periferik sinir yaralanmaları
• travma
• sıçan modeli

References

1. Daneyemez M, Solmaz I, Izci Y. Prognostic factors for the surgical management of peripheral nerve lesions. Tohoku J Exp Med. 2005. 205(3): p. 269-275.
2. Seçer Hİ, Daneyemez MK. Periferik sinir yaralanmaları ve cerrahisi. İçinde: Temel Nöroşirürji. Ankara: Türk Nöroşirürji Derneği Yayınları; 2010. p. 1763- 1764.
3. Zhang P, Xue F, Zhao F, Lu Hu, et al. The immunohistological observation of proliferation rule of Schwann cell after sciatic nerve injury in rats Artif Cells Blood Substit Immobil Biotechnol. 2008. 36(2): p. 150-155.
4. Gonçalves BMF, Salvador JAR, Marín S, Cascante M. Synthesis and biological evaluation of novel asiatic acid derivatives with anticancer activity. Eur J Med Chem. 2016. 6(5): p. 3967-3985.
5. Tang L, He R, Yang G, et al. Asiatic acid inhibits liver fibrosis by blocking TGFbeta/Smad signaling in vivo and in vitro. PLoS One, 2012. 7(2).
6. Zhang X, Wu J, Dou Y, et al. Asiatic acid protects primary neurons against C2-ceramideinduced apoptosis. Eur J Pharmacol. 2012. 679(1-3): p. 51-59.
7. Meng Z, Li H, Si C, et al. Asiatic acid inhibits cardiac fibrosis throughNrf2/HO-1 and TGF-β1/Smads signaling pathways in spontaneous hypertension rats. Int Immunopharmacol. 2019. 74: p. 105712.
8. Çakır M, Zengin S, Çalıkoğlu Ç, et al. Protective Role of İnterferon-B in experimentally ınduced peripheral nerve ınjury in rats. Acta Medica Mediterranea. 2015. p.973-979

9. Faroni A, Mobasseri SA, Kingham PJ, et al. Peripheral nerve regeneration: experimental strategies and future perspectives. Adv Drug Deliv Rev. 2015. 82: p. 160-167.
10. Yarar E, Kuruoglu E, Kocabıcak E, et al. Electrophysiological and histopathological effects of mesenchymal stem cells in treatment of experimental rat model of sciatic nerve injury. Int J Clin Exp Med. 2015. 8(6): p. 8776
11. Zeynal M. Ratlarda Periferik Sinir Enjeksiyon Hasarı Modelinde Siyatik Sinirin Değerlendirilmesi. Atatürk Üniversitesi Tıp Fakültesi, Tıpta Uzmanlık Tezi, 2017, Erzurum (Danışman: Prof. Dr. H. Hadi Kadıoğlu)
12. Ehmedah A, Nedeljkovic P, Dacic S, et al. Vitamin B complex treatment attenuates local inflammation after peripheral nerve injury. Molecules, 2019. 24(24): p. 4615. 273. Scheib J, Höke A. Advances in peripheral nerve regeneration. Nat Rev Neurol. 2013. 9(12): p. 668-676.
13. Benli K. Periferik sinir cerrahisinin önemi, içinde Türk Nöroşirurji Dergisi 2005. p. 15:196-197.
14. Burnett MG, Zager EL. Pathophysiology of peripheral nerve injury: a brief review. Neurosurg Focus. 2004. 16(5): p. 1-7.
15. Mishra P, Stringer M. Sciatic nerve injury from intramuscular injection: a persistent and global problem. Int J Clin Pract Suppl. 2010. 64(11): p. 1573-1579.
16. Scheib J, Höke A. Advances in peripheral nerve regeneration. Nat Rev Neurol. 2013. 9(12): p. 668-676.
17. Kurtoglu Z, Ozturk AH, Bagdatoglu C, et al. Effects of trapidil after crush injury in peripheral nerve. Acta Med Okayama. 2005. 59(2): p. 37-44
18. McLachlan EM, Hu P. Inflammation in dorsal root ganglia after peripheral nerve injury: effects of the sympathetic innervation. Auton Neurosci. 2014. 182: p. 108-117.
19. Zhang L, Johnson D, Johnson JA. Deletion of Nrf2 impairs functional recovery, reduces clearance of myelin debris and decreases axonal remyelination after peripheral nerve injury. Neurobiol Dis. 2013. 54: p. 329-338.
20. Lanza C, Raimondo S, Vergani L, et al. Expression of antioxidant molecules after peripheral nerve injury and regeneration. J Neurosci Res. 2012. 90(4): p. 842-848.
21. Wang H, Ding XG, Li SW, et al. Role of oxidative stress in surgical cavernous nerve injury in a rat model. J Neurosci Res. 2015. 93(6): p. 922-929.
22. Qian Y, Han Q, Zhao X, et al. 3D melatonin nerve scaffold reduces oxidative stress and inflammation and increases autophagy in peripheral nerve regeneration. J Pineal Res. 2018. 65(4): p. e12516. 131
23. Renno WM, Benov L, Khan KM. Possible role of antioxidative capacity of epigallocatechin-3-gallate treatment in morphological and neurobehavioral recovery after sciatic nerve crush injury. J Neurosurg Spine. 2017. 27(5): p. 593-613.
24. Mietto BS, Kroner A, Girolami EI, et al. Role of IL-10 in resolution of inflammation and functional recovery after peripheral nerve injury. J Neurosci. 2015. 35(50): p. 16431- 16442.
25. Ydens E, Demon D, Lornet G, et al. Nlrp6 promotes recovery after peripheral nerve injury independently of inflammasomes. Journal of neuroinflammation, 2015. 12(1): p. 1-14. 296. Said G. Examination and clinical care of the patient with neuropathy. Handbook of Clinical Neurology, 2013. 115: p. 235-244.
26. Dubový P, Jančálek R, Kubek T. Role of Inflammation and Cytokines in Peripheral Nerve Regeneration, Tissue Engineering of the Peripheral Nerve, Volume 108, Int Rev Neurobiol. Academic Press;2013. p. 173-206.