Co-administration of ascorbic acid and α-tocopherol modifies ascorbic acid and attenuates p38, Akt, and TNF-α expression in spinal cord of rats with neuropathic pain



p38 and Akt plays an important role in neuropathic pain. p38 appears to regulate the SVCT-2 expression, a specific transporter for ascorbic acid (an important antioxidant and neuromodulator). Vitamins C and E induce antinociception and appear to affect p38, Akt, and SVCT-2, but it is unknown their effect on these molecules in spinal cord of rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. So, we investigated the effect of vitamins C + E on the expression of p38, Akt, and transporter SVCT-2 and ascorbic acid content in the lumbosacral spinal cord of rats with CCI. TNF-α mRNA level was also determined.


Placement of four loose chromic thread ligatures around the sciatic nerve produced CCI. Ascorbic acid (vitamin C 30 mg/kg/day) + α-tocopherol (vitamin E 15 mg/kg/day) or vehicle (saline containing 1% Tween 80) were administrated (intraperitoneally) daily after CCI for 3 or 10 days. At the end of these periods, lumbosacral spinal cord was dissected out and used for assays.


The vitamins prevented the increase in phosphorylated p38 and phosphorylated Akt and the decrease in SVCT-2 expressions, which were found in vehicle-treated CCI rats at days 3 and 10. Akt expression was reduced only at day 10. Vitamins also prevented the increase in ascorbic acid content and TNF-α level, which was found in vehicle-treated CCI rats.


The changes in p-p38, p-Akt, ascorbic acid, SVCT-2, and TNF-α may be contributing to antinociception induced by vitamins C + E in rats with CCI.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Data availability

All data generated or analyzed in this study are included in this published article.



Ascorbic acid


Protein kinase B


Analysis of variance


Chronic constriction injury


Ethics Committee of the Universidade Federal do Rio Grande do Sul




Glyceraldehyde-3-phosphate dehydrogenase

I.P. :



Potassium chloride




Mitogen-activated protein kinases


Protein kinase B in phosphorylated form


p38 in phosphorylated form


Sodium dodecyl sulfate–polyacrylamide gel electrophoresis


Sodium/vitamin C co-transporter isoform 2


Tumor necrosis factor-alpha


  1. 1.

    Kumar A, Harshpreet K, Singh A. Neuropathic pain models caused by damage to central or peripheral nervous system. Pharmacol Rep. 2018;70:206–16.

    PubMed  Google Scholar 

  2. 2.

    Colloca L, Ludman T, Bouhassira D, Baron R, Dickenson AH, Yarnitsky D, et al. Neuropathic pain. Nat Rev Dis Primers. 2017;3:17002.

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Wawrzyniak A, Górnicka M, Hamułka J, Gajewska M, Drywień M, Pierzynowska G-SA. α-Tocopherol, ascorbic acid, and β-carotene protect against oxidative stress but reveal no direct influence on p53 expression in rats subjected to stress. Nutr Res. 2013;33:868–75.

    CAS  PubMed  Google Scholar 

  4. 4.

    Salazar K, Martinez F, Pérez-Martin M, Cifuentes M, Triqueros L, Ferrada L, et al. SVCT2 expression and function in reactive astrocytes is a common event in different brain pathologies. Mol Neurobiol. 2017;23:23.

    Google Scholar 

  5. 5.

    Okubo M, Yamanaka H, Kobayashi K, Kanda H, Dai Y, Noguchi K. Upregulation of platelet-activating factor synthases and its receptor in spinal cord contribute to development of neuropathic pain following peripheral nerve injury. Mol Pain. 2012;8:8.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Schencking M, Sandholzer H, Frese T. Intravenous administration of vitamin C in the treatment of herpetic neuralgia: two case reports. Med Sci Monit. 2010;16:58–61.

    Google Scholar 

  7. 7.

    Bruno RS, Leonard SW, Atkinson J, Montine TJ, Ramakrishnan R, Bray TM. Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation. Free Radic Biol Med. 2006;40:689–97.

    CAS  PubMed  Google Scholar 

  8. 8.

    Meymandi MS, Sepehri G, Izadi G, Zamiri Z. Evidence for antinociceptive effects of combined administration of vitamin E and celecoxib in tail-flick and formalin test in male rats. Pharmacol Rep. 2019;71:457–64.

    Google Scholar 

  9. 9.

    Naziroglu M, Özgül C. Vitamin E modulates oxidative stress and protein kinase C activator (PMA)-induced TRPM2 channel gate in dorsal root ganglion of rats. J Bioenerg Biomembr. 2013;45:541–9.

    CAS  PubMed  Google Scholar 

  10. 10.

    Garelnabi N, Veledar E, White-Welkley J, Santanam N, Abramson J, Weintraub W, et al. Vitamin E differentially affects short term exercise induced changes in oxidative stress, lipids, and inflammatory markers. Nut Metab Cardiovasc Dis. 2012;22:907–13.

    CAS  Google Scholar 

  11. 11.

    Wu D, Hayek MG, Meydani S. Vitamin E and macrophage cyclooxygenase regulation in the aged. J Nutr. 2001;131:382S–8S.

    CAS  PubMed  Google Scholar 

  12. 12.

    Riffel APK, de Souza JA, do CQ SM, Horst A, Scheid T, Kolberg C, et al. Systemic administration of vitamins C and E attenuates nociception induced by chronic constriction injury of the sciatic nerve in rats. Brain Res Bull. 2016;121:169–77.

    CAS  PubMed  Google Scholar 

  13. 13.

    Jaggi AS, Jain V, Singh N. Animal models of neuropathic pain. Fundam Clin Pharmacol. 2011;25:1–28.

    CAS  PubMed  Google Scholar 

  14. 14.

    Huang YN, Lai CC, Chiu CT, Lin JJ, Wang JY. L-ascorbate attenuates the endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation and NF- κB translocation in cortical neurons/glia cocultures. PLoS One. 2014;9:1–12.

    Google Scholar 

  15. 15.

    Giraldo E, Lloret A, Fuchsberger T, Viña J. Aβ and tau toxicities in Alzheimer’s are linked via oxidative stress-induced p38 activation: protective role of vitamin E. Redox Biol. 2014;2:873–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Zakharova IO, Sokolova TV, Vlasova YA, Bayunova LV, Rychkova MP, Avrova NF. α-tocopherol at nanomolar concentration protects cortical neurons against oxidative stress. Int J Mol Sci. 2017;18:216.

    PubMed Central  Google Scholar 

  17. 17.

    Moretti M, Budni J, Ribeiro CM, Rieger DK, Leal RB, Rodriques ALS. Subchronic administration of ascorbic acid elicits antidepressant-like effect and modulates cell survival signaling pathways in mice. J Nutr Biochem. 2016;38:50–6.

    CAS  PubMed  Google Scholar 

  18. 18.

    Guo J, Wang H, Jin X, Jia D, Zhou X, Tao Q. Effect and mechanism of inhibition of PI3K / Akt / mTOR signal pathway on chronic neuropathic pain and spinal microglia in a rat model of chronic constriction injury. Oncotarget. 2017;8:52923–34.

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Jiang L, Pan CL, Wang CL, Liu BQ, Han Y, Hu L, et al. Selective suppression of the JNK-MMP2/9 signal pathway by tetramethylpyrazine attenuates neuropathic pain in rats. J Neuroinflamm. 2017;14:174.

    Google Scholar 

  20. 20.

    Tian H, Ye X, Hou X, Yang X, Yang J, Wu C. SVCT2, a potential therapeutic target, protects against oxidative stress during ethanol-induced neurotoxicity via JNK/p38 MAPKs, NF-κB and miRNA 125a-5p. Free Radic Biol Med. 2016;96:362–73.

    CAS  PubMed  Google Scholar 

  21. 21.

    Covarrubias-Pinto A, Acuña AI, Beltrán FA, Torres-Díaz L, Castro MA. Old things new view: ascorbic acid protects the brain in neurodegenerative disorders. Int J of Mol Sci. 2015;16:28194–217.

    CAS  Google Scholar 

  22. 22.

    Horst A, Souza JA, Santos MCQ, Riffel APK, Kolberg C, Partata WA. Effects of N-acetylcysteine on spinal cord oxidative stress biomarkers in rats with neuropathic pain. Braz J Med Biol Res. 2017;50:12.

    Google Scholar 

  23. 23.

    Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988;33:87–107.

    CAS  PubMed  Google Scholar 

  24. 24.

    Roe JH, Kuether CA. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid. J Biol Chem. 1943;147:399–407.

    CAS  Google Scholar 

  25. 25.

    Ding Y, Yao P, Hong T, Li H, Zhu Y, Han Z, et al. The analgesic effect of early hyperbaric oxygen treatment in chronic constriction injury rats and its influence on nNOS and iNOS expression and inflammatory factor production. Mol Pain. 2018;14:1–11.

    Google Scholar 

  26. 26.

    Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–9.

    CAS  PubMed  Google Scholar 

  27. 27.

    Lu R, Kallenborn-Gerhardt W, Geisslinger G, Schmidtko A. Additive antinociceptive effects of a combination of vitamin C and vitamin E after peripheral nerve injury. PLoS One. 2011;6:1–9.

    CAS  Google Scholar 

  28. 28.

    Hu JY, Li CH, Wang YW. Intrathecal administration of triptolide, a T lymphocyte inhibitor, attenuates chronic constriction injury-induced neuropathic pain in rats. Brain Res. 2012;1436:122–9.

    CAS  PubMed  Google Scholar 

  29. 29.

    Piao JM, Zhong-Xi WW, Yu-Zhen Y, Luo LQ, Yu J. MicroRNA-381 favors repair of nerve injury through regulation of the SDF-1/ CXCR4 signaling pathway via LRRC4 in acute cerebral ischemia after cerebral lymphatic blockage. Cell Physiol Biochem. 2018;46:890–906.

    CAS  PubMed  Google Scholar 

  30. 30.

    Tsuda M, Masuda T, Tozaki-Saitoh H, Inoue K. Microglial regulation of neuropathic pain. J Pharm Sci. 2013;121:89–94.

    CAS  Google Scholar 

  31. 31.

    Watkins LR, Milligan ED, Maier SF. Glial activation: a driving force for pathological pain. Trends Neurosci. 2001;24:450–1.

    CAS  PubMed  Google Scholar 

  32. 32.

    Sumizono M, Sakakima H, Otsuka S, Terashi T, Nakanishi K, Ueda K, et al. The effect of exercise frequency on neuropathic pain and pain-related cellular reactions in the spinal cord and midbrain in a rat sciatic nerve injury model. J Pain Res. 2018;11:281–91.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Mika J, Osikowicz M, Rojewska E, Korostynski M, Wawrzczak-Bargiela A, Przewlocki R, et al. Differential activation of spinal microglial and astroglial cells in a mouse model of peripheral neuropathic pain. Neuropharmacol Analgesia. 2009;623:65–72.

    CAS  Google Scholar 

  34. 34.

    Portugal CC, Socodato R, Canedo T, Silva CM, Martins T, Coreixas VSM, et al. Caveolin-1–mediated internalization of the vitamin C transporter SVCT2 in microglia triggers an inflammatory phenotype. Sci Signal. 2017;10:2005.

    Google Scholar 

  35. 35.

    VanHook A. Vitamin C prevents microglia activation. Science. 2017;355:1386–8.

    PubMed  Google Scholar 

  36. 36.

    Egger T, Schuligoi R, Wintersperger A, Amann R, Malle E, Sattler W. Vitamin E (α-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortalized murine BV-2 microglia. Biochem J. 2003;370:459–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Lee G, Grovey B, Furnish T, Wallace M. Medical Cannabis for neuropathic pain. Cur Pain Headache Rep. 2018;22:1–8.

    Google Scholar 

  38. 38.

    Li R, Shen L, Yu X, Ma C, Huang Y. Vitamin C enhances the analgesic effect of gabapentin on rats with neuropathic pain. Life Sci. 2016;15:25–31.

    Google Scholar 

  39. 39.

    Li Z, Wei H, Piirainen S, Chen Z, Kalso E, Pertovaara A, et al. Spinal versus brain microglial and macrophage activation traits determine the differential neuroinflammatory responses and analgesic effect of minocycline in chronic neuropathic pain. Brain Behav Immun. 2016;58:107–17.

    CAS  PubMed  Google Scholar 

  40. 40.

    Acuña A, Esparza M, Kramm C, Beltra FA, Parra AV, Cepeda C, et al. A failure in energy metabolismo and antioxidante uptake precede symptoms of Huntington’s disease in mice. Nat Commun. 2013;4:2917.

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Liu W, Lv Y, Ren F. PI3K/Akt pathway is required for spinal central sensitization in neuropathic pain. Cell Mol Neurobiol. 2018;38:747–55.

    CAS  PubMed  Google Scholar 

  42. 42.

    Meacham K, Shepherd A, Mohapatra DP, Haroutounian S. Neuropathic pain: central vs. peripheral mechanisms. Curr Pain Headache Rep. 2017;21:1–6.

    Google Scholar 

  43. 43.

    Park JM, Kim CK, Lee HC, Jung H, Choi KU, Hong SW, et al. Antiallodynic effects of vitamin C and vitamin E in chronic post-ischemia pain rat model. Korean J. 2013;65(5):442–8.

    CAS  Google Scholar 

  44. 44.

    Grace PM, Gaudet AD, Staikopoulos V, Maier SF, Hutchinson MR, Salvemini D, et al. Nitroxidative signaling mechanisms in pathological pain. Trends Neurosci. 2016;39:862–79.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Riffel APK, Santos MCQ, Souza JA, Scheid T, Horst A, Kolberg C, et al. Treatment with ascorbic acid and α-tocopherol modulates oxidative-stress markers in the spinal cord of rats with neuropathic pain. Braz J Med Biol Res. 2018;51:1–11.

    Google Scholar 

  46. 46.

    Ferreira NR, Ledo A, Laranjinha J, Gerhardt GA, Barbosa RM. Simultaneous measurements of ascorbate and glutamate in vivo in the rat brain using carbono fiber nanocomposite sensors and microbiosensor arrays. Bioelectrochemistry. 2018;121:142–50.

    CAS  PubMed  Google Scholar 

  47. 47.

    Desrumaux CM, Mansuy M, Lemaires S, Przybilski J, Le Guern N, Givalois L, et al. Brain vitamin D deficiency during development is associated with increased glutamate levels and anxiety in adult mice. Front Behav Neurosci. 2018;12:310.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Scheid T, Bosco LD, Guedes RP, Pavanato MA, Belló-Klein A, Partata WA. Sciatic nerve transection modulates oxidative parameters in spinal and supraspinal regions. Neurochem Res. 2013;38:935–42.

    CAS  PubMed  Google Scholar 

  49. 49.

    Goecks CS, Horst A, Moraes MS, Scheid T, Kolberg C, Belló-Klein A, et al. Assessment of oxidative parameters in rat spinal cord after constriction of the sciatic nerve. Neurochem Res. 2012;37(9):1952–8.

    CAS  PubMed  Google Scholar 

Download references


This work was supported by grants from Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS—process 2010/1007354) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—process 870582/1997-5).

Author information




A.P.K.R and W.A.P. were responsible for the study concept and design and they prepared and wrote the manuscript. A.P.K.R., J.A.S., M.C.Q.S., A.K., E.M.S.S., T.S., A.H., C.K., and G.M. performed the experiments. L.S.F and M.F.M.R. helped in the Western blotting procedure and analysis. All authors approved the final manuscript.

Corresponding author

Correspondence to Ana Paula Konzen Riffel.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

This study was approved by the Ethics Committee of the Universidade Federal do Rio Grande do Sul (CEUA-UFRGS #23352).

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Riffel, A.P.K., de Souza, J.A., Santos, M. et al. Co-administration of ascorbic acid and α-tocopherol modifies ascorbic acid and attenuates p38, Akt, and TNF-α expression in spinal cord of rats with neuropathic pain. Nutrire 45, 9 (2020).

Download citation


  • Phosphorylated p38
  • Phosphorylated Akt
  • Chronic constriction injury
  • Sciatic nerve