Sodium nitrate preconditioning prevents progression of the neuropathic pain in streptozotocin-induced diabetes Wistar rats

  • 2 Accesses



The purpose of the study was to evaluate the possible protective effects of low dose sodium nitrate preconditioning on the peripheral neuropathy in streptozotocin (STZ)-induced diabetic model.


Male Wistar rats were randomly divided into five groups: control (no intervention), control treated sodium nitrate (100 mg/L in drinking water), diabetic (no intervention), diabetic treated NPH insulin (2-4 U), and diabetic treated sodium nitrate (100 mg/L in drinking water). Diabetes was induced by intraperitoneal injection of STZ (60 mg/kg). All interventions were done for 60 days immediately following diabetes confirmation. Thermal and mechanical algesia thresholds were measured by means of hot-plate test, von Frey test, and tail-withdrawal test before the diabetic induction and after diabetes confirmation. At the end of the experiment, serum NOx level and serum insulin level were assessed. Blood glucose concentration and body weight have recorded at the base and duration of the experiment.


Both hypoalgesia, hyperalgesia along with allodynia developed in diabetic rats. Significant alterations including, decrease in tail withdrawal latency (30th day), decreased mechanical threshold (60th day), and an increase in hot plate latency (61st day) were displayed in diabetic rats compared to control rats. Nitrate and insulin preconditioning produced protective effects against diabetes-induced peripheral neuropathy. Data analysis also showed a significant increase in glucose level as well as a considerable reduction in serum insulin and body weight of diabetic rats, which restored by both insulin and nitrate preconditioning.


Sodium nitrate preconditioning produces a protective effect in diabetic neuropathy, which may be mediated by its antihyperglycemic effects and increased serum insulin level.

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

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Organization WH. Global report on diabetes. World Health Organization 2016. 2017.

  2. 2.

    Urbancic-Rovan V. Causes of diabetic foot lesions. Lancet. 2005;366(9498):1675–6.

  3. 3.

    Pirart J. Diabetes mellitus and its degenerative complications: a prospective study of 4,400 patients observed between 1947 and 1973. Diabetes Care. 1978;1(3):168–88.

  4. 4.

    Cermenati G, Abbiati F, Cermenati S, Brioschi E, Volonterio A, Cavaletti G, et al. Diabetes-induced myelin abnormalities are associated with an altered lipid pattern: protective effects of LXR activation. J Lipid Res. 2012;53(2):300–10.

  5. 5.

    Vinik AI. Diabetic neuropathies. Controversies in treating diabetes. Springer 2008. p. 135–156.

  6. 6.

    Rojas DR, Kuner R, Agarwal N. Metabolomic signature of type 1 diabetes-induced sensory loss and nerve damage in diabetic neuropathy. arXiv preprint arXiv:180306740 2018.

  7. 7.

    Han JW, Sin MY, Y-s Y. Cell therapy for diabetic neuropathy using adult stem or progenitor cells. Diabetes Metab J. 2013;37(2):91–105.

  8. 8.

    Nowicki M, Kosacka J, Serke H, Blüher M, Spanel-Borowski K. Altered sciatic nerve fiber morphology and endoneural microvessels in mouse models relevant for obesity, peripheral diabetic polyneuropathy, and the metabolic syndrome. J Neurosci Res. 2012;90(1):122–31.

  9. 9.

    Fioretto P, Dodson PM, Ziegler D, Rosenson RS. Residual microvascular risk in diabetes: unmet needs and future directions. Nat Rev Endocrinol. 2010;6(1):19–25.

  10. 10.

    Oates PJ. Polyol pathway and diabetic peripheral neuropathy. Int Rev Neurobiol. 2002;50:325–92.

  11. 11.

    Du X-L, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, et al. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci. 2000;97(22):12222–6.

  12. 12.

    Eichberg J. Protein kinase C changes in diabetes: is the concept relevant to neuropathy? Int Rev Neurobiol. 2002;50:61–82.

  13. 13.

    Mišur I, Žarković K, Barada A, Batelja L, Miličević Z, Turk Z. Advanced glycation endproducts in peripheral nerve in type 2 diabetes with neuropathy. Acta Diabetol. 2004;41(4):158–66.

  14. 14.

    Miyauchi Y, Shikama H, Takasu T, Okamiya H, Umeda M, Hirasaki E, et al. Slowing of peripheral motor nerve conduction was ameliorated by aminoguanidine in streptozocin-induced diabetic rats. Eur J Endocrinol. 1996;134(4):467–73.

  15. 15.

    Albers JW, Herman WH, Pop-Busui R, Feldman EL, Martin CL, Cleary PA, et al. Effect of prior intensive insulin treatment during the Diabetes Control and Complications Trial (DCCT) on peripheral neuropathy in type 1 diabetes during the epidemiology of Diabetes Interventions, and Complications (EDIC) study. Diabetes Care. 2010.

  16. 16.

    Jin HY, Lee KA, Song SK, Liu WJ, Choi JH, Song CH, et al. Sulodexide prevents peripheral nerve damage in streptozotocin induced diabetic rats. Eur J Pharmacol. 2012;674(2–3):217–26.

  17. 17.

    Wang L, Chopp M, Szalad A, Lu X, Jia L, Lu M, et al. Tadalafil promotes the recovery of peripheral neuropathy in type II diabetic mice. PLoS One. 2016;11(7):e0159665.

  18. 18.

    Wang L, Chopp M, Szalad A, Jia L, Lu X, Lu M, et al. Sildenafil ameliorates long term peripheral neuropathy in type II diabetic mice. PLoS One. 2015;10(2):e0118134.

  19. 19.

    Gally JA, Montague PR, Reeke GN, Edelman GM. The NO hypothesis: possible effects of a short-lived, rapidly diffusible signal in the development and function of the nervous system. Proc Natl Acad Sci. 1990;87(9):3547–51.

  20. 20.

    Moncada S, Higgs E. The discovery of nitric oxide and its role in vascular biology. Br J Pharmacol. 2006;147(S1):S193–201.

  21. 21.

    Garry P, Ezra M, Rowland M, Westbrook J, Pattinson K. The role of the nitric oxide pathway in brain injury and its treatment—from bench to bedside. Exp Neurol. 2015;263:235–43.

  22. 22.

    Lundberg JO, Weitzberg E. NO-synthase independent NO generation in mammals. Biochem Biophys Res Commun. 2010;396(1):39–45.

  23. 23.

    Muenzel T, Daiber A. Inorganic nitrite and nitrate in cardiovascular therapy: a better alternative to organic nitrates as nitric oxide donors? Vasc Pharmacol. 2018;102:1–10.

  24. 24.

    Keyhanmanesh R, Hamidian G, Alipour MR, Oghbaei H. Benefiial treatment effects of dietary nitrate supplementation on testicular injury in streptozotocin-induced diabetic male rats. Reprod BioMed Online. 2019.

  25. 25.

    Lee-Kubli CA, Mixcoatl-Zecuatl T, Jolivalt CG, Calcutt NA. Animal models of diabetes-induced neuropathic pain. Behavioral Neurobiology of Chronic Pain. Springer 2014. p. 147-.

  26. 26.

    Oghbaei H, Asl NA, Sheikhzadeh F, Alipour MR. The effect of regular moderate exercise on miRNA-192 expression changes in kidney of streptozotocin-induced diabetic male rats. Adv Pharm Bull. 2015;5(1):127–32.

  27. 27.

    Oghbaei H, Asl NA, Sheikhzadeh F. Can regular moderate exercise lead to changes in miRNA-146a and its adapter proteins in the kidney of streptozotocin-induced diabetic male rats? Endocr Regul. 2017;51(3):145–52.

  28. 28.

    Keyhanmanesh R, Hamidian G, Alipour MR, Ranjbar M, Oghbaei H. Protective effects of sodium nitrate against testicular apoptosis and spermatogenesis impairments in streptozotocin-induced diabetic male rats. Life Sci. 2018;211:63–73.

  29. 29.

    Oghbaei H, Alipour MR, Hamidian G, Ahmadi M, Ghorbanzadeh V, Keyhanmanesh R. Two months sodium nitrate supplementation alleviates testicular injury in streptozotocin-induced diabetic male rats. Exp Physiol. 2018;103(12):1603–17.

  30. 30.

    Oghbaei H, Alipour MR, Mohaddes G, Hamidian GR, Keyhanmanesh R. Evaluation of ameliorative effect of sodium nitrate in experimental model of streptozotocin-induced diabetic neuropathy in male rats. Endocr Regul. 2019;53(1):14–25.

  31. 31.

    Dobson C, Tohyama Y, Diksic M, Hamel E. Effects of acute or chronic administration of anti-migraine drugs sumatriptan and zolmitriptan on serotonin synthesis in the rat brain. Cephalalgia. 2004;24(1):2–11.

  32. 32.

    Kubo K, Nishikawa K, Ishizeki J, Hardy-Yamada M, Yanagawa Y, Saito S. Thermal hyperalgesia via supraspinal mechanisms in mice lacking glutamate decarboxylase 65. J Pharmacol Exp Ther. 2009;331(1):162–9.

  33. 33.

    Zeng P, Li S, Zheng Y-h, Liu F-Y, Wang J-l, Zhang D-l, et al. Ghrelin receptor agonist, GHRP-2, produces antinociceptive effects at the supraspinal level via the opioid receptor in mice. Peptides. 2014;55:103–9.

  34. 34.

    Oyenihi AB, Ayeleso AO, Mukwevho E, Masola B. Antioxidant strategies in the management of diabetic neuropathy. Biomed Res Int. 2015;2015.

  35. 35.

    Grote CW, Wright DE. A role for insulin in diabetic neuropathy. Front Neurosci. 2016;10:581.

  36. 36.

    Cellek S. Point of NO return for nitrergic nerves in diabetes: a new insight into diabetic complications. Curr Pharm Des. 2004;10(29):3683–95.

  37. 37.

    Sasaki T, Yasuda H, Maeda K, Kikkawa R. Hyperalgesia and decreased neuronal nitric oxide synthase in diabetic rats. Neuroreport. 1998;9(2):177.

  38. 38.

    Gheibi S, Jeddi S, Carlström M, Gholami H, Ghasemi A. Effects of long-term nitrate supplementation on carbohydrate metabolism, lipid profiles, oxidative stress, and inflammation in male obese type 2 diabetic rats. Nitric Oxide. 2018;75:27–41.

  39. 39.

    Nyström T, Ortsäter H, Huang Z, Zhang F, Larsen FJ, Weitzberg E, et al. Inorganic nitrite stimulates pancreatic islet blood flow and insulin secretion. Free Radic Biol Med. 2012;53(5):1017–23.

  40. 40.

    Henstridge DC, Duffy SJ, Formosa MF, Ahimastos AA, Thompson BR, Kingwell BA. Oral nitrate therapy does not affect glucose metabolism in healthy men. Clin Exp Pharmacol Physiol. 2009;36(11):1086–92.

  41. 41.

    Bahadoran Z, Ghasemi A, Mirmiran P, Azizi F, Hadaegh F. Beneficial effects of inorganic nitrate/nitrite in type 2 diabetes and its complications. Nutr Metab. 2015;12(1):16.

  42. 42.

    Gheibi SBF, Jeddi S, Farrokhfall K, Zardooz H, Ghasemi A. Nitrite increases glucose-stimulated insulin secretion and islet insulin content in obese type 2 diabetic male rats. Nitric Oxide. 2017;1(64):39–51.

  43. 43.

    Varzandi T, Abdollahifar MA, Rohani SAH, Piryaei A, Zadeh-Vakili A, Jeddi S, et al. Effect of long-term nitrite administration on browning of white adipose tissue in type 2 diabetic rats: a stereological study. Life Sci. 2018;207:219–26.

  44. 44.

    Henstridge D, Kingwell BA, Formosa MF, Drew B, McConell GK, Duffy S. Effects of the nitric oxide donor, sodium nitroprusside, on resting leg glucose uptake in patients with type 2 diabetes. Diabetologia. 2005;48(12):2602–8.

  45. 45.

    Gilchrist M, Winyard PG, Aizawa K, Anning C, Shore A, Benjamin N. Effect of dietary nitrate on blood pressure, endothelial function, and insulin sensitivity in type 2 diabetes. Free Radic Biol Med. 2013;60:89–97.

  46. 46.

    Kobayashi J. Nitric oxide and insulin resistance. Immunoendocrinology. 2015;2.

  47. 47.

    Ogur R, Coskun O, Korkmaz A, Oter S, Yaren H, Hasde M. High nitrate intake impairs liver functions and morphology in rats; protective effects of α-tocopherol. Environ Toxicol Pharmacol. 2005;20(1):161–6.

  48. 48.

    Chaoui A, Zaki A, Talibi A, Chait A, Derouiche A, Aboussaouira T, et al. Effects of inorganic nitrates on thyroid gland activity and morphology in female rats. Therapie. 2004;59(4):471–5.

  49. 49.

    Seethalakshmi L, Menon M, Diamond D. The effect of streptozotocin-induced diabetes on the neuroendocrine-male reproductive tract axis of the adult rat. J Urol. 1987;138(1):190–4.

  50. 50.

    Assmann TS, Brondani LA, Boucas AP, Rheinheimer J, de Souza BM, Canani LH, et al. Nitric oxide levels in patients with diabetes mellitus: a systematic review and meta-analysis. Nitric Oxide. 2016;61:1–9.

  51. 51.

    Schmetterer L, Findl O, Fasching P, Ferber W, Strenn K, Breiteneder H, et al. Nitric oxide and ocular blood flow in patients with IDDM. Diabetes. 1997;46(4):653–8.

  52. 52.

    Bryan NS, Fernandez BO, Bauer SM, Garcia-Saura MF, Milsom AB, Rassaf T, et al. Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nat Chem Biol. 2005;1(5):290–7.

  53. 53.

    Ashmore T, Roberts LD, Morash AJ, Kotwica AO, Finnerty J, West JA, et al. Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism. BMC Biol. 2015;13(1):110.

  54. 54.

    Barrot M. Tests and models of nociception and pain in rodents. Neuroscience. 2012;211:39–50.

  55. 55.

    Khan G, Chen S-R, Pan H-L. Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats. Neuroscience. 2002;114(2):291–9.

  56. 56.

    Shir Y, Seltzer ZE. A-fibers mediate mechanical hyperesthesia and allodynia and C-fibers mediate thermal hyperalgesia in a new model of causalgiform pain disorders in rats. Neurosci Lett. 1990;115(1):62–7.

  57. 57.

    Wang X-L, Zhang Q, Zhang Y-Z, Liu Y-T, Dong R, Wang Q-J, et al. Downregulation of GABAB receptors in the spinal cord dorsal horn in diabetic neuropathy. Neurosci Lett. 2011;490(2):112–5.

  58. 58.

    Calcutt NA. Potential mechanisms of neuropathic pain in diabetes. Int Rev Neurobiol. 2002;50:205–28.

  59. 59.

    Gong Y-H, Yu X-R, Liu H-L, Yang N, Zuo P-P, Huang Y-G. Antinociceptive effects of combination of tramadol and acetaminophen on painful diabetic neuropathy in streptozotocin-induced diabetic rats. Acta Anaesthesiol Taiwanica. 2011;49(1):16–20.

  60. 60.

    Yagihashi S, Mizukami H. Diabetic Neuropathy. Diabetes and aging-related complications: Springer; 2018. p. 31–43.

  61. 61.

    Calcutt NA, Jorge MC, Yaksh TL, Chaplan SR. Tactile allodynia and formalin hyperalgesia in streptozotocin-diabetic rats: effects of insulin, aldose reductase inhibition and lidocaine. Pain. 1996;68(2–3):293–9.

  62. 62.

    Gilliatt R, Willison R. Peripheral nerve conduction in diabetic neuropathy. J Neurol Neurosurg Psychiatry. 1962;25(1):11.

  63. 63.

    Prnova MS, Svik K, Bezek S, Kovacikova L, Karasu C, Stefek M. 3-Mercapto-5H-1, 2, 4-Triazino [5, 6-b] Indole-5-acetic acid (Cemtirestat) alleviates symptoms of peripheral diabetic neuropathy in Zucker diabetic fatty (ZDF) rats: a role of aldose reductase. Neurochem Res. 2019:1–9.

  64. 64.

    Chen Y-W, Hsieh P-L, Chen Y-C, Hung C-H, Cheng J-T. Physical exercise induces excess hsp72 expression and delays the development of hyperalgesia and allodynia in painful diabetic neuropathy rats. Anesth Analg. 2013;116(2):482–90.

  65. 65.

    Ismail CAN, Aziz CBA, Suppian R, Long I. Imbalanced oxidative stress and pro-inflammatory markers differentiate the development of diabetic neuropathy variants in streptozotocin-induced diabetic rats. J Diabetes Metab Disord. 2018;1.

  66. 66.

    Brussee V, Cunningham FA, Zochodne DW. Direct insulin signaling of neurons reverses diabetic neuropathy. Diabetes. 2004;53(7):1824–30.

Download references


This article is a part of database from the investigation entitled “The effect of inorganic nitrate on the prevention and treatment of peripheral diabetic neuropathy in typ-1 diabetic male rats” written by Hajar Oghbaei, Faculty of Medicine, Tabriz University of Medical Sciences and was funded by the student research committee of Tabriz University of Medical Sciences (No. IR.TBZMED. REC.1395.960), Tabriz, Iran.

Author information

Correspondence to Rana Keyhanmanesh.

Ethics declarations

Ethical issues

All of protocols were approved by the Ethics Committee of Animal Research of Tabriz University of Medical Sciences (No. IR.TBZMED.REC.1395.960).

Conflict of interests

Authors declare no conflict of interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


• Diabetes decreased mechanical threshold (mechanical allodynia)

• Diabetes caused the first reduction and then a secondary increase in tail withdrawal and hot plate latency

• Sodium nitrate preconditioning decreased the blood glucose and increased the serum insulin level in diabetic rats.

• Sodium nitrate preconditioning reduced allodynia and thermal algesia in diabetic rats

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Oghbaei, H., Mohaddes, G., Hamidian, G. et al. Sodium nitrate preconditioning prevents progression of the neuropathic pain in streptozotocin-induced diabetes Wistar rats. J Diabetes Metab Disord (2020) doi:10.1007/s40200-019-00481-4

Download citation


  • Nitrate preconditioning
  • Diabetes
  • Peripheral nerve
  • Thermal algesia
  • Mechanical algesia