The Effect of Intrauterine Acute Ethanol Exposure on Developing Sciatic Nerves and Their Myelination: A Stereological Study

Abstract

Ethanol (ETH) delays myelination in the sciatic nerve of rat following fetal exposure. It has been suggested that melatonin (MLT), a naturally occurring indole, may be a potential protective agent for ETH toxicity, possibly due to its protective effects against free radical damage in experimental models. However, the precise mechanism of MLT protection in the development of the rat sciatic nerves and their myelination is unclear. In this study, the effect of ETH, MLT and ETH+MLT on the myelin thickness, axon number and axonal area of rat sciatic nerve were investigated. Pregnant rats were injected with either 25% ETH (dosage 2g/kg body weight), 25% ETH plus MLT (dosage 2g/kg and 10 mg/kg, respectively), MLT (dosage 10 mg/kg) or physiological saline (dosage 1 ml/kg) at 7, 15 and 20 days post-conception (first, second and third trimesters, respectively). At postnatal day 14, sciatic nerves of offspring were dissected out and processed for stereological analyses. Our results demonstrated that the myelin thickness was increased by exogenous MLT, and decreased by acute ETH, during first and second trimesters. It was found that MLT was protective to developing myelin thickness following fetal exposure during the first trimester, whereas it was toxic to myelin thickness during the third trimester. The presented study is the first stereological study indicating that exogenous MLT has a toxic effect on the myelin thickness of rat sciatic nerves during the third trimester. Based on our findings, we suggested that exogenous MLT, given during the third trimester as an antioxidant agent, as well as acute ETH, given in the first and second trimesters, may alter the normal development of sciatic nerve myelination.

Keywords

• Ethanol, Melatonin, Sciatic nerve, Stereology, IntrauterineRat

References

1. Agar E, Demir S, Amanvermez R, Bosnak M, Ayyildiz M, Celik C. The effects of ethanol consumption on the lipid peroxidation and glutathione levels in the right and left brains of rats. Int J Neurosci 2003; 113: 1643-52.
2. Antonio AM, Druse MJ. Antioxidants prevent ethanol-associated apoptosis in fetal rhombencephalic neurons. Brain Res 2008; 1204: 16-23.
3. Baydas G, Tuzcu M. Protective effects of melatonin against ethanol-induced reactive gliosis in hippocampus and cortex of young and aged rats. Exp Neurol 2005; 194: 175-81.
4. Das SK, Vasudevan DM. Alcohol-induced oxidative stress. Life Sci 2007; 81: 177-87.
5. Druse MJ, Hofteig JH. The effect of chronic maternal alcohol consumption on the development of central nervous system myelin subfractions in rat offspring. Drug Alcohol Depend 1977; 2: 421-9.
6. Genovese T, Mazzon E, Muia C, Bramanti P, De Sarro A, Cuzzocrea S. Attenuation in the evolution of experimental spinal cord trauma by treatment with melatonin. J Pineal Res 2005; 38: 198-208.
7. Geuna S, Tos P, Guglielmone R, Battiston B, Giacobini-Robecchi MG. Methodological issues in size estimation of myelinated nerve fibers in peripheral nerves. Anat Embryol (Berl) 2001; 204: 1-10.
8. Heaton MB, Madorsky I, Paiva M, Siler-Marsiglio KI. Vitamin E amelioration of ethanol neurotoxicity involves modulation of apoptotis-related protein levels in neonatal rat cerebellar granule cells. Brain Res Dev Brain Res 2004; 150: 117-24.

9. Heaton MB, Paiva M, Madorsky I, Shaw G. Ethanol effects on neonatal rat cortex: comparative analyses of neurotrophic factors, apoptosis-related proteins, and oxidative processes during vulnerable and resistant periods. Brain Res Dev Brain Res 2003; 145: 249-62.
10. Hofteig JH, Druse MJ. Central nervous system myelination in rats exposed to ethanol in utero. Drug Alcohol Depend 1978; 3: 429-34.
11. Jan JE, Wasdell MB, Freeman RD, Bax M. Evidence supporting the use of melatonin in short gestation infants. J Pineal Res 2007; 42: 22-7.
12. Jessen KR, Brennan A, Morgan L, Mirsky R, Kent A, Hashimoto Y, et al. The Schwann cell precursor and its fate: a study of cell death and differentiation during gliogenesis in rat embryonic nerves. Neuron 1994; 12: 509-27.
13. Jessen KR, Mirsky R. The origin and development of glial cells in peripheral nerves. Nat Rev Neurosci 2005; 6: 671-82.
14. Juknat AA, Mendez Mdel V, Quaglino A, Fameli CI, Mena M, Kotler ML. Melatonin prevents hydrogen peroxide-induced Bax expression in cultured rat astrocytes. J Pineal Res 2005; 38: 84-92.
15. Kaplan S, Canan S, Aslan H, Unal B, Sahin B. A simple technique to measure the movements of the microscope stage along the x and y axes for stereological methods. J Microsc 2001; 203: 321-5.
16. Kaplan S, Gokyar A, Unal B, Tunc AT, Bahadir A, Aslan H. A simple technique for localizing consecutive fields for disector pairs in light microscopy: application to neuron counting in rabbit spinal cord following spinal cord injury. J Neurosci Methods 2005; 145: 277-84.
17. Kodituwakku PW, Kalberg W, May PA. The effects of prenatal alcohol exposure on executive functioning. Alcohol Res Health 2001; 25: 192-8.
18. Koh SH, Kim SH, Kwon H, Park Y, Kim KS, Song CW, et al. Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase/Akt and glycogen synthase kinase-3. Brain Res Mol Brain Res 2003; 118: 72-81.
19. Kozan R, Ayyildiz M, Bas O, Kaplan S, Agar E. Short-term ethanol administration does not change the total pyramidal neuron number of the rat hippocampus: a stereologic study. Adv Ther 2007; 24: 231-8.
20. Lancaster FE. Alcohol and white matter development--a review. Alcohol Clin Exp Res 1994; 18: 644-7.
21. Lancaster FE, Phillips SM, Patsalos PN, Wiggins RC. Brain myelination in the offspring of ethanol-treated rats: in utero versus lactational exposure by crossfostering offspring of control, pairfed and ethanol treated dams. Brain Res 1984; 309: 209- 16.
22. Larsen JO. Stereology of nerve cross sections. J Neurosci Methods 1998; 85: 107-18.
23. Maldonado MD, Murillo-Cabezas F, Terron MP, Flores LJ, Tan DX, Manchester LC, et al. The potential of melatonin in reducing morbidity-mortality after craniocerebral trauma. J Pineal Res 2007; 42: 1-11.
24. Montoliu C, Valles S, Renau-Piqueras J, Guerri C. Ethanol-induced oxygen radical formation and lipid peroxidation in rat brain: effect of chronic alcohol consumption. J Neurochem 1994; 63: 1855-62.
25. Mooney SM, Miller MW. Time-specific effects of ethanol exposure on cranial nerve nuclei: gastrulation and neuronogenesis. Exp Neurol 2007; 205: 56-63.
26. Nordmann R, Ribiere C, Rouach H. Ethanol-induced lipid peroxidation and oxidative stress in extrahepatic tissues. Alcohol Alcohol 1990; 25: 231-7.
27. Nordmann R, Ribiere C, Rouach H. Implication of free radical mechanisms in ethanol-induced cellular injury. Free Radic Biol Med 1992; 12: 219-40.
28. Okatani Y, Okamoto K, Hayashi K, Wakatsuki A, Tamura S, Sagara Y. Maternal-fetal transfer of melatonin in pregnant women near term. J Pineal Res 1998; 25: 129-34.
29. Ozdemir D, Tugyan K, Uysal N, Sonmez U, Sonmez A, Acikgoz O, et al. Protective effect of melatonin against head trauma- induced hippocampal damage and spatial memory deficits in immature rats. Neurosci Lett 2005; 385: 234-9.
30. Pei Z, Ho HT, Cheung RT. Pre-treatment with melatonin reduces volume of cerebral infarction in a permanent middle cerebral artery occlusion stroke model in the rat. Neurosci Lett 2002; 318: 141-4.
31. Reiter RJ. Oxidative damage in the central nervous system: protection by melatonin. Prog Neurobiol 1998; 56: 359-84.
32. Reiter RJ, Acuna-Castroviejo D, Tan DX, Burkhardt S. Free radical-mediated molecular damage. Mechanisms for the protective actions of melatonin in the central nervous system. Ann N Y Acad Sci 2001; 939: 200-15.
33. Reiter RJ, Tan DX, Poeggeler B, Menendez-Pelaez A, Chen LD, Saarela S. Melatonin as a free radical scavenger: implications for aging and age-related diseases. Ann N Y Acad Sci 1994; 719: 1-12.
34. Riethmacher D, Sonnenberg-Riethmacher E, Brinkmann V, Yamaai T, Lewin GR, Birchmeier C. Severe neuropathies in mice with targeted mutations in the ErbB3 receptor. Nature 1997; 389: 725-30.
35. Riley EP, McGee CL, Sowell ER. Teratogenic effects of alcohol: a decade of brain imaging. Am J Med Genet C Semin Med Genet 2004; 127: 35-41.
36. Roebuck TM, Mattson SN, Riley EP. A review of the neuroanatomical findings in children with fetal alcohol syndrome or prenatal exposure to alcohol. Alcohol Clin Exp Res 1998; 22: 339-44.
37. Sowell ER, Mattson SN, Kan E, Thompson PM, Riley EP, Toga AW. Abnormal cortical thickness and brain-behavior correlation patterns in individuals with heavy prenatal alcohol exposure. Cereb Cortex 2008; 18: 136-44.
38. Tan DX, Manchester LC, Reiter RJ, Qi WB, Karbownik M, Calvo JR. Significance of melatonin in antioxidative defense system: reactions and products. Biol Signals Recept 2000; 9: 137-59.
39. Tunc AT, Aslan H, Turgut M, Ekici F, Odaci E, Kaplan S. Inhibitory effect of pinealectomy on the development of cerebellar granule cells in the chick: a stereological study. Brain Res 2007; 1138: 214-20.
40. Turgut M, Uyanikgil Y, Baka M, Tunc AT, Yavasoglu A, Yurtseven ME, et al. Pinealectomy exaggerates and melatonin treatment suppresses neuroma formation of transected sciatic nerve in rats: gross morphological, histological and stereological analysis. J Pineal Res 2005a; 38: 284-91.
41. Turgut M, Uysal A, Pehlivan M, Oktem G, Yurtseven ME. Assessment of effects of pinealectomy and exogenous melatonin administration on rat sciatic nerve suture repair: an electrophysiological, electron microscopic, and immunohistochemical study. Acta Neurochir (Wien) 2005b; 147: 67-77; discussion 77.
42. Watjen W, Michels G, Steffan B, Niering P, Chovolou Y, Kampkotter A, et al. Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis. J Nutr 2005; 135: 525-31.
43. Ziskind-Conhaim L. Physiological and morphological changes in developing peripheral nerves of rat embryos. Brain Res 1988; 470: 15-28.