IJAR.2025.128
Type of Article: Original Research
Volume 13; Issue 2 (June 2025)
Page No.: 9204-9219
DOI: https://dx.doi.org/10.16965/ijar.2025.128
The Possible Protective Effect of Vitamin C on the Developmental Toxicity of Polytherapy in Epilepsy in Pregnant Albino Mice
Miriam Ramzy Riad *1, Mennat Allah Abdefattah Saqr 2.
*1 Department of Human Anatomy and Emb zjryology, Alexandria University, Alexandria, Egypt. ORCiD: https://orcid.org/0009-0007-0993-6306
2 Department of Human Anatomy and Embryology, Alexandria University, Alexandria, Egypt and Beirut Arab University, Beirut, Lebanon. ORCiD: https://orcid.org/ 0009-0000-3009-0383
Corresponding Author: Miriam Ramzy Riad, PhD., Department of Human Anatomy and Embryology, Alexandria University, Alexandria, Egypt. E-Mail: MEREYAM.RIYADH@ALEXMED.edu.eg
ABSTRACT
Background: Epilepsy is one of the most prevalent chronic neurologic disorders. Antiepileptic drugs (AEDs) may impact fetal development, however pregnant women are unable to discontinue using them to avoid seizures that could endanger the mother and the fetus. Forty percent of women need polytherapy to control seizures which make their fetus at a higher risk to have deformities than for those who receive monotherapy. Both Lamotrigine and Gabapentin are among the new AEDs, that are relatively safe however when used combined there is increasing evidence to have adverse reproductive effects especially neurological congenital malformations. Vitamin C, a hydrophilic potent antioxidant, can cross the placental and blood brain barrier.
Aim: This research aims to assess the antioxidant effect of VitC on AEDs polytherapy.
Material and Methods: Forty-eight pregnant mice were categorized into 3 groups of 8 pregnant mice each: group I (control), group II (Lamotrigine 20 mg/kg and Gabapentin 200 mg/kg), group III (Vitamin C 40mg/kg with Lamotrigine and Gabapentin) from gestational days (GD) 6-15. On GD 18 they were sacrificed and the uteri and placentae examined and blood collected to assess oxidant markers while fetuses were removed for gross, skeletal and histopathological examinations.
Results: AEDs polytherapy caused significant signs of developmental toxicity as decreased maternal body weight, decrease in absolute and relative placental weight. Also, it caused significant decrease in number of implantations, live fetuses, fetal weights and increase in number of resorptions. MDA was increased, while SOD decreased thus oxidative stress. Gross examination showed reduced size of fetuses with not well-developed limbs. Stereomicroscopic findings of the skeleton revealed incomplete ossification of parts of the skeleton. Light microscopic examination of the frontal cortex showed loss of neurons with dark pyknotic nuclei and vacuolization. Ultrastructural examination revealed the nucleus with chromatin aggregates, clumps and crescents, vacuolated organelles in cytoplasm. Administration of Vitamin C caused significant increase in maternal and fetal parameters, decrease in oxidative stress, accompanied by marked improvements in gross and histological findings.
Conclusion: Vitamin C regimen appeared to be protective against AEDs induced developmental toxicity, therefore intake of Vitamin C as adjunctive therapy to AEDs should be mandatory in pregnant epileptic women.
Key words: Antiepileptic drugs, Vitamin C, Oxidative stress, developmental toxicity, congenital malformations, Electron Microscopy of the brain, Neuroprotection.
REFERENCES
[1]. Annegers JF, Dubinsky S, Coan SP, Newmark ME, Roht L. The incidence of epilepsy and unprovoked seizures in multiethnic, urban health maintenance organizations. Epilepsia 1999; 40(4):502-506.
https://doi.org/10.1111/j.1528-1157.1999.tb00748.x
PMid:10219279
[2]. Li Y, Meador KJ. Epilepsy and Pregnancy. Continuum (Minneap Minn) 2022; 28(1):34-54.
https://doi.org/10.1212/CON.0000000000001056
PMid:35133310 PMCid:PMC9642109
[3]. Barrett C, Richens A. Epilepsy and pregnancy: Report of an Epilepsy Research Foundation Workshop. Epilepsy Res 2003; 52(3):147-187.
https://doi.org/10.1016/S0920-1211(02)00237-1
PMid:12536051
[4]. Harden CL. Pregnancy and epilepsy. Continuum (Minneap Minn) 2014; 20(1 Neurology of Pregnancy):60-79.
https://doi.org/10.1212/01.CON.0000443837.95260.af
PMid:24492811 PMCid:PMC10563908
[5]. Etemad L, Moshiri M, Moallem SA. Epilepsy drugs and effects on fetal development: Potential mechanisms. J Res Med Sci 2012; 17(9):876-881.
[6]. Perucca E. Birth defects after prenatal exposure to antiepileptic drugs. Lancet Neurol 2005; 4(11):781-786.
https://doi.org/10.1016/S1474-4422(05)70224-6
PMid:16239185
[7]. Soltani D, Ghaffar Pour M, Tafakhori A, Sarraf P, Bitarafan S. Nutritional Aspects of Treatment in Epileptic Patients. Iran J Child Neurol 2016; 10(3):1-12.
[8]. Harden CL, Meador KJ, Pennell PB, et al. Practice parameter update: management issues for women with epilepsy–focus on pregnancy (an evidence-based review): teratogenesis and perinatal outcomes: report of the Quality Standards Subcommittee and Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology and American Epilepsy Society. Neurology 2009; 73(2):133-141.
https://doi.org/10.1212/WNL.0b013e3181a6b312
[9]. National Clinical Guideline Centre. The Epilepsies: The Diagnosis and Management of the Epilepsies in Adults and Children in Primary and Secondary Care. London: Royal College of Physicians; 2012.
[10]. de Groot MCH, Schuerch M, de Vries F, et al. Antiepileptic drug use in seven electronic health record databases in Europe: a methodologic comparison. Epilepsia 2014; 55(5):666-673.
https://doi.org/10.1111/epi.12557
PMid:24575970
[11]. Landmark CJ, Fossmark H, Larsson PG, Rytter E, Johannessen SI. Prescription patterns of antiepileptic drugs in patients with epilepsy in a nation-wide population. Epilepsy Res 2011; 95(1-2):51-59.
https://doi.org/10.1016/j.eplepsyres.2011.02.012
PMid:21435840
[12]. de Jong J, Garne E, de Jong-van den Berg LT, Wang H. The Risk of Specific Congenital Anomalies in Relation to Newer Antiepileptic Drugs: A Literature Review. Drugs Real World Outcomes 2016; 3(2):131-143.
https://doi.org/10.1007/s40801-016-0078-1
PMid:27398292 PMCid:PMC4914544
[13]. Dong Y, Wang S, Zhang T, et al. Ascorbic acid ameliorates seizures and brain damage in rats through inhibiting autophagy. Brain Res 2013; 1535:115-123.
https://doi.org/10.1016/j.brainres.2013.08.039
PMid:23994218
[14]. Pritchett-Corning KR, Chang FT, Festing MF. Breeding and housing laboratory rats and mice in the same room does not affect the growth or reproduction of either species. J Am Assoc Lab Anim Sci. 2009;48(5):492-8.
[15]. Behringer R, Gertsenstein M, Nagy KV, Nagy A. Selecting Female Mice in Estrus and Checking Plugs. Cold Spring Harb Protoc 2016; 2016(8).
https://doi.org/10.1101/pdb.prot092387
PMid:27480722
[16]. Chen Y, He X, Sun Q, Fang Z, Zhou L. Effect of lamotrigine on seizure development in a rat pentylenetetrazole kindling model. Brain Behav 2017; 7(7):e00727.
https://doi.org/10.1002/brb3.727
PMid:28729934 PMCid:PMC5516602
[17]. Traa BS, Mulholland JD, Kadam SD, Johnston MV, Comi AM. Gabapentin neuroprotection and seizure suppression in immature mouse brain ischemia. Pediatr Res 2008; 64(1):81-85.
https://doi.org/10.1203/PDR.0b013e318174e70e
PMid:18391849 PMCid:PMC2565570
[18]. Fernández I, Peña A, Del Teso N, Pérez V, Rodríguez-Cuesta J. Clinical biochemistry parameters in C57BL/6J mice after blood collection from the submandibular vein and retroorbital plexus. J Am Assoc Lab Anim Sci 2010; 49(2):202-206.
[19]. Wise LD, Beck SL, Beltrame D, et al. Terminology of developmental abnormalities in common laboratory mammals (version 1). Teratology 1997; 55(4):249-292.
https://doi.org/10.1002/(SICI)1096-9926(199704)55:4<249::AID-TERA5>3.0.CO;2-W
[20]. Aliesfehani T. Modified double skeletal staining protocols with Alizarinred and Alcian blue in laboratory animals. Ann Military Health Sci Res 2015; 13(2):76-81.
[21]. Menegola E, Broccia ML, Giavini E. Atlas of rat fetal skeleton double stained for bone and cartilage. Teratology 2001; 64(3):125-133.
https://doi.org/10.1002/tera.1055
PMid:11514942
[22]. Kimmel CA, Trammell C. A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals. Stain Technol 1981; 56(5):271-273.
https://doi.org/10.3109/10520298109067325
PMid:6171056
[23]. Gavaia PJ, Sarasquete C, Cancela ML. Detection of mineralized structures in early stages of development of marine Teleostei using a modified alcian blue-alizarin red double staining technique for bone and cartilage. Biotech Histochem 2000; 75(2):79-84.
https://doi.org/10.3109/10520290009064151
PMid:10941510
[24]. Suvarna S, Layton C, Bancroft J. The hematoxylins and eosin. In Banchroft’s Theory and practice of histological techniques. 7th edition. Edited by Suvarna S, Layton C, Bancroft J. London, UK: Churchil Livingstone; 2013: 123-189.
[25]. Bozzola JJ. Conventional specimen preparation techniques for transmission electron microscopy of cultured cells. Methods Mol Biol 2007; 369:1-18.
https://doi.org/10.1007/978-1-59745-294-6_1
PMid:17656743
[26]. Gedzelman ER, Meador KJ. Neurological and psychiatric sequelae of developmental exposure to antiepileptic drugs. Front Neurol 2012; 3:182.
https://doi.org/10.3389/fneur.2012.00182
PMid:23293628 PMCid:PMC3530733
[27]. Palac S, Meador KJ. Antiepileptic drugs and neurodevelopment: an update. Curr Neurol Neurosci Rep 2011; 11(4):423-427.
https://doi.org/10.1007/s11910-011-0194-y
PMid:21465150 PMCid:PMC6339508
[28]. Meador KJ, Pennell PB, Harden CL, et al. Pregnancy registries in epilepsy: a consensus statement on health outcomes. Neurology 2008; 71(14):1109-1117.
https://doi.org/10.1212/01.wnl.0000316199.92256.af
PMid:18703463
[29]. Vajda FJ, O’Brien T J, Hitchcock A, et al. Critical relationship between sodium valproate dose and human teratogenicity: results of the Australian register of anti-epileptic drugs in pregnancy. J Clin Neurosci 2004; 11(8):854-858.
https://doi.org/10.1016/j.jocn.2004.05.003
PMid:15519862
[30]. Cunnington MC, Weil JG, Messenheimer JA, Ferber S, Yerby M, Tennis P. Final results from 18 years of the International Lamotrigine Pregnancy Registry. Neurology 2011; 76(21):1817-1823.
https://doi.org/10.1212/WNL.0b013e31821ccd18
PMid:21606453
[31]. Neligan A, Renganathan R, Sweeney BJ. Management of epilepsy in the community. Ir Med J 2006; 99(2):52-54.
[32]. Vajda FJ, Graham J, Roten A, Lander CM, O’Brien TJ, Eadie M. Teratogenicity of the newer antiepileptic drugs–the Australian experience. J Clin Neurosci 2012; 19(1):57-59.
https://doi.org/10.1016/j.jocn.2011.08.003
PMid:22104350
[33]. Holmes LB, Harvey EA, Coull BA, et al. The teratogenicity of anticonvulsant drugs. N Engl J Med 2001; 344(15):1132-1138.
https://doi.org/10.1056/NEJM200104123441504
PMid:11297704
[34]. Adab N. Therapeutic monitoring of antiepileptic drugs during pregnancy and in the postpartum period: is it useful? CNS Drugs 2006; 20(10):791-800.
https://doi.org/10.2165/00023210-200620100-00001
PMid:16999450
[35]. Prakash, Prabhu LV, Rai R, et al. Teratogenic effects of the anticonvulsant gabapentin in mice. Singapore Med J 2008; 49(1):47-53.
[36]. Margulis AV, Hernandez-Diaz S, McElrath T, et al. Relation of in-utero exposure to antiepileptic drugs to pregnancy duration and size at birth. PLoS One 2019; 14(8):e0214180.
https://doi.org/10.1371/journal.pone.0214180
PMid:31381574 PMCid:PMC6681941
[37]. Afshar M, Hassanzadeh-Taheri MM, Moallem SA, Tamizi A, Golalipour MJ. Teratogenic effects of gabapentin on the skeletal system of Balb/C mice fetuses. Neurosciences (Riyadh) 2009; 14(3):239-244.
[38]. Quintero G C. Review about gabapentin misuse, interactions, contraindications and side effects. J Exp Pharmacol. 2017 Feb 9:9:13-21.
https://doi.org/10.2147/JEP.S124391
PMid:28223849 PMCid:PMC5308580
[39]. Alsanie WF, Abdelrahman S, Alhomrani M, et al. Prenatal Exposure to Gabapentin Alters the Development of Ventral Midbrain Dopaminergic Neurons. Front Pharmacol 2022; 13:923113.
https://doi.org/10.3389/fphar.2022.923113
PMid:35942222 PMCid:PMC9356305
[40]. Bittigau P, Sifringer M, Genz K, et al. Antiepileptic drugs and apoptotic neurodegeneration in the developing brain. Proc Natl Acad Sci U S A 2002; 99(23):15089-15094.
https://doi.org/10.1073/pnas.222550499
PMid:12417760 PMCid:PMC137548
[41]. Ullah I, Badshah H, Naseer MI, Lee HY, Kim MO. Thymoquinone and vitamin C attenuates pentylenetetrazole-induced seizures via activation of GABAB1 receptor in adult rats cortex and hippocampus. Neuromolecular Med 2015; 17(1):35-46.
https://doi.org/10.1007/s12017-014-8337-3
PMid:25429759
[42]. Furugen A, Ishiguro Y, Kobayashi M, et al. Involvement of l-type amino acid transporter 1 in the transport of gabapentin into human placental choriocarcinoma cells. Reprod Toxicol 2017; 67:48-55.
https://doi.org/10.1016/j.reprotox.2016.11.002
PMid:27818298
[43]. Charest PL, Vrolyk V, Herst P, et al. Histomorphologic Analysis of the Late-term Rat Fetus and Placenta. Toxicol Pathol 2018; 46(2):158-168.
https://doi.org/10.1177/0192623318755135
PMid:29400254
[44]. Hafez S. Comparative Placental Anatomy: Divergent Structures Serving a Common Purpose. Prog Mol Biol Transl Sci 2017; 145:1-28.
https://doi.org/10.1016/bs.pmbts.2016.12.001
PMid:28110748
[45]. Tetro N, Hamed R, Berman E, Eyal S. Effects of antiseizure medications on placental cells: Focus on heterodimeric placental carriers. Epilepsy Res 2021; 174:106664.
https://doi.org/10.1016/j.eplepsyres.2021.106664
PMid:34022522
[46]. Wong WT, Wong RO. Changing specificity of neurotransmitter regulation of rapid dendritic remodeling during synaptogenesis. Nat Neurosci 2001; 4(4):351-352.
https://doi.org/10.1038/85987
PMid:11276221
[47]. Stefovska VG, Uckermann O, Czuczwar M, et al. Sedative and anticonvulsant drugs suppress postnatal neurogenesis. Ann Neurol 2008; 64(4):434-445.
https://doi.org/10.1002/ana.21463
PMid:18991352
[48]. Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407(6805):770-776.
https://doi.org/10.1038/35037710
PMid:11048727
[49]. Venters HD, Dantzer R, Kelley KW. A new concept in neurodegeneration: TNFalpha is a silencer of survival signals. Trends Neurosci 2000; 23(4):175-180.
https://doi.org/10.1016/S0166-2236(99)01533-7
PMid:10717677
[50]. Bittigau P, Sifringer M, Ikonomidou C. Antiepileptic drugs and apoptosis in the developing brain. Ann N Y Acad Sci 2003; 993:103-114; discussion 123-104.
https://doi.org/10.1111/j.1749-6632.2003.tb07517.x
PMid:12853301
[51]. Ikonomidou C, Turski L. Antiepileptic drugs and brain development. Epilepsy Res 2010; 88(1):11-22. https://doi.org/10.1016/j.eplepsyres.2009.09.019.
https://doi.org/10.1016/j.eplepsyres.2009.09.019
PMid:19825509
[52]. Kośmider K, Kamieniak M, Czuczwar SJ, Miziak B. Second Generation of Antiepileptic Drugs and Oxidative Stress. Int J Mol Sci 2023; 24(4).
https://doi.org/10.3390/ijms24043873
PMid:36835284 PMCid:PMC9964930
[53]. Finsterer J. Toxicity of Antiepileptic Drugs to Mitochondria. Handb Exp Pharmacol 2017; 240:473-488.
https://doi.org/10.1007/164_2016_2
PMid:27590225
[54]. Waldbaum S, Patel M. Mitochondria, oxidative stress, and temporal lobe epilepsy. Epilepsy Res 2010; 88(1):23-45.
https://doi.org/10.1016/j.eplepsyres.2009.09.020
PMid:19850449 PMCid:PMC3236664
[55]. Miura S, Ishida-Nakajima W, Ishida A, et al. Ascorbic acid protects the newborn rat brain from hypoxic-ischemia. Brain Dev 2009; 31(4):307-317.
https://doi.org/10.1016/j.braindev.2008.06.010
PMid:18682317
[56]. Fuchs M, Viel C, Lehto A, Lau H, Klein J. Oxidative stress in rat brain during experimental status epilepticus: effect of antioxidants. Front Pharmacol 2023; 14:1233184.
https://doi.org/10.3389/fphar.2023.1233184
PMid:37767398 PMCid:PMC10520702
[57]. Santos LF, Freitas RL, Xavier SM, Saldanha GB, Freitas RM. Neuroprotective actions of vitamin C related to decreased lipid peroxidation and increased catalase activity in adult rats after pilocarpine-induced seizures. Pharmacol Biochem Behav 2008; 89(1):1-5.
https://doi.org/10.1016/j.pbb.2007.10.007
PMid:18096215
[58]. Sawicka-Glazer E, Czuczwar SJ. Vitamin C: a new auxiliary treatment of epilepsy? Pharmacol Rep 2014; 66(4):529-533. https://doi.org/10.1016/j.pharep.2014.02.016.
https://doi.org/10.1016/j.pharep.2014.02.016
PMid:24948051
[59]. Tomé Ada R, Ferreira PM, Freitas RM. Inhibitory action of antioxidants (ascorbic acid or alpha-tocopherol) on seizures and brain damage induced by pilocarpine in rats. Arq Neuropsiquiatr 2010; 68(3):355-361.
https://doi.org/10.1590/S0004-282X2010000300005
PMid:20602035
[60]. Huang HY, Appel LJ, Croft KD, Miller ER, 3rd, Mori TA, Puddey IB. Effects of vitamin C and vitamin E on in vivo lipid peroxidation: results of a randomized controlled trial. Am J Clin Nutr 2002; 76(3):549-555.
https://doi.org/10.1093/ajcn/76.3.549
PMid:12197998
[61]. Liu J, Wang A, Li L, Huang Y, Xue P, Hao A. Oxidative stress mediates hippocampal neuron death in rats after lithium-pilocarpine-induced status epilepticus. Seizure 2010; 19(3):165-172.
https://doi.org/10.1016/j.seizure.2010.01.010
PMid:20149694
[62]. Mahmoudabadi MM, Rahbar AR. Effect of EPA and vitamin C on superoxide dismutase, glutathione peroxidase, total antioxidant capacity and malondialdehyde in type 2 diabetic patients. Oman Med J 2014; 29(1):39-45.
https://doi.org/10.5001/omj.2014.09
PMid:24498481 PMCid:PMC3910411
