Original Contributions Valproic acid-induced alterations in growth and neurotrophic factor G. D. Bennett a , B. Wlodarczyk a,b , J. A. Calvin c , J. C. Craig a , R. H. Finnell a, * a Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, TX 77843, USA b National Veterinary Research Institute, Pulawy, Poland c Department of Statistics, Texas A&M University, College Station, TX 77843, USA Received 10 November 1998; received in revised form 10 August 1999; accepted 5 September 1999 Abstract Although the teratogenicity of valproic acid (VPA) has been well established, the mechanism(s) by which this anticonvulsant drug induces malformations remains controversial. Using the combined molecular techniques of in situ-transcription (IST) and antisense RNA (aRNA) amplification we analyzed VPA-induced alterations in the gene expression for 10 genes within the neural tubes of embryos from two murine strains that have been shown to differ in their susceptibility to VPA-induce neural tube defects (NTD). Pregnant dams from both SWV (susceptible) and LM/Bc (resistant) strains were either treated with saline (control) or VPA (600 mg/kg) on gestational day (GD) 8:12 (day:hour). Neural tubes were isolated from control or VPA exposed embryos at three gestational time points, which represented the beginning (GD 8:18), middle (GD 9:00), and end (GD 9:12) of neural tube closure (NTC) in both of these murine strains. Using univariant statistics we demonstrated that in LM/Bc embryos with NTDs, the expression of bdnf, ngf, and trk, ngf-R were significantly elevated at all three time points, and the cytokine, cntf was significantly decreased at GD 9:00. In contrast, the major gene alterations observed in SWV embryos were a significant increase in tfg and tgf1–3 at GD 9:00. In an effort to better define the more intricate interactions between VPA exposure and the expression of these genes, we analyzed our data using Principal Component Analysis. The results from this analysis demonstrated that embryos from these two stains behaved differently, not only in response to a VPA exposure, but also under control conditions, which may explain the multifactorial nature of NTDs in these mice. © 2000 Published by Elsevier Science Inc. All rights reserved. Keywords: Valproic acid; Neural tube defects; Animal model; In situ-transcription; Antisense RNA amplification; Gene expression; Principal Component Analysis 1. Introduction Valproic acid (VPA) is a widely prescribed anticonvul- sant drug that has been demonstrated to be efficacious in a wide spectrum of convulsive disorders. Although it is con- sidered to be relatively safe, VPA has been linked to rare but serious hepatotoxicity as well as to specific developmental malformations [1– 4]. In humans, in utero exposure to VPA has been associated with neural, craniofacial, cardiovascu- lar, and skeletal defects [5–7]. The developing nervous system seems to be particularly sensitive to this anticonvul- sant drug, for it has been estimated that 1 to 2% of the infants exposed to VPA in utero will develop a neural tube defect (NTD), specifically spina bifida, which is 10 to 20 times the occurrence rate of this defect within the general population [5,8]. Humans are not unique in their response to VPA, for this drug has also been shown to induce various neural, renal, cardiac, urogenital, and/or skeletal anomalies in numerous laboratory animals, including rodents [9 –14], rabbits [15], and nonhuman primates [16,17]. Despite the occurrence of growth retardation, skeletal and soft tissue anomalies, and an increased incidence of embryolethality in these labora- tory animals, murine embryos remain the only animal model that is susceptible in vivo to VPA-induced NTDs. Even though in utero VPA exposure has been associated with an increased risk for NTDs in both humans and ro- dents, a clear understanding of the mechanism(s) of how VPA initiates the cascade of molecular and biochemical events that ultimately lead to aberrant neurulation remains unknown. The process of neurulation involves a series of morpho- * Corresponding author. Tel.: +001-402-559-4056; fax: +001-402- 559-5397. E-mail address: rfinnell@UNMC.edu (R.H. Finnell) www.elsevier.com/locate/reprotox Reproductive Toxicology 14 (2000) 1–11 0890-6238/00/$ – see front matter © 2000 Published by Elsevier Science Inc. All rights reserved. PII: S0890-6238(00)00064-7