1. Histological brain alterations following prenatal methamphetamine exposure in rats Chun Cui, Hiromi Sakata-Haga, Ken-ichi Ohta, Manami Nishida, Mikio Yashiki, Kazuhiko Sawada and Yoshihiro Fukui Department of Anatomy and Developmental Neurobiology, Institute of Health Biosciences, The University of Tokushima Graduate School Journal compilation © 2006 Japanese Teratology Society; 46, 180–187

2. Methamphetamine is a central nervous system stimulant and one of the most abused drugs in the USA and some Asian countries including Japan. Recently, its use by pregnant women has increased ( Vega et al. 1993 ). Since methamphetamine readily goes through the placenta ( Burchfield et al. 1991; Won et al. 2001 ), it has been suspected to disturb the normal development of fetuses in mothers who abuse this drug during pregnancy ( Marwick 2000 ).

4. Exposure to amphetamine or methamphetamine in utero can cause physical and behavioral abnormalities in infants, such as Growth reduction ( dixon & bejar 1989; plessinger 1998 ) Cleft palate ( thomas 1995; plessinger 1998 ) Cardiac defects ( nora et al. 1970 ) Aggressive behavior ( eriksson et al. 1989 ) Peerrelated problems ( eriksson & zetterstrom 1994 ) Low IQ ( billing et al. 1985 ) Learning problems ( cernerud et al. 1996 )

5. The potential toxicity of methamphetamine for fetuses were found after prenatal exposure to amphetamine or methamphetamine in animal models Exencephaly ( nora et al. 1965; kasirsky 1971; fein et al. 1987; yamamoto et al. 1992 ), Eye abnormalities ( nora et al. 1965; kasirsky 1971; fein et al. 1987; yamamoto et al. 1992; acuff-smith et al. 1996 ), Cardiovascular defects ( nora et al. 1970; fein et al. 1987; inoue et al. 2004), Skeletal malformations ( nora et al. 1965; kasirsky 1971; fein et al. 1987; yamamoto et al. 1992 )

6. The developing brain seems to be one of the primary targets of developmental toxicities of amphetamine and methamphetamine. Severe brain malformations in a human infant exposed to amphetamine in utero, such as Exencephaly, anomalies of gyria and hemorrhage ( matera et al. 1968 ). Decrease of head circumference( little et al. 1988; dixon & bejar 1989 ) Volume decrease of subcortical area ( chang et al. 2004 ) Abnormal energy metabolism in children ( smith et al. 2001 )

7. in vitro studies using rat whole embryo culture technique have indicated that amphetamines can disrupt brain morphogenesis resulting in: Microcephaly Neural tube defects Tortuous spinal cord Derangement Necrosis of the neuroepithelial tissue ( Yamamoto et al. 1995, 1998 ).

8. MATERIALS AND METHODS Animals and methamphetamine treatment Ten-week-old female Sprague- Dawley rats were purchased from Japan-SLC ( Hamamatsu, Japan ). Methamphetamine hydrochloride ( Philopon, Dainippon Pharma- ceuticals, Osaka, Japan ) was dissolved in saline at 5 mg/ml and then subcutaneously administered at a dose of 5 mg/kg/day to rats, and their blood was collected at 1, 3, 12, and 24 h after subcutaneous injection. Serum methamphetamine concentrations were determined by gas chromatography-mass spec- trometry.

9. As the control, the second group [SAL] was injected with saline vehicle only at a dose of 1 ml/ kg/day. Pregnant rats of both groups received a daily injection at around 10.00 am from GD 10 to GD 20, and they were weighed just before given the daily injection. On GD 21, pregnant rats were anesthetized with ether, and then their fetuses were removed by cesarean section and weighed. Fetal brains were removed from the skull, immersed in Bouin’s fluid without acetic acid overnight, and weighed. Other fetal brains were quickly frozen in liquid nitrogen, and then kept at -80°C until the Western blot analysis.

10. Histology Fixed fetal brains were dehydrated with ethanol series, embedded in paraffin, and serially sectioned in a frontal plane at 5 µm thickness. Every 20 sections (100 µm thickness) were stained with hematoxylin and eosin (HE staining) and checked for histological alterations using a light microscope. When histological changes were found, neighboring sections were examined by both HE staining and immunostaining analysis to obtain an overall grasp of the malformation.

11. For immunohistochemical examinations, sections were irradiated with microwaves for 5 min in 10 mM citrate buffer (pH 6.0), and then incubated with antibody against laminin ( 1:200, Sigma [Product No. L9393], St. Louis, MO ) overnight at 4°C. Immunoreactions were visualized using an immunoperoxidase method employing avidin-biotin-peroxidase complex ( elite ABC kit, Vector, Burlingame, CA ). For fluorescent staining, sections were incubated with 1:200 dilution of anti-rabbit IgG linked to Alexafluor 594 ( Molecular Probe, Eugene, OR ) at 37°C for two hours following incubation with the primary antibody.

12. Western blotting Frozen fetal brains were homogenized in 10 mM HEPES containing protease inhibitor cocktail (Compete, Roche, Penzberg, Ger- many) on ice and centrifuged at 5000 g for 10 min at 4°C, after which the supernatant was collected. The protein concentration of each sample was determined using BCA protein assay ( Pierce, Rockford, IL )....... Band intensity was quan- tified by scanning film and comparing average pixel intensity of each band between two experimental groups using NIH image (version 1.62) software (Research Service Branch of the NIMH, National Institutes of Health, Bethesda, MD).

13. RESULTS Maternal body weight gain and litter size

15. Histological changes in the brain We found various histological brain alterations in MA subjects, including microgyria, ectopic cell cluster, and hemorrhage. Microgyria, abnormal folding of the cortical surface, was found solitarily or multifocally.

16. Frontal section with H&E staining Marginal zone of the cortex leptomeninx

17. Cerebral cortex

18. Immunostaining of laminin H&E staining

19. Immunostaining of laminin H&E staining

20. Immunostaining of laminin H&E staining

21. Immunostaining of laminin H&E staining

22. Hemorrhage cerebral cortex subependymal zone

23. lateral ventricles

24. Western blotting There was no difference in laminin expression between the two groups.

25. DISCUSSION Corresponding to previous studies ( Acuff-Smith et al. 1996; Slam- berova et al. 2006 ), methamphetamine treatment during pregnancy resulted in lower body weight gain. It could be caused by the pharmacological action of methamphetamine, which increases the metabolism and decreases appetite, although we did not check food intake in the present study

26. The lower body weight gain of dams during pregnancy predicted the lower body weight of their fetuses, and in fact our MA fetuses showed significantly lower body weight compared to SAL controls of either sex. Some reports have shown a reduction of head circumference in prenatally methamphetamine- exposed human individuals ( Little et al. 1988; Dixon & Bejar 1989 ).

27. The histological abnormalities of brain seen in our MA subjects, such as microgyria, ectopia are commonly observed in developmental disorders. For example, rodent models for perinatal exposure to ethanol ( Kotkoskie & Norton 1988; Sakata-Haga et al. 2002 ) Methylmercury ( Kakita et al. 2001 ) have shown similar brain malformations.

28. Prenatal or early postnatal administration of 6-hydroxydopamine (6-OHDA), a well-known neurotoxin for dopaminergic neurons, also induces meningeal cell death resulting in ectopias sprouting from the brain surface ( Lidov & Molliver 1982 ) and fusions of cerebellar folia ( Sievers et al. 1981 ) in rats.

29. The brain hemorrhage reported among human amphetamines abusers ( Moriya & Hashimoto 2002; Inamasu et al. 2003 ) might be due to the cardiovascular effects of amphetamines, resulting from high blood pressure or vasoconstriction. Brain hemorrhage has also been reported in children prenatally exposed to methamphetamine ( Dixon & Bejar 1989 ) and caudal hematomas were found in chick embryos after amphetamine exposure ( Kolesari & Kaplan 1979 ).

30. Interestingly, Stek and colleagues reported that maternal metham- phetamine administration in sheep induced an increase in blood pressure not only in dams but also in their fetuses ( Stek et al. 1995 ). In the present study, various types of hemorrhage were found in the MA fetuses. Our findings confirmed that brain hemorrhage is one of the characteristic aspects following prenatal exposure to methamphetamine.

31. In conclusion, prenatal methamphetamine exposure can induce histological brain alterations, which might be related to the psychiatric or behavioral abnormalities seen in children with a history of methamphetamine exposure in utero. Although abnormality of the leptomeninx could contribute to cortical dysplasia, the exact mechanism of morphological anomalies after prenatal exposure to methamphetamine remains unclear.

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