Correlation of axon size and myelin occupancy in rats prenatally exposed to methamphetamine

Abstract

The abuse of methamphetamine (MA) and other psychostimulants is a social and medical problem. In particular, the use of these drugs by pregnant women results in an increased number of children exposed prenatally to psychostimulants. Our previous work has demonstrated that prenatal exposure to MA affects the normal development of the rat visual system due to alterations of biochemical mechanisms and oxidative stress. It was also demonstrated that prenatal exposure to MA affects the dopaminergic system of the rat retina and optic nerve (ON) myelination. The present work was conducted to evaluate the effects of prenatal exposure to MA on the development of the ON in terms of axon growth and the myelin sheath. Pregnant female rats were given 5 mg/kg/day MA, subcutaneously (s.c.), in 0.9% saline from gestational day (GD) 8 to 22. The pair-fed control group was injected s.c. with an isovolumetric dose of 0.9% saline. Qualitative analysis was performed using representative electron ultramicrographs. Quantitative analysis was performed at an electron microscopic level on ON cross sections; parameters measured included myelinated/unmyelinated ratio, outer axon mean area, inner axon mean area, myelin mean area, myelin occupancy and distribution of axons by size. The ON of prenatally MA-exposed rats presented a higher rate of deformed axons and slighter lamellar separation. At PND 21, the average outer axon area of MA-treated males was significantly reduced. The average inner axon area only showed a significant difference between MA and control males for axons with an area of less than 0.3 µm². The average myelin area of MA-treated males was significantly reduced, and in MA-treated females was only significantly reduced in axons with an area of less than 0.3 µm². The percentage of myelin occupancy was significantly affected in MA-treated males, and in MA-treated females in the group of axons with an area of more than 0.3 µm². At PND 14 no significant differences were found between MA and control groups. The spectrum of ON myelinated axon size of MA-treated animals was shifted to the left at PND 14 and PND 21 for both genders. These results are in agreement with previous animal studies of prenatal and perinatal exposure to drugs of abuse. Taken together, these data indicate that the ON is vulnerable to early exposure to MA which causes developmental changes and may interfere with the functioning of the visual system.

Introduction

Illicit psychostimulant drugs, such as cocaine and amphetamines, represent a highly addictive class of compounds, and in recent years there has been a dramatic increase in the use of some of these drugs. The 2006 World Drug Report of the Office of Drug and Crime of the United Nations states that, worldwide, around 35 million people abuse amphetamine and amphetamine-like substances (Hammond, 2006). It is well known that exposure to drugs and alcohol during development of the central nervous system (CNS) can have profound effects on morphological, physiological, and behavioral parameters. Methamphetamine (MA), like other psychostimulants, acts indirectly in the sympathomimetic system, causing a massive release of dopamine in the brain. This is achieved by inhibition of both monoamine oxidase activity and dopamine reuptake. Dopamine reacts with molecular oxygen to form reactive oxygen species, such as superoxide, hydroxyl free radicals and hydrogen peroxide (Graham, 1978). High doses of MA are toxic to dopaminergic neurons, resulting in dopaminergic terminal degeneration, dopamine depletion, and a decrease in dopamine transporter numbers (Giros et al., 1996, Golembiowska and Zylewska, 1998, Graham, 1978). MA also induces an acute release of serotonin, followed by a reduction of its synthesis and metabolism (Frost and Cadet, 2000).

MA is increasingly used as a drug of abuse due to its relatively uncomplicated production and its low price compared with other drugs, such as cocaine and heroin (Marwick, 2000, Vega et al., 1993). Social differences with regard to drug and alcohol habits become even more relevant when gender is taken into account. Women of reproductive age must be considered as uniquely at risk for drug- and/or alcohol-induced birth defects. Indeed, women who abuse drug or alcohol during pregnancy give birth to children who show a pattern of adverse effects, which in their most profound manifestation are known as fetal alcohol syndrome or “drug syndrome” (Stromland and Pinazo-Duran, 2002). Statistics show that only 17% of American women who take illicit drugs are primary MA-abusers, but 38% had used it during pregnancy (Marwick, 2000). The reason why women often replace other drugs with MA during pregnancy might be related to its anorectic property coupled with its ability to increase energy. The use of MA by pregnant women has led several research groups to investigate the effects of this psychostimulant drug on the perinatal development of the offspring (Frost and Cadet, 2000, Melo et al., 2005, Melo et al., 2006a, Melo et al., 2006b, Rodrigues et al., 2006, Silva-Araujo and Tavares, 1996, Slamberova et al., 2006, Summavielle et al., 2000). The abuse of MA during pregnancy is associated with developmental alterations in children, including low birth weight, cleft palate, reduced head circumference and cerebral hemorrhage (Plessinger, 1998, Smith et al., 2003, Smith et al., 2001). Children prenatally exposed to amphetamine may develop neurobehavioral alterations, including slowed learning, impaired performance on psychometric tests, aggressive behavior and poor social adjustment (Cernerud et al., 1996, Eriksson et al., 2000).

The development of the human visual system is extremely vulnerable to exposure to neuroactive drugs (Dominguez et al., 1991, Good et al., 1992, Hajnal et al., 2004); for example, mild-to-severe eye defects, such as folded retina and anophthalmia, have been reported to occur in the offspring of rodents after prenatal exposure to MA (Acuff-Smith et al., 1996). Previous work from our group has demonstrated that MA exposure during development induces changes in the rat eye due to alterations in biochemical mechanisms and increased oxidative stress (Melo et al., 2005). We have also shown that the dopaminergic system of the rat retina is affected by early exposure to MA (Melo et al., 2006b, Rodrigues et al., 2006), together with the myelination of the rat optic nerve (Melo et al., 2006a).

The optic nerve is a suitable structure to analyze developmental changes in the visual system after prenatal exposure to MA. It is a well-characterized CNS structure, which has been used in numerous studies (Crespo et al., 1985, Tennekoon et al., 1977). The optic nerve is simple, relatively homogeneous and is a particularly vulnerable structure to insults such as drugs of abuse and alcohol (Harris et al., 2000, Melo et al., 2006b, Pinazo-Duran et al., 1997).

In the present study, we examined the effect of MA (5 mg/kg/day) administrated during gestation on the development of the rat optic nerve. In addition to analyzing qualitative changes in the optic nerve of prenatally MA-exposed rats, we also performed quantitative analysis. The quantitative data function supports the qualitative findings but importantly also provides a baseline pattern to the optic nerve damage, which will lead to a better understanding of the ophthalmological alterations found in children born from MA-abusing women.