[1] 12w Myelination changes in the rat optic nerve after prenatal exposure to methamphetamine

[1] 305w The use of psychostimulants during adolescence and early adult life has increased in recent years. It is known that these substances affect the sensory systems, and the optic nerve has been shown to be a target tissue. This work was conducted to evaluate the effects of prenatal exposure to methamphetamine (MA) on the developmental pattern of the rat optic nerve. Pregnant female rats were given 5 mg/kg body weight/day MA, s.c., in 0.9% saline from gestational days 8 to 22. The control group was injected with an isovolumetric dose of 0.9% saline. Animal model parameters, such as gestational body weight evolution, food intake and pups parameters were registered. The offspring were sacrificed at postnatal days (PND) 7, 14 and 21. Morphometric analyses were performed at light and electron microscopic levels on optic nerve cross sections; parameters measured included optic nerve diameter and area, axonal density, total number of axons and myelin thickness. Myelin basic protein (MBP) was measured by western blotting in optic nerve samples at PND14 and PND21. The animal model parameters, such as maternal and pup weight, showed no significant differences between MA and control groups. Optic nerve diameter was smaller at PND7 in the male MA group and in both male and female MA groups at PND21. The mean cross-sectional area was smaller at PND14 in the male MA group and in both male and female groups at PND21. The total number of myelinated axons did not vary between groups at any of the studied ages. The myelin thickness of the axons in MA-treated females was thinner when compared with the respective control group at PND21. No other differences were found concerning myelin thickness. There was a reduction of MBP protein expression in MA-injected females at PND14 and PND21. The combined results suggest that prenatal exposure to MA affects the myelination process.

[1] 85w Exposure to illicit drugs of abuse and alcohol during pregnancy is known to induce a range of adverse effects on the offspring and the central nervous system (CNS) is a particular target of these substances. Psychostimulants elevate the extracellular dopamine level by inhibiting its reuptake by dopamine transporters and also, in the case of damphetamine, by promoting reverse transport of dopamine. Some of the neurological complications related to exposure to psychostimulants have been attributed to dopamine-mediated vasoconstriction effects (Volkow et al., 1997;Wang et al., 1990).

[2] 238w The use of methamphetamine (MA) is becoming increasingly widespread in many developed countries because of its relatively uncomplicated production and low price compared with other drugs such as cocaine and heroin (Marwick, 2000). MA acts indirectly on the symphathomimetic system, causing a massive release of dopamine in the brain, while simultaneously preventing dopamine degradation by inhibiting monoamine oxidase activity and dopamine uptake (Golembiowska and Zylewska, 1998;Graham, 1978). Dopamine reacts with molecular oxygen to form reactive oxygen species, such as superoxide and hydroxyl-free radicals and hydrogen peroxide (Graham, 1978), which are known to be neurotoxic. Chronic MA administration has been shown to cause a long-term deficit in the dopamine system in 60-dayold rats (Pu and Vorhees, 1993) and to have a moderately persistent effect in 20-day-old rats (Kokoshka et al., 2000). In adults, MA also induces an acute, massive release of serotonin, followed by a reduction in the synthesis and metabolism of serotonin and its transporters (cf. Frost and Cadet, 2000). In addition, MA exacerbates the effect of other toxic substances, such as kainic acid in the adult rat retina (Rodrigues et al., 2004). A single administration of MA has been shown to cause loss of dopamine and dopamine-related markers (Fleckenstein et al., 1997;Melega et al., 1997;Seiden, 1985;Seiden and Sabol, 1996). Moreover, biochemical and behavior changes have been observed in rodents in many MA studies, which sometimes persist longterm (Davidson et al., 2005;Ricaurte et al., 1982;Sonsalla et al., 1996).

[3] 177w The effects of substituted amphetamines during pregnancy have not been well characterized in humans, even though the use of some of these drugs, for example MA, increased dramatically during the 1990s (Johnston et al., 2004a,b). The increased use of MA and other psychostimulants has led several researchers to utilize a variety of experimental models in order to investigate the effects of these psychotropic substances on the perinatal development of the offspring (Frost and Cadet, 2000;Gomes-da-Silva et al., 1998, 2002;Melo et al., 2005;Silva et al., 1995;Silva-Araujo et al., 1991, 1996a;Silva-Araujo and Tavares, 1996;Slamberova et al., 2005;Summavielle et al., 2000Summavielle et al., , 2002;;Tavares et al., 1996). Information generated about the effects of prenatal exposure to MA is still quite limited and results are not always consistent. Several factors have to be considered when comparing different studies, such as the experimental models used, the doses and periods of exposure to MA and the animal strains employed. MA exposure during pregnancy induces changes in rat eye and retinal development probably by biochemical mechanisms and oxidative stress (Melo et al., 2005).

[4] 132w In humans, the use of amphetamines and particularly MA during pregnancy is associated with developmental defects in children, including low birth weight, cleft palate, reduced head circumference and cerebral hemorrhage (Plessinger, 1998;Smith et al., 2001Smith et al., , 2003)). Children exposed prenatally to amphetamine may exhibit neurobehavioral alterations such as slowed learning, impaired performance in psychometric tests, aggressive behavior and poor social adjustment (Cernerud et al., 1996;Eriksson et al., 2000a,b). Within this context, it has also been demonstrated that the developing visual system is extremely vulnerable to the effects of prenatal exposure to neuroactive drugs (Dominguez et al., 1991;Good et al., 1992;Hajnal et al., 2004). Eye defects, such as anophthalmia and folded retina, have been reported in rodent offspring after early and late exposure occurs to MA (Acuff- Smith et al., 1996).

[5] 181w The optic nerve is an ideal structure to analyze when testing the hypothesis that maternal exposure to MA induces changes in the development of visual circuits. Several aspects of the optic nerve make it suitable for developmental analysis: (a) it is a well-characterized CNS structure, which has been studied in numerous qualitative and quantitative studies (Crespo et al., 1985;Tennekoon et al., 1977); (b) its structure is laid out in a simple and relatively homogeneous pattern (Crespo et al., 1985;Forrester and Peters, 1967;Kuwabara, 1975;Tennekoon et al., 1977); (c) it is particularly vulnerable to toxic and metabolic insults such as alcohol (Pinazo-Duran et al., 1997;Samorajski et al., 1986) and undernutrition (Bedi and Warren, 1983;Wiggins and Ruiz, 1990); (d) at birth, the entire optic nerve is unmyelinated, yet in the adult, most optic nerve axons are myelinated (Forrester and Peters, 1967;Kuwabara, 1975;Tennekoon et al., 1977); and (e) during the early postnatal development there is a reduction in the number of optic nerve axons (Forrester and Peters, 1967;Kuwabara, 1975;Tennekoon et al., 1977), a phenomenon that also occurs in other CNS zones (Cowan et al., 1984).

[6] 70w In the present study, performed in rats, we investigated the effects of chronic maternal exposure to MA on the development of the optic nerve in offspring. Specifically, we studied the effects of prenatal MA exposure on axon maturation and myelination, using morphological, morphometrical and biochemical approaches. In this study, we have tried to provide a basis for understanding the pattern of visual alterations found in children born from MA-abusing mothers.

[1] 102w Optic nerve development starts at an early stage following gastrulation and is completed in the postnatal period of life with the maturation of the cell lineages and the end of myelination (Hirose and Bass, 1973;Takayama et al., 1991). Different xenobiotics, including amphetamines and alcohol, can alter the morphogenesis of the visual system at particular time points of ontogeny (Pinazo-Duran et al., 1997;Silva-Araujo et al., 1991;Stromland and Pinazo-Duran, 1994). It is known that exposure to psychostimulants during early stages of development is capable of producing deficits and distortions in the organization of the CNS, including in the myelination process (Wiggins et al., 1984).

[2] 193w In the present study, an experimental regime of 5 mg/kg/ day MA was chosen. We selected this dose based on the findings of Weissman and Coldecott-Hazard, who used the same concentration and showed altered locomotor and exploratory behaviors (Weissman and Caldecott-Hazard, 1993). The dose selected is in the range of doses modeled for MA users. The results should therefore have some applicability to humans (Cho et al., 2001). Several previous prenatal studies used extremely high doses of MA (up to 50 mg/kg as free base) leading, for instance, to eye defects (i.e., anophthalmia, microphthalmia and folded retina) and a high incidence of delivery failure and maternal deaths relative to controls (Acuff- Smith et al., 1992Smith et al., , 1996)); these effects were unwanted in this study. It is likely that the relatively low dose of MA we used together with the pair-fed control contributed to us being unable to find any significant differences between groups on pups body weight. There are reports of impairments in weight of pups after prenatal exposure to 5 mg/kg/day of methamphetamine, but these studies were performed without a pairfed control group (Martin et al., 1976;Slamberova et al., 2005).

[3] 67w Fig. 3 -Developmental expression of myelin basic protein (MBP) (19 kDa) in optic nerves of control rats and rats prenatally exposed to methamphetamine (MA). F21C-21-day-old control female rat; M21C-21-day-old control male rat; F21MA-21-day-old MA female rat; M21MA-21-day-old MA male rat; F14C-14-day-old control female rat; M21C-14-day-old control male rat; F14MA-14-day-old MA female rat; M14MA-14-day-old MA male rat. The results represent the mean ± standard deviation (*P < 0.01).

[4] 173w Table 4 -Mean myelin thickness of both large and small axons of optic nerves from male and female rats prenatally exposed to methamphetamine on PND14 and PND21 and the respective controls The impairment in cerebellum weight after prenatal exposure to MA shown in this study is in agreement with previous studies on prenatal exposure to cocaine and amphetamine (Tavares and Silva, 1996). This study demonstrates that prenatal exposure to even a low dose of MA induces developmental changes in the optic nerve, with alterations in the cross section and the mean diameter of the optic nerve between exposed and control groups. These differences start to be evident in the second week of the postnatal period. In rats prenatally exposed to ethanol, this impairment is observed from the late prenatal period (embryonic day 21) until PND28 (Pinazo-Duran et al., 1997). In mice prenatally treated with a single dose of ethanol, at GD11 or GD12, the same pattern is observed but differences are only significant at week 15 (Parson and Sojitra, 1995;Parson et al., 1995).

[5] 121w In this study, it is shown that prenatal exposure to MA did not affect significantly the total number of myelinated axons. These results are in agreement with data published on neonatal exposure to cocaine and amphetamine in rats (Silva-Araujo et al., 1991) but are contrary to experiments which investigated prenatal exposure to ethanol, where impairment of axonal number was observed. In fact, there was a significant reduction in the number of myelinated optic axons at different postnatal ages (PND14, PND21 and PND28) (Pinazo-Duran et al., 1997). Other studies on pre-or postnatal exposure of rats to ethanol (Lancaster et al., 1982;Melcer et al., 1995), or even late prenatal exposure in humans (Zoeller et al., 1994), also revealed a significant decrease in myelination.

[6] 123w Elaboration of myelin occurs later in development than neuronal proliferation and migration of neuronal proliferation (Miller and Ono, 1998). Malnutrition can affect myelination, and it is more evident in the late gestational period in humans and postnatally in rodents. Alterations in myelination processes can have a persistent effect resulting from early developmental malnutrition or an alteration of the thyroid normal function even though somatic growth may recover (Wiggins, 1982). Effects on myelination are observed after malnutrition or endocrine alterations during the developmental peak, which occurs in the second week of postnatal life in rats, or during the last trimester in humans. It is known that myelination continues until adolescence in both rodents (Wiggins, 1982) and humans (Hunter et al., 1997;Paus et al., 1999).

[7] 138w It is known that myelinogenesis in the rat optic nerve starts around PND7. Differentiation of oligodendrocytes follows axogenesis and is believed to be mediated by complex trophic signals between neurons. Essentially, oligodendrocytes insulate and myelinate axons of neurons in the CNS. After PND7, a rapid increase in myelinization is observed (Dangata and Kaufman, 1997). In this study, almost all axons were unmyelinated at PND7, and between PND14 and PND21 we observed a rapid raise in myelin thickness. The morphometric analysis in this study showed an impairment of myelin production in the optic nerve in the female group at PND21. Other studies, using experimental models of prenatal exposure of ethanol or cocaine in rats, also suggest a decrease in the optic nerve myelin ensheathment, but without gender differences (Harris et al., 2000;Pinazo-Duran et al., 1997, 2005;Silva-Araujo et al., 1995a).

[8] 79w In the present study, support for the myelin thickness results comes from the western blot experiments, where a significantly lower MBP content was seen at postnatal day 21 in the female MA group. We suggest that this impairment starts even earlier, at PND14, where a lower MBP content was also evident in the female MA group. Pinazo-Duran and colleagues (1993) also reported a lower MBP content in rat optic nerve after prenatal exposure to ethanol (Pinazo-Duran and colleagues, 1993).

[9] 78w The significance to humans of the optic nerve hypoplasia and the reduction of the myelin ensheathment observed in rats after prenatal exposure to MA are not yet clear. In the best-characterized fetal alcohol syndrome, the optic nerve head hypoplasia and visual impairment observed in children prenatally exposed to alcohol (Stromland, 1985;Stromland et al., 1991) are supported by the reduced optic nerve cross-sectional area and the delayed myelination observed in rats (Harris et al., 2000;Pinazo-Duran et al., 1997, 2005).

[10] 53w In conclusion, the data herein support the view that the structural organization of the optic nerve is affected by early chronic exposure to methamphetamine, as has been reported earlier for cocaine and amphetamine (Silva-Araujo et al., 1991). These alterations to optic nerve development may interfere with the normal functioning of the visual system.

[11] 86w Quantitative studies were performed in 12 animals (6 males and 6 females) per group and treatment (PND7 control-C, PND14 C, PND21 C, PND7 MA, PND14 MA and PND21 MA), selected at random from 3 different litters. Each optic nerve cross section (at least 4 per animal) was considered. Nerves were photographed with a 20× objective of an Olympus Light Microscope BX50, captured by an Olympus digital camera and processed using the DP soft software. Optic nerve cross-sectional areas were obtained discarding the outline of the perineurium.

[12] 30w For transmission electron microscopy, for each animal, 8 photomicrographs (×3400) at a final amplification (×10,200) were obtained; other 4 photomicrographs were taken (×13,000) and used at a final amplification (×39,000).

[13] 32w Myelinated nerve fiber densities were determined by counting the number of myelinated fibers in three different 8 μm 2 squares in each of the 8 photomicrographs (×3400) with the final amplification (×10,200).

[14] 37w Nerve fiber number was estimated by multiplying nerve fibers density by the optic nerve cross section. The number of myelinated axons was estimated relying on a proven systematic random sampling (Parson and Sojitra, 1995;Parson et al., 1995).

[15] 43w Myelin thickness was obtained, in both large (axons diameter larger than 0.3 μm) and small (axons diameter smaller than 0.3 μm) axons, using the mean of 4 myelin thickness measurements performed in each axon. At least six axons were counted in each photomicrographs.

[1] 84w Both the maternal body weight gain during pregnancy and the offspring body weight gain in the first 21 days of postnatal life did not differ between control and MA groups (Table 1). Brain weight did not show significant differences between control and MA groups at any of the measured age groups postnatal days (PND) 7, 14 and 21. When the cerebellum was weighed separately, a significantly decreased weight in MA-treated animals was found on PND14 and PND21 for both males and females (Table 2).

[2] 54w The results of western blotting analysis are shown in Fig. 3, where it can be seen that at postnatal days 14 and 21 the female group treated with MA showed a lower myelin basic protein (MBP) level when compared with the respective agematched control group. No differences were found between control and MA-treated males.

[3] 77w The optic nerves from pups of the same age, gender and litter were homogenized together in 10 volumes of lysis buffer with an Ultra Turrax. The protein content of each sample was measured using the Bicinchoninic Acid kit for protein determination from Sigma-Aldrich, and samples (10 μg of protein) were resolved in a 4-12% NuPage gel (Invitrogen). Western blots were performed using the western breeze kit (Invitrogen) with monoclonal anti-myelin basic protein (MBP) clone 14 from Sigma-Aldrich.

[4] 73w Statistical determinations, defined by measures of parameters in different times, were made using a fixed-effect factorial ANOVA by means of the general linear modeling procedures. Main effects were treatment group, sex and day, and these were all treated as within-subjects factors. The experimental unit was the litter. When differences were detected, comparisons were made using the Tukey HSD test for unequal N. The statistical level of significance was considered at P < 0.05.

[1] 47w Representative optic micrographs of semi-thin sections taken through the optic nerves of rats from the various groups are shown in Fig. 1. Analysis showed that for PND7 MA-treated males, the mean diameter of the optic nerve was significantly smaller when compared with males from the control group.

[2] 45w For the female group, the difference did not reach significance. At PND14, for both males and females, no significant differences were observed. At PND21, the optic nerve mean diameter was smaller, both for males and females, when MA animals were compared with controls (Table 3).

[3] 80w No significant differences were found in the optic nerve crosssectional area at PND7, both for males and females. At PND14, there was a significant reduction in the optic nerve crosssectional area for the MA male group compared to the control group, but no differences were found in females between the MA and control groups. The optic nerve cross-sectional area at PND21 was significantly smaller in the MA groups of both males and females compared to the control groups (Table 3).

[4] 78w Representative electron ultramicrographs of sections taken through the optic nerves from rats in the various PND ages are shown in Fig. 1. In both groups, there was a rapid agedependent increase in the number of myelinated axons. In younger rats, large numbers of unmyelinated axons were present, but at PND21 around 80% of the total axon counts were myelinated. No significant differences were found in myelinated axon counts among groups at any of the studied ages (Table 3).

[5] 27w Myelination progressed throughout development but was especially marked between postnatal days 14 and 21, with an increase in the myelin thickness of the optic axons (Fig. 2).

[6] 57w No differences were found between control and MA groups regarding the time of onset of myelination. The differences in myelin thickness were only apparent at a latter age, such as PND21, and only in the female group. In this group, there was a significant reduction in the myelin thickness of both large and small axons (Table 4).

[7] 114w 4.1. Experimental design and drug administration The animals used in this study were Wistar rats bred in the Institute for Molecular and Cell Biology, Porto, Portugal. Experiments were performed in accordance with the Association for Research into Vision and Ophthalmology (ARVO) Statement for the Use of Animal in Ophthalmic and Vision Research and Portuguese law (Decret No. 1005/92). Nulliparous female rats approximately two months old were housed in conditions of constant temperature and humidity with a 12-h light/dark cycle. These females were caged overnight with males and checked for sperm in the next morning. The presence of copulatory plugs or sperm in the vaginal cytology was considered to indicate pregnancy (Gestational day 1; GD1

[8] 207w ).The experimental model used in the current study followed previous reports on the experimental exposure to psychostimulants (Gomes-da- Silva et al., 1998;Melo et al., 2005;Silva et al., 1995;Silva-Araujo et al., 1995a,b;Silva-Araujo and Tavares, 1996;Silva-Araujo et al., 1996a). Nine different dams were used for each treatment group (control and MA) giving a total of 18 animals. At GD8, pregnant females were separated and randomly assigned to the different age groups. Pregnant rats of MA groups were injected subcutaneously with a total of 5 mg/kg body weight/ day of MA (d-N,∝-dimethylphenethylamine; Sigma), in 0.9% saline, from GD8 to GD22. The schedule and volume for injecting saline in the control group were the same as for the MA dams. In a pair-fed control group, the food amount given to the pregnant female was similar to that consumed by MA group at the same GD (Melo et al., 2005). By using the pairfed, it was possible to mimic the anorectic effects of MA in the control group. The animals were assigned to three different age groups, which were sacrificed at different PND (7, 14 and 21). From each litter, two males and two females were used for the morphometric analysis and two males and two females for the western blotting analysis.

[9] 70w Changes in maternal body weight and food consumption during pregnancy were registered, as well as the weight of each pup in the day after delivery (PND1). At PND1, the litters were culled to eight pups: four males and four females. Animals were identified by painting their paws, head or trunk with unwashable ink according to a coding system. The pups were weighed every day at 09:00 AM to avoid variation.

[10] 56w Maternal weight gain was determined by calculating the daily difference between body weights, each day, from GD8 to GD22 (Silva-Araujo et al., 1996b;Spear et al., 1989). The body weight of the pups was measured from PND1 to PND21. The rat brains and cerebellum were weighed at the end of each experimental period (PND7, PND14 and PND21).

[11] 176w At PND7, PND14 or PND21, each animal was anesthetized by an intraperitoneal injection of ketamine (75 mg/kg body weight) and medetomidine (1 mg/kg body weight) in 0.9% saline. Animals were transcardially perfused with fixative (1% glutaraldehyde and 1% paraformaldehyde in 0.12 M phosphate buffer at pH 7.4). After fixation, the brain was removed and dissected. The optic nerves were immediately dissected 1 mm behind the eye globe. Optic nerves were immersed in the same fixative for 2 h, rinsed for 2 h in cacodylate buffer and postfixed for 2 h in 1% osmium tetroxide in cacodylate buffer. Osmicated tissues were dehydrated in ascending concentrations of ethanol followed by mixtures of resin with propylene oxide and embedded in Epon (Silva-Araujo et al., 1991). From each optic nerve, semithin sections (∼1.5 μm) were cut perpendicular to the long axis of the nerve and stained with 1% toluidine blue for light microscopy. For transmission electron microscopy, optic nerve ultrathin cross sections were cut and picked up on copper grids and subsequently double stained with uranyl acetate and lead citrate.