Life Sciences 67 (2000) 1759Ð1772
Prenatal exposure to low levels of carbon monoxide alters sciatic nerve myelination in rat offspring M.R. Carrat•a, R. Cagianoa, S. Desantisb, M. Labateb, M. Tattolia, L. Trabacea, V. Cuomoa,* a
Department of Pharmacology and Human Physiology, Medical School, Faculty of Biological Sciences, University of Bari, Policlinico, Piazza G. Cesare, 70124 Bari, Italy b Department of Zoology, Faculty of Biological Sciences, University of Bari, 70124 Bari, Italy
Abstract Prenatal exposure to low concentrations of carbon monoxide (CO, 75 and 150 ppm from day 0 to day 20 of gestation), resulting in maternal blood HbCO concentrations equivalent to those maintained by human cigarette smokers, leads to subtle myelin alterations in the sciatic nerve of male rat offspring. The rapid growth spurt in pup body weight was related to the period of maximal increase in myelin sheath thickness in both control and CO-exposed animals. A signiÞcant reduction in myelin sheath thickness of sciatic nerve Þbers, paralleled by changes in the frequency distribution, occurred in both 40- and 90-day-old rats exposed in utero to CO (75 and 150 ppm). Myelin deÞcit observed in 75 and 150 ppm CO-exposed animals showed up only after the major spurt in myelination but not early during development. The subtle myelin alterations observed in CO-exposed offspring were not accompanied by changes in developmental pattern of axon diameters and did not result in a gross impairment of motor activity. These results suggest that the myelination process is selectively targeted by a prenatal exposure model simulating the CO exposure observed in human cigarette smokers. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Prenatal carbon monoxide exposure; Rat; Sciatic nerve; Myelin; Motor activity
Introduction The developing central nervous system has been shown to be extremely susceptible to the reduction in oxygen availability produced by carbon monoxide (CO), one of the constituents of cigarette smoke [1]. In this regard, both neurobehavioral and neurochemical alterations have been found in male rat offspring exposed to low levels of CO during gestation [2Ð6]. Although the developing brain has been considered the primary target for CO-toxicity, recent studies have shown that a prenatal exposure model which simulates the CO exposure * Corresponding author. Tel.: 139 80 5478439; fax: 139 80 5478444. E-mail address:
[email protected] (V. Cuomo) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 6 1 -X
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observed in human cigarette smokers produces adverse effects on developing peripheral tissues as well. In fact, gestational exposure to CO impairs vascular reactivity in rat offspring at different stages of neurogenesis [7]. Moreover, alterations of ion channels responsible for the passive and active membrane electrical properties occur in skeletal muscle Þbers of rats exposed to low CO levels during pregnancy [8]. In agreement with the results obtained in skeletal muscle Þbers, changes in voltage-dependent properties of Na channels were also observed in peripheral myelinated nerve Þbers of rat offspring prenatally exposed to this gas [9]. On the basis of these last Þndings showing an impairment of ion channel maturation in peripheral nerves, it was of interest to investigate the possible consequences of maternal CO (75 and 150 ppm) exposure on myelination in the sciatic nerve of rat offspring at times ranging from 3 to 90 days of age. To date, both morphological and molecular aspects of peripheral nerve myelination have been extensively studied in rodents at different developmental stages and during nerve regeneration following crush injury [10Ð13]. Moreover, myelinogenesis has been shown to be altered by environmental agents, such as trialkyllead and tellurium, and by nutritional deÞciency [14]. The aim of the present study was to investigate whether maternal CO inhalation during gestation can impair sciatic nerve myelination and motor function in male rat offspring. For this purpose, the following endpoints were evaluated: myelin thickness, axon diameter, and motor activity. In particular, our experiments were focused on possible changes in the correlation between myelin thickness and axon diameter. In fact, according to Schršder et al. [15] this ratio should be considered as a reliable indicator of hypomyelination responsible for an infantile or juvenile peripheral neuropathy. Methods Animals and exposure conditions This study was performed in accordance with the Italian Ethics governing these experiments (Ministry of Health, D.L. 116/92). Primiparous Wistar female rats (Morini Laboratories, S. Polo dÕEnza, Italy) weighing 250Ð280 g were used. Animals were allowed free access to food and water, were housed at constant room temperature (20Ð228C) and exposed to a light cycle of 12 h/day (08:00 hÐ 20:00 h) for 1 week before the experiment. Pairs of females were placed with single male rats in the late afternoon. Vaginal smears were taken the following morning at 09:00 h. The day on which sperm were present was designated day 0 of gestation. Pregnant rats were then exposed to 0, 75 or 150 ppm of CO mixed with air from day 0 to day 20 of pregnancy. Each group consisted of 20 pregnant rats whose litters were reduced (six male pups per litter) and used for computer-assisted morphometric analysis and motor activity recording as described below. In another series of experiments pregnant rats were exposed to CO (0, 75 and 150 ppm) and used for spectrophotometric measurements of carboxyhaemoglobin (HbCO) on gestational days (GD) 10 and 20. The number of pregnant rats for each exposure level is reported in Table 1. Exposures were conducted in stainless steel and glass 0.73 m3 chambers (A.&L.CO.Industries, Segrate, Milan, Italy) with the airßow maintained at 120 l min21. Concentrations of CO
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Table 1 HbCO levels in pregnant rats exposed to CO CO concentrations
GD
HbCO %
0 ppm (n 5 6) 0 ppm (n 5 5) 75 ppm (n 5 4) 75 ppm (n 5 4) 150 ppm (n 5 6) 150 ppm (n 5 4)
10 20 10 20 10 20
0.97 6 0.02 1.62 6 0.10* 7.20 6 0.12** 7.43 6 0.62** 14.42 6 0.52** 16.08 6 0.88**
Values are mean 6 SEM; GD: gestational day. * P , 0.01 vs GD 10; ** P , 0.001 vs controls (two-tail StudentÕs t-test with BonferroniÕs correction).
in each exposure chamber were monitored every 15 min for a 5 min period by an infrared CO detector (CO 11M, Environment SA, Poissy, France) at a wavelength of 4.67 mm. Actual concentration was recorded on a digital display every 3 s for the 5 min period. Moreover, CO concentrations were also continuously monitored by a multi-pen recorder. Chamber concentrations of CO deviated by less than 3% from the stated value. Mean temperature values in the exposure chambers ranged from 21 to 238C, and the light dark cycle was 12 h light (08:00 hÐ 20:00 h) and 12 h dark (20:00 hÐ08:00 h). Litter sizes at birth were as follows: CO 0 ppm 12 6 0.37 (n 5 18); CO 75 ppm 12.36 6 0.51 (n 5 19); CO 150 ppm 11.61 6 0.49 (n 5 18). Due to the cannibalism of some dams, the number of litters available for testing was reduced by one (75 ppm CO-exposed group) or two units (0 and 150 ppm CO-exposed groups). An overall one-way ANOVA showed that litter size at birth was not affected by prenatal exposure to CO (F 5 0.65, df 5 2/52, n.s.). All litters were reduced to a standard size of six male pups per litter (when possible) within 24 h after birth. Litters from the control group (0 ppm of CO) or CO-exposed groups (75 and 150 ppm) were then assigned (six pups per litter) to non-exposed mothers whose pups were born on the same day. Data were collected only from male pups whose mothers were exposed either to 0 ppm CO or to CO (75 and 150 ppm) during pregnancy. Pups were weaned at 21 days of age. One pup per litter from eight (computer-assisted morphometric analysis) or six (motor activity analysis) different litters for each tested age per treatment group was used. As observed previously [4], prenatal CO (75 and 150 ppm) exposure does not affect dam weight gain, number of dams giving birth, pregnancy length, and postnatal mortality (data not shown). Spectrophotometric measurements of maternal carboxyhaemoglobin (HbCO) Subgroups of pregnant CO (0, 75 and 150 ppm)-exposed rats were implanted under anesthesia (Equithesin, 3 ml kg21 i.p.) with catheters in the abdominal aorta. Maternal HbCO levels were measured on both gestational days (GD) 10 and 20 by the spectrophotometric method described by Rodkey et al. [17]. Blood samples (10 ml) were taken with a heparinized syringe, diluted 1000 fold in a deionized solution containing Na2S2O4 (2 mg ml21) and analysed for their absorbance in the Soret region (390Ð440 nm) using a UV/VIS spectrophotometer (Perkin Elmer).
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Computer-assisted morphometric analysis Male rats (1 pup per litter from eight different litters per age and per treatment group) exposed in utero to CO (0, 75, 150 ppm) were sacriÞced on postnatal days (PND) 3, 8, 18, 40 and 90. Sciatic nerves were quickly removed and processed for morphometric analysis according to the procedures described by Schafer and Friede [18]. The nerve specimens were Þxed in a cold solution of 3% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.4) from 2 to 4 hours, then postÞxed in 2% osmium tetroxide in cacodylate buffer (1 hour), dehydrated and embedded in araldite 502. Semithin sections (1 mm) were cut and stained with Toluidine blue. The light microscopic measurements were performed by computer-assisted method. Cross sections were examined under a light microscope (Diaplan, Leitz) equipped with an automated image analysis system (Quantimet 500, Leica). The microscope used a 3100 NPL objective lens to achieve a Þnal magniÞcation at the monitor display (DC 101P video camera, Sony) of 31440. Myelin sheath thickness and axon diameter measurements were performed in nerve Þbers included in four standard areas each of 2372 pixels corresponding to 7526.65 mm2 (1 pixel 5 0.134 mm). Motor activity Motor activity was recorded in Macrolon cages by infrared monitoring, according to the technique previously described by Tamborini et al. [19] and modiÞed as follows. Brießy, a passive infrared sensor (RK 2000 QPC) was placed 23 cm above the center of each cage (59 3 38.5 3 20 cm) which was covered with stainless steel wire lids. The infrared sensors were connected, via an interface, to an Amstrad PC 1640 which was programmed to check all sensors at 1 s intervals. When a sensor was turned on, i.e. registering no movement, it gave a score of 0 for this interval; when a sensor was turned off, i.e. registering a movement, it gave a score of 1. These points were added and registered over 5 min blocks. Therefore, the theoretical range of activity for 5 min was 0Ð300. All tests were carried out in a 3 3 2 3 2 m sound attenuating cabin illuminated by a 20 W white light. Background noise of 46 dB was produced by a fan. Immediately after each test, the apparatus was thoroughly cleaned by cotton pads wetted with 96% ethanol solution. Male rats were subjected to a 30 min session at 40 and 90 days of age. Each group consisted of six animals (1 pup per litter from six different litters per age and per treatment group). Statistical analysis Litter sizes at birth were analysed by an overall one-way ANOVA. Statistical analysis of HbCO levels in pregnant rats was performed by two-tail StudentÕs t-test with BonferroniÕs correction. Data of morphometric analysis were pooled and grouped per age and per treatment, and analysed by two-way ANOVA. Post-hoc comparisons were made by two-tail DunnettÕs t test. The regression analysis was performed to analyse the developmental change of myelin thickness and axon diameter with both age and body weight gain. Differences in the slope of the regression lines were evaluated by the test for parallelism. Statistical analysis of motor activity was performed by an overall one-way ANOVA for each age.
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Results Maternal HbCO levels Data are shown in Table 1. A dose-dependent increase in maternal HbCO levels was produced by exposure to CO (75 and 150 ppm) on both GDs 10 and 20. Moreover, HbCO levels in air-exposed pregnant rats were signiÞcantly increased on GD 20 with respect to those found on GD 10. These data conÞrm that HbCO concentrations tend to be somewhat elevated during gestation, reßecting increased endogenous CO production [20Ð22]. Offspring body weight gain Prenatal exposure to CO (75 and 150 ppm) did not signiÞcantly affect pup weight gain (Fig. 1). Two-way ANOVA showed the following differences: (i) between treatments: F 5 0.38, df 2/105, n.s.; (ii) between times: F 5 7661.47, df 4/105, P , 0.0001; (iii) between treatments 3 times: F 5 0.14; df 8/105, n.s. Morphometric measurements The light-microscopic measurements of myelin sheath thickness were performed on 1 mm Toluidine blue-stained sections of the sciatic nerve from 3-, 8-, 18-, 40- and 90-day-old rats. Examples of sciatic nerve cross sections from control (a), 75 (b) and 150 (c) ppm CO-exposed rats (PND 40) are shown in Fig. 2. Two-way ANOVA for myelin sheath thickness showed the following signiÞcant differences: (i) between treatments: F 5 60.62, df 2/1485, P , 0.0001; (ii)
Fig. 1. Body weight gain in rats born to dams exposed to CO (0, 75 and 150 ppm) throughout gestation. Data are expressed as mean 6 s.e. mean.
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Fig. 2. Toluidine blue-stained cross sections (1 mm) of the sciatic nerve from 40-day-old rats born to dams exposed to 0 (a), 75 (b) and 150 ppm (c) of CO throughout gestation. Note the difference in myelin sheath thickness between control and CO-exposed rats. MagniÞcation: 3480.
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between times: F 5 468.04, df 4/1485, P , 0.0001; (iii) between treatments 3 times: F 5 15.07, df 8/1485, P , 0.0001. Individual between group comparisons (two-tail DunnettÕs t test) revealed a signiÞcant (P , 0.01) decrease in myelin sheath thickness of the sciatic nerve Þbers from CO (75 and 150 ppm)-exposed rats on PNDs 40 and 90 with respect to controls (Fig. 3). Although a signiÞcant decrease in myelin sheath thickness was detected in rat offspring on PNDs 40 and 90, the regression analysis has revealed a linear relationship between myelin thickness and age in both air- and CO-exposed animals: 0 ppm: r 5 0.97, slope 5 0.01 6 0.001, df 3, P , 0.01; 75 ppm: r 5 0.96, slope 5 0.007 6 0.001, df 3, P , 0.01; 150 ppm: r 5 0.93, slope 5 0.006 6 0.001, df 3, P , 0.05. By comparing the slopes of the regression lines (test for parallelism) between air- and CO-exposed rats, the following differences were observed: controls vs 75 ppm CO-exposed rats: t 5 2.036, df 6, n.s.; controls vs 150 ppm CO-exposed rats: t 5 2.21, df 6, n.s. Moreover, data obtained with regression analysis have shown a linear relationship between body weight gain and myelin sheath thickness (0 ppm: r 5 0.99, slope 5 0.0003 6 0.00002, df 3, P , 0.01; 75 ppm: r 5 0.98, slope 5 0.00016 6 0.000017, df 3, P , 0.01; 150 ppm: r 5 0.95, slope 5 0.00015 6 0.000026, df 3, P , 0.05). Test for parallelism has shown the following differences in the slope of the regression lines: 75 ppm vs 0 ppm: t 5 3.57, df 6, P , 0.01; 150 ppm vs 0 ppm: t 5 3.43, df 6, P,0.01.
Fig. 3. Myelin sheath thickness of the sciatic nerve Þbers from 3-, 8-, 18-, 40- and 90-day-old rats born to dams exposed to CO (0, 75, and 150 ppm) throughout gestation. Each column represents pooled data (grouped per age and per treatment) expressed as mean 6 s.e. mean. * P,0.01, ** P,0.01 vs controls (two-tail DunnettÕs t test).
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CO-induced hypomyelination was further conÞrmed by the frequency distribution analysis of myelin sheath thickness shown in Fig. 4. No differences were observed between control and CO (75 and 150 ppm)-exposed rats on PNDs 3, 8 and 18. On PND 40, the frequency distribution in control rats was typically bimodal with two peaks at 0.6 and 1 mm; conversely, frequency distribution in 75 and 150 ppm CO-exposed rats was unimodal showing the main peak in the region of 0.6 mm and 0.4 mm, respectively. On PND 90, the frequency distribution in control rats showed the main peak in the region of 1.2 mm; conversely, frequency distribution in 75 and 150 ppm CO-exposed rats showed the main peak in the region of 0.8 mm and 0.6 mm, respectively. In order to compare the rate of myelin deposition in developing sciatic nerves of air- and CO-exposed rats, the daily increase in myelin sheath thickness was calculated from means shown in Fig. 1. In both air- and CO (75 and 150 ppm)-exposed rats myelin deposition occurred at high rate during the Þrst days of postnatal life and declined thereafter (Fig. 5). However, the daily increase in myelin thickness was lower in CO-exposed rats in comparison with air-exposed ones. Unlike myelin sheath thickness, the developmental pattern of axonal diameters was not signiÞcantly affected by CO (75 and 150 ppm) exposure (data not shown). Two-way ANOVA
Fig. 4. Frequency distribution of myelin sheath thickness in the sciatic nerve Þbers from 3-, 8-, 18-, 40- and 90day-old rats born to dams exposed to 0, 75 and 150 ppm of CO throughout gestation. Nerve Þbers are grouped in 0.2 mm amplitude classes according to the thickness of their myelin sheath. Ordinate: absolute number of myelinated nerve Þbers. Abscissa: class amplitude.
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Fig. 5. Average speed of myelin deposition in rats born to dams exposed to CO (0, 75 and 150 ppm) throughout gestation expressed as daily increase in myelin sheath thickness (mm/die). Calculations are based on mean myelin thickness shown in Fig. 3.
for axon diameters yielded the following results: (i) between treatments: F 5 2.04, df 2/1485, n.s.; (ii) between times: F 5 280.27, df 4/1485, P , 0.0001; (iii) between treatments 3 times: F 5 1.74, df 8/1485, n.s. A linear relationship between axon diameters and age was observed in both air- and COexposed rats. The regression analysis yielded the following results: 0 ppm : r 5 0.96, slope 5 0.042 6 0.007, df 3, P , 0.01; 75 ppm: r 5 0.97, slope 5 0.039 6 0.006, df 3, P , 0.01; 150 ppm: r 5 0.97, slope 0.04 6 0.006, df 3, P , 0.01. The slopes of the regression lines did not differ signiÞcantly (test for parallelism): controls vs 75 ppm CO-exposed rats: t 5 0.35, df 6, n.s.; controls vs 150 ppm CO-exposed rats: t 5 0.24, df 6, n.s. No differences were observed in frequency distribution of axonal diameters between airand CO (75 and 150 ppm)-exposed rats from PND 3 up to PND 90 (data not shown). Moreover, a linear relationship between myelin sheath thickness and axon diameter was observed within each treatment group (Fig. 6); the regression analysis yielded the following results: 0 ppm: r 5 0.99, slope 5 4.13 6 0.20, df 3, P , 0.01; 75 ppm: r 5 0.98, slope 5 5.82 6 0.66, df 3, P , 0.01; 150 ppm: r 5 0.99, slope 5 6.52 6 0.56, df 3, P , 0.01. Test for parallelism revealed the following differences in the slope of the regression lines between controls and CO-exposed rats: controls vs 75 ppm CO-exposed rats: t 5 2.74, df 6, P , 0.01; controls vs 150 ppm CO-exposed rats: t 5 4.42, df 6, P , 0.01. Motor activity Prenatal exposure to CO (75 and 150 ppm ) did not signiÞcantly affect motor activity in both 40- and 90-day-old rats (data not shown). In fact, overall one-way ANOVAs for activity counts showed the following differences: (i) day 40 of age (F 5 0.22, df 2/15, n.s.); (ii) day 90 of age (F 5 0.17, df 2/15, n.s.).
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Fig. 6. Relationship between myelin sheath thickness and axon diameter in rats born to dams exposed to CO (0, 75 and 150 ppm) throughout gestation. Each symbol refers to data pooled per age from PND 3 to PND 90 (mean values).
Discussion The results of the present study show that prenatal exposure to low concentrations of CO induces subtle myelin alterations in the sciatic nerve of rat offspring. The myelin deÞcit, which shows up only after the major spurt in myelination, is induced by CO concentrations below those associated with maternal toxicity, gross malformations and/or overt neurotoxic effects in the offspring. The CO-induced myelin deÞcit is not related to changes in growth patterns since no differences in body weight between air- and CO-exposed rats were found. The developmental increase in myelin sheath thickness is linearly related to both age and body weight gain. In particular, our data show that the rapid growth spurt in body weight relates to the period of maximal increase in myelin sheath thickness in both control and CO-exposed rats. The developmental changes of myelin sheath thickness observed in our study show the same pattern described in the rat sciatic nerve [23] and in the human sural nerve [15,24]. Moreover, the rate of myelin deposition is strikingly similar to the rate of incorporation of 14C acetate into lipids of the developing rat sciatic nerve showing a peak during the Þrst days of postnatal life followed by a gradual decline [25]. The results of the present study show that myelination is not altered during the early stages of postnatal life when the maximum rate of myelin deposition occurs. By contrast, a signiÞcant reduction in myelin sheath thickness is observed in 40- and 90-day-old rats prenatally exposed to CO. Obviously, myelin thickness reßects the myelin formation around the axon and cannot account for the overall increase in deposition and accumulation of myelin during development. In this regard, it should be pointed out that myelin deposition in rats continues at a considerable rate through the life because, as the rat grows rapidly, the myelin internodes increase in length to keep pace with the lengthening hind limb and sciatic nerve [23]. Therefore, the CO-induced myelin deÞcit could be interpreted as a dysregulation of myelin formation around the axon.
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As far as the delayed appearance of myelin deÞcit (PNDs 40 and 90) following prenatal CO exposure is concerned, this could be explained, at least partially, taking into account the onset and rate of myelination in peripheral nerves of rodents. Myelination is a highly regulated process that begins postnatally and must be completed within the proper Òdevelopmental windowÓ. Most morphologic and biochemical events occur during this vulnerable period (Þrst 30 days of postnatal life), although myelin continues to expand in all planes during growth and development [see 26 for references]. Therefore, it could be hypothesized that, since myelination is a postnatal event, differences in myelin sheath thickness between control and CO-exposed rats can be detected at the time when a sufÞcient amount of myelin is already formed. Moreover, on the basis of the developmental pattern of myelin, a more severe deÞcit could be expected following CO exposure during the well deÞnied Òdevelopmental windowÓ. In this regard, literature data recently reviewed by Harry and Toews [26] show that toxic insults or nutritional deÞciences from birth to postweaning period can severely affect the process of myelination with a consequent decrease of the normal accumulation of myelin during development. The results of the present study showing a myelin deÞcit which persists until 90 days of age, indicate that continuous maternal CO inhalation during gestation induces a long lasting impairment of myelin accumulation in the sciatic nerve of male rat offspring. In this regard, it should be pointed out that our previous studies have shown also permanent cognitive deÞcits in rats born to dams exposed to this gas during gestation [3]. In particular, prenatal CO exposure signiÞcantly impairs the acquisition of a two-way active avoidance task in 3-month-old male rats as well as the acquisition and reacquisition of this schedule in 18-month-old rats. As far as the possible involvement of CNS myelin deÞcit in cognitive dysfunctions is concerned, very little is known since the available literature data are based on acute exposure to extremely high concentrations of CO. However, a delayed neurological deterioration with brain white matter injury has been observed in humans after acute exposure to this gas during the late gestational period [27], childhood [28] and adulthood [29]. As far as the possible mechanisms involved in CO-induced myelination disorders is concerned, it may be argued that the moderate fetal hypoxia induced by continuous maternal exposure to low levels of this gas would play an important role. In fact, myelin formation imposes an enormous burden on the metabolism of rapidly myelinating glia. It has been estimated that, on the average, each cell makes an amount of myelin per day that is more than three times its own weight [16]. Much of the myelin is formed during a vulnerable period of development corresponding to the Þrst 15Ð30 days of postnatal life in rats. During this period most of the metabolic and synthesizing activities are involved in myelinogenesis. Just before this rapid deposition of myelin, there is a burst of Schwann cell proliferation [16,26]. Therefore, the CO-induced hypomyelination could be due to inßuences on cell proliferation as well as on metabolic and synthesizing activities during myelin growth. As far as cell proliferation is concerned, the present prenatal exposure model simulating the CO exposure observed in human cigarette smokers is not expected to inßuence this parameter. In fact, literature data show that tobacco smoke inhalation by pregnant mice on gestational days 6Ð8 produces persistent surface changes (suggestive of depressed metabolic activity) in cells of the embryonic neural plate without altering the total cell number or number of dead cells or the mitotic index [30].
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With regard to the synthesizing activity, our Þndings indicate that prenatal CO (150 ppm) does produce alterations in the pattern of protein synthesis. In fact, the results of our recent experiments (unpublished data) demonstrate changes in the electrophoretic proÞle of myelin proteins in rats exposed to CO (150 ppm). In particular, a reduction in the MBP synthesis associated with the expression of a novel low-molecular-mass protein occurs. These results are in agreement with literature data showing a speciÞc inhibition of MBP synthesis and a dramatic induction of Òde novoÓ synthesis of a novel low-molecular-mass protein in neonatal rat oligodendrocytes exposed to increasingly hypoxic culture conditions [31]. On the basis of these experimental evidences it could be hypothesized that myelin forming cells, which undergo very active metabolism during the rapid myelination stage, are particularly vulnerable to hypoxic conditions, and that an impairment in synthesizing activities can, at least partially, explain the developmental myelin deÞcit induced by prenatal CO-induced hypoxia. Moreover, it should be pointed out that the CO-induced myelin deÞcit mainly involves the larger diameter Þbers. Literature data show that myelination usually begins when axons have reached a caliber of about 1 mm, so that those Þbers that are the Þrst to be myelinated correspond to the largest Þbers in mature nerves [24]. Moreover, myelination of motor roots is known to precede that of sensory ones [32]. Therefore, nerve Þbers which Þrst undergo myelination and mostly represent motor axons could have been mainly affected by prenatal CO exposure. Consequently, the CO-induced hypomyelination would be expected to be associated with functional deÞcits. Conversely, the results of the present study show that maternal CO inhalation during gestation does not impair motor activity in rat offspring. In this regard, it should be underlined that nerve impulse propagation is severely impaired only when myelin thickness is uniformely reduced to less than 2Ð7% of its normal value [33]. Furthermore, a proportionate inßuence of the myelin sheath thickness on conduction velocity is inconsistent with the experimental data showing that maximal conduction velocity is achieved when axonal diameters have reached their maximal values and myelin sheaths are still increasing in thickness [see 15 for references]. Therefore, on the basis of these data, it can be suggested that the extent of COinduced myelin deÞcit is not sufÞcient to induce a gross impairment of motor activity. Finally, the results of the present study show that prenatal CO exposure does not affect the developmental pattern of axonal diameter, thus suggesting that myelination process is selectively targeted by the low-level exposure to this gas. These Þndings can further contribute to explain the lack of marked functional deÞcits induced by prenatal CO, taking into account that the axon size plays an important role in nerve impulse propagation. In sum, these Þndings provide the Þrst evidence that subtle myelin alterations occur in male rat offspring prenatally exposed to low CO concentrations resulting in maternal HbCO levels of about 7% (75 ppm) and 16% (150 ppm). Interestingly, cigarette smokers show HbCO levels ranging from 1 to 16%, depending on the occupation, smoking habits (frequency and inhalation patterns) and ambient CO concentrations [34Ð36]. Acknowledgments This study was supported by MURST (Cofinanziamento 1997) and CNR (97.04632.CT13; 98.00730.PS13) grants.
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