TY - JOUR AU - Duffard,, Ricardo AB - Abstract The purpose of this study was to determine whether 2,4-dichlorophenoxyacetic acid (2,4-D), which is an herbicide used to control the growth of broadleaf weeds, had a direct or an indirect (mediated by undernutrition) hypomyelinating effect. We also proposed to analyze the effect of 2,4-D on undernourished (UN) pups. Four experimental rat groups were used: well-nourished (WN) pups, litters with eight offsprings; UN pups, litters with fourteen offsprings; WN pups whose mother received 70 mg/kg/day of 2,4-D from postnatal day (PND) 9 to 21 (WN70 pups); and UN pups whose mother received 70 mg/kg/day of 2,4-D from PND 9 to 21 (UN70 pups). In this work, we demonstrated that (1) myelin proteins (analyzed by Western blot and/or immunohistochemical study) showed a significant decrease in WN70, UN, and UN70 with respect to control group; (2) there is a good correlation between these myelin-specific protein expression with the degree of myelin compaction detected by electron microscopy in groups exposed to 2,4-D; (3) a decreased and normal number of myelin sheets were detected in UN and 2,4-D exposed pups, respectively; and (4) undernourishment sensitized pups to the hypomyelinating effect of 2,4-D. According to this and besides the fact that WN70 group have no body weight changes, these results are indicating that 2,4-D and undernourishment are two independent hypomyelinating factors. 2,4-dichlorophenoxyacetic acid, herbicide, brain development, myelin proteins, myelin ultrastructure, neurotoxicity, neonate rat Brain development is a sequential anatomical process characterized by specific well-defined stages of growth and maturation. One of the fundamental and necessary events in the normal development of the central nervous system (CNS) in vertebrates is the formation of myelin sheaths by oligodendrocyte cells (Salvati et al., 2000). Myelination is a major metabolic and structural event that occurs during a relatively brief but precisely defined period in the normal progression of events involved in nervous system development. During these “vulnerable periods,” the process of myelination is especially susceptible to perturbations such as toxic insult, nutritional deficiencies, genetic disorders of metabolism, viral infections and other environmental factors. Early malnutrition produces extensive alterations in the composition of the various membranes present in the rat's CNS. Specifically, early postnatal starvation depresses myelin synthesis to about the same extent in all major brain regions from 18 to 21 days of age (Wiggins and Fuller, 1978). Morphological and biochemical studies have shown that rats subjected to malnutrition during early stages of life—up to 21 days of age, on the peak of myelination—developed irreversible brain damage (Benton et al., 1966; Davison and Dobbing, 1966; Dobbing, 1972; Ghittoni and Faryna de Raveglia, 1972; Krigman and Hogan, 1976; Pasquini et al., 1981). 2,4-Dichlorophenoxyacetic acid (2,4-D) is a phenoxyherbicide used for selective control of broadleaf weeds, which causes disruption of plant hormone responses. Plant injuries include growth and reproduction abnormalities followed by their death. Unfortunately, 2,4-D produces different harmful effects on mammals, including humans, which range from embryotoxicity to neurotoxicity (Barnekow et al., 2000; Blakley et al., 1989; Charles et al., 2001; Osaki et al., 2001; Rosso et al., 2000a; Sulik et al., 1998). Surprisingly, very little is known about the cellular mechanisms underlying its neurotoxicity. We have previously described that the exposure to the herbicide 2,4-D through mother's milk during the period of rapid myelination (from the ninth to the 25th postnatal days [PND]) results in a myelin deficit in the pup's brain. This myelin deficit was also found in pups nursed only from the PND 15 to 25 period by dams treated with 2,4-D (Duffard et al., 1996). The aim of this study was to determine whether the brain myelin deficit previously detected in 2,4-D exposed pups is a direct or an indirect effect, mediated by undernourishment and additionally, if this myelin deficit is similar to that observed in undernourishment. MATERIALS AND METHODS Experimental animals. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication No. 86-23 revised 1986) and were accepted by the Institutional Animal Care and Use Committee. Nulliparous female (Wistar origin) rats were separately placed with fertile males on the proestrus night and the presence of spermatozoa was checked in the vaginal smear the following morning. This day was denoted as gestation day 0. At this time, pregnant females were individually housed in plastic breeding cages in a temperature-controlled nursery (22–24°C) and maintained on a 12-h light/dark cycle. Food (Cargill pellets, Buenos Aires, Argentina) and water were available ad libitum. Twenty-four hours following parturition (parturition was PND 0), litters were examined and pups were housed with their dams. As parturition approached, dams were checked for birth twice a day. Two experimental designs were selected: (1) litters with eight pups to ensure good nutrition through lactation or well-nourished pups (WN pups) and (2) litters with 14 pups to ensure undernourishment according to Miller's (1992) and Ferri et al. (2003) procedure, to obtain undernourished pups (UN pups). This undernourishment method was chosen because pups began to receive less milk immediately after birth and dams were never subject to nutritional stress, which could alter mothering. Handling of pups was, by far, less than in some procedures involving forced feeding of pups or restriction of access to mothers. There was no sex selection in the litter but—whenever it was possible—equal representation of sex within litter was done. All dams were intraperitoneally injected with the herbicide or with the solvent used (dimethyl sulfoxide, DMSO) once a day, from PND 9 to 22, in volumes of 1 ml/kg body weight, according to previous works (Duffard et al., 1996; Ferri et al., 2003). Four experimental groups were assessed. Dams of WN pups were treated with DMSO (group 1, WN—control) or with 70 mg 2,4-D/kg body weight (group 2, WN70). Dams of UN pups were administered with DMSO (group 3, UN—control) or 70 mg 2,4-D/kg body weight (group 4, UN70). In addition, as dams received 2,4-D dissolved in DMSO (WN and UN groups of rats received only DMSO) other two groups of WN and UN dams, which received neither 2,4-D nor DMSO were used only to test the effect of DMSO on the ultrastructural organization of the CNS myelin. An n = 5 litters per each group were evaluated in each determination. Herbicide treatment and dose were selected based on previous studies, which demonstrated behavioral changes and alterations in neurotransmitters levels in adult rats exposed to the herbicide according to Evangelista de Duffard et al. (1990) and in neonatal rats (Ferri et al., 2003). Pups were weaned at PND 22, at the end of the experimental protocol. The body weight of all animals was recorded throughout the experiment. Histochemical and immunohistochemical studies. Five 22-day-old rats from each group (one of each litter) were ip anaesthetized with 75 mg/kg sodium pentobarbital and transcardially perfused with 4% paraformaldehyde (wt/vol), in 0.1M phosphate buffer, pH 7.4. Prior to fixation, a saline solution (0.9% wt/vol NaCl with 10 μl of 0.4M NaNO2 and 50 IU of heparin) was passed through their circulatory system. Brains were kept in the same fixative solution for 2–4 h and immersed in 20% sucrose at 4°C overnight or until they sank. Control and exposed 40-μm-thick brain sections were cut with a cryostat. Immunohistochemical stainings were performed according to Sternberger's peroxidase-antiperoxidase (PAP) technique (Sternberger et al., 1970), using polyclonal anti-myelin basic protein (MBP) antibody (1:1000), mouse monoclonal anti-proteolipid protein (PLP) antibody (1:1000) and mouse monoclonal anti 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) antibody (1:1000). Development of peroxidase activity was performed with diaminobenzidine (DAB)/hydrogen peroxide in phosphate buffer. After the enzymatic incubation step, sections were mounted on gelatin-coated standard glass slides, dehydrated and coverslipped for light microscopy observation. The histochemical myelin staining was performed according to the Schmued (1990) staining technique, using a 0.2% solution of gold chloride, modified for free floating sections. Transmission electron microscopy. Four 22-day-old rats from each group were perfused-fixed with 4% paraformaldehyde (wt/vol), 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.4 in the same procedure described above (see histochemical and immunohistochemical studies). Then, 1-mm-thick sections of brains containing corpus callosum were selected for postfixation with 1.5% wt/vol osmium tetroxide in 0.1mM phosphate buffer pH 7.4 for 2 h at 4°C. The sections were contrasted with 2% wt/vol uranyl acetate, dehydrated and embedded in Epon 812 resin. Ultrathin sections were obtained with an ultramicrotome Porter Bloom MT 1 and collected in copper grids. The ultrathin sections were contrasted with uranyl acetate and stained with Reynolds solution; photographed in a Zeiss 10C Electron Microscope using 35-mm Kodak Technical Pan Profesional 2415 films (for morphometrical studies) and also 4485 EM films (to obtain the photomicrographs). Computerized image analysis. Images of the 35-mm negatives were scanned using a Nikon coolscan II. For each experimental condition, twelve electron microscopic images of each animal (primary magnification 31,500× and 100,000×) were processed using Optimas 6.0 software). Thickness of major dense line (MDLt), interperiod distance (ID), and number of sheets in the myelin sheath around the axon were measured. We used 5-nm Au particles as unit of measurement and for calibration the Optimas 6.0 software at each magnification. Myelin isolation. At PND 22, pups were decapitated, their brains removed and myelin was isolated by centrifugation in a sucrose gradient as described by Norton and Poduslo (1973). In brief, brains were homogenized in ice-cold 0.32M sucrose (wt/vol) in a glass homogenizer and centrifuged in a sucrose step gradient using an Optima XL-100K Beckman ultracentrifuge. Myelin was recovered from the 0.32 and 0.85M interface and further purified by two rounds of hypo-osmotic shock by resuspension in a large volume of ice-cold water, followed by a second round of centrifugation in a sucrose step gradient. Purified myelin was collected from the interface, washed twice with ice-cold water, resuspended in a small volume of water, and frozen in small aliquots at −80°C. Western blot analysis. For Western blotting, aliquots of each sample containing equal amounts of protein (measured by Lowry et al. 1951) were mixed with sample buffer containing sodium dodecyl sulfate (SDS) and resolved on SDS–polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970). After SDS-PAGE, the separated polypeptides were transferred to nitrocellulose membranes. Membranes were blocked in phosphate buffered saline/3% milk powder/0.1% triton X100 at 4°C and incubated with primary antibody diluted in blocking buffer overnight. Blots were incubated with appropriate alkaline phosphatase–labeled secondary antibody for 1 h, and then visualized by incubating the membrane for 15 min in solution containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. Semiquantification was performed using the Gel-Pro 3 system. Lipid chemical analysis. Total lipids were extracted with chloroform:methanol in a ratio of 2:1(vol/vol) according to the method of Folch et al. (1957). The lower phases were washed twice with “theoretical” upper phase chloroform:methanol:water (3:48:47, vol/vol/vol). The lower phases were dried under a stream of nitrogen. These total lipid extracts were resuspended in chloroform:methanol (2:1, vol/vol), and aliquots were taken for cholesterol, phospholipids, and galactolipids determinations. Total lipids were determined in myelin aliquots by a colorimetric method using the sulfuric acid-vanillin reaction (Cantarow and Trumper, 1962). Phospholipid phosphorus was determined according to the Dodge and Phillips (1967) method. Cholesterol was determined in aliquots of lower phases by the method of Searcy et al. (1960). Galactolipids were purified in aliquots of lower phases by liquid chromatography in Florisil column and hexoses quantified by the anthrona reaction (Yem and Willis, 1954). Drugs and reagents. All drugs, solvents, 2,4-D, and standards used in this study were of analytical grade from Sigma Chemical Co. (St Louis, MO) or Merck Co. (Buenos Aires, Argentina). Monoclonal anti-CNPase, antimouse IgG, mouse PAP complex, and DAB hydrochloride were purchased from Sigma Chemical Co. Monoclonal anti-PLP antibody was purchased from Chemicon (Temecula, CA). Polyclonal anti-MBP antibody was raised in rabbits in our laboratory (Rosso et al., 2000b). Statistical analysis. Data were expressed as means ± SEM. Statistical differences between groups were assessed using ANOVA. In all cases, p values less than 0.05 were considered to indicate a significant difference. For semiquantitative electron microscopic study four to six grids of each group were used to take twelve photomicrographs of each sample. The reported values in Table 2 represent the means ± SEM of each measurement. Differences among the means of the groups were statistically analyzed by Tukey-Kramer multiple comparison test, one-way ANOVA followed by Student-Newman-Keuls multiple comparison test and, when differences were detected between the groups, a two-tailed Student's t-test was performed for each pair of results. RESULTS WN and WN70 pups gained uniformly body weight and no difference in the body weight profile between them was observed during the exposure period. However, in both UN groups of pups (UN and UN70), a significant delay in body weight gain was observed from PND 2 and, from PND 11 (2 days after the 2,4-D dam's treatment began), the body weight of UN70 pups was significant already lower than UN pup group. A significant decrease in body weight in UN (26%); UN70 (32%) with respect to WN and in UN70 (11%) with respect to UN at PND 22 was determined at the end of the treatment (Fig. 1). FIG. 1. Open in new tabDownload slide Body weight profile of WN (litters of eight pups) and UN (litters of 14 pups) pups. The values of body weight profile among different pups treatments are expressed as mean ± SEM, n = 5 litters. ap < 0.01 UN and UN70 relative to control (WN). bp < 0.01 UN70 relative to UN. FIG. 1. Open in new tabDownload slide Body weight profile of WN (litters of eight pups) and UN (litters of 14 pups) pups. The values of body weight profile among different pups treatments are expressed as mean ± SEM, n = 5 litters. ap < 0.01 UN and UN70 relative to control (WN). bp < 0.01 UN70 relative to UN. The histochemical study showed a decreased myelin staining in 2,4-D exposed pups corpus callosum (Fig. 2). CNPase, PLP, and MBP immunostaining showed decreased corpus callosum staining in 2,4-D exposed pups, similar to that observed in the histochemical study. Although a decreased MBP and PLP staining in UN pups was observed with respect to WN pups, no difference in CNP immunostaining between WN and UN pups was detected. (Fig. 3 only shows PLP staining). FIG. 2. Open in new tabDownload slide Gold chloride histochemical staining of control and exposed pups corpus callosum at PND 22, WN (A), WN70 (B), UN (C), and UN70 (D). Calibration bar: 130 μm. Note the decreased myelinated fiber density in 2,4-D exposed pups. FIG. 2. Open in new tabDownload slide Gold chloride histochemical staining of control and exposed pups corpus callosum at PND 22, WN (A), WN70 (B), UN (C), and UN70 (D). Calibration bar: 130 μm. Note the decreased myelinated fiber density in 2,4-D exposed pups. FIG. 3. Open in new tabDownload slide PLP immunohistochemistry of WN (A), WN70 (B), UN (C), and UN70 (D) pups corpus callosum at PND 22. Note the decreased PLP immunostaining in UN and 2,4-D exposed pups. Calibration bar: 130 μm. FIG. 3. Open in new tabDownload slide PLP immunohistochemistry of WN (A), WN70 (B), UN (C), and UN70 (D) pups corpus callosum at PND 22. Note the decreased PLP immunostaining in UN and 2,4-D exposed pups. Calibration bar: 130 μm. Western blot (WB) analysis of myelin proteins showed significant differences between WN70, UN, and UN70 with respect to control group, and between WN70 and UN70 with respect to UN. CNPase was decreased in UN pups (15%), in WN70 pups (38%) and in UN70 pups (57%) with respect to WN pups. In addition, a diminution of CNPase in UN70 pups with respect to UN pups (50%) was observed (Fig. 4). MBP showed a different diminution degree in its isoforms. MBP decreased in UN pups (29, 49, 30, and 52%), in WN70 pups (44, 50, 50, and 47%) and in UN70 pups (64, 72, 64, and 73% accordingly) with respect to WN pups (Fig. 5). FIG. 4. Open in new tabDownload slide The effect of 2,4-D and undernourishment on CNPase myelin expression. (A) WB for CNPase were performed with myelin isolated from brains of control and exposed pups at PND 22. Note the decreased CNPase expression in UN and 2,4-D exposed pups. (B) Semiquantification was performed with respect to the control group (WN). The values are expressed as mean ± SEM. ap < 0.001 relative to Control (WN). FIG. 4. Open in new tabDownload slide The effect of 2,4-D and undernourishment on CNPase myelin expression. (A) WB for CNPase were performed with myelin isolated from brains of control and exposed pups at PND 22. Note the decreased CNPase expression in UN and 2,4-D exposed pups. (B) Semiquantification was performed with respect to the control group (WN). The values are expressed as mean ± SEM. ap < 0.001 relative to Control (WN). FIG. 5. Open in new tabDownload slide The effect of 2,4-D and undernourishment on MBP myelin expression. (A) WBs for MBP were performed with myelin isolated from brains of control and exposed pups at PND 22. Note the decreased MBP expression in UN and 2,4-D exposed pups and the different diminution degree in its isoforms. (B) Semiquantification was performed with respect to control group (WN). The values are expressed as mean ± SEM. ap < 0.001 relative to control (WN). FIG. 5. Open in new tabDownload slide The effect of 2,4-D and undernourishment on MBP myelin expression. (A) WBs for MBP were performed with myelin isolated from brains of control and exposed pups at PND 22. Note the decreased MBP expression in UN and 2,4-D exposed pups and the different diminution degree in its isoforms. (B) Semiquantification was performed with respect to control group (WN). The values are expressed as mean ± SEM. ap < 0.001 relative to control (WN). Differences in isolated myelin composition were detected. All 2,4-D exposed pups (WN and UN) showed a significant decrease of total protein, total lipid, cholesterol, phospholipid, and galactolipid levels. However, a significant decrease in total protein, cholesterol and galactolipid contents in UN group with respect to WN pups was observed (Table 1). Ratios differences among the different chemical components were not found. TABLE 1 Myelin Chemical Composition Total proteins (mg/g brain) Total lipids (mg/g brain) Cholesterol (mg/g brain) Galactolipids (mg/g brain) Phospholipids phosphorus (nm P/g brain) WN 6.0 ± 0.7 683.6 ± 117.2 3.2 ± 0.2 1.0 ± 0.3 9.1 ± 1.6 WN70 4.3 ± 0.3* 478.6 ± 71.2** 2.0 ± 0.3** 0.6 ± 0.1** 4.9 ± 1.4* UN 4.5 ± 0.6* 526.2 ± 94.6 1.9 ± 0.2** 0.5 ± 0.2* 6.8 ± 2.1 UN70 3.4 ± 1.0***,† 218.3 ± 121.1***,† 1.7 ± 0.2** 0.3 ± 0.2* 4.4 ± 1.9*** Total proteins (mg/g brain) Total lipids (mg/g brain) Cholesterol (mg/g brain) Galactolipids (mg/g brain) Phospholipids phosphorus (nm P/g brain) WN 6.0 ± 0.7 683.6 ± 117.2 3.2 ± 0.2 1.0 ± 0.3 9.1 ± 1.6 WN70 4.3 ± 0.3* 478.6 ± 71.2** 2.0 ± 0.3** 0.6 ± 0.1** 4.9 ± 1.4* UN 4.5 ± 0.6* 526.2 ± 94.6 1.9 ± 0.2** 0.5 ± 0.2* 6.8 ± 2.1 UN70 3.4 ± 1.0***,† 218.3 ± 121.1***,† 1.7 ± 0.2** 0.3 ± 0.2* 4.4 ± 1.9*** Note. Chemical composition of myelin isolated from brains of control and exposed pups at PND 22. The values are expressed as mean ± SEM, n = 5 litters. **p < 0.05, *p < 0.01, and ***p < 0.001 significantly different from control group (WN). †p < 0.001 significantly different from UN group. Open in new tab TABLE 1 Myelin Chemical Composition Total proteins (mg/g brain) Total lipids (mg/g brain) Cholesterol (mg/g brain) Galactolipids (mg/g brain) Phospholipids phosphorus (nm P/g brain) WN 6.0 ± 0.7 683.6 ± 117.2 3.2 ± 0.2 1.0 ± 0.3 9.1 ± 1.6 WN70 4.3 ± 0.3* 478.6 ± 71.2** 2.0 ± 0.3** 0.6 ± 0.1** 4.9 ± 1.4* UN 4.5 ± 0.6* 526.2 ± 94.6 1.9 ± 0.2** 0.5 ± 0.2* 6.8 ± 2.1 UN70 3.4 ± 1.0***,† 218.3 ± 121.1***,† 1.7 ± 0.2** 0.3 ± 0.2* 4.4 ± 1.9*** Total proteins (mg/g brain) Total lipids (mg/g brain) Cholesterol (mg/g brain) Galactolipids (mg/g brain) Phospholipids phosphorus (nm P/g brain) WN 6.0 ± 0.7 683.6 ± 117.2 3.2 ± 0.2 1.0 ± 0.3 9.1 ± 1.6 WN70 4.3 ± 0.3* 478.6 ± 71.2** 2.0 ± 0.3** 0.6 ± 0.1** 4.9 ± 1.4* UN 4.5 ± 0.6* 526.2 ± 94.6 1.9 ± 0.2** 0.5 ± 0.2* 6.8 ± 2.1 UN70 3.4 ± 1.0***,† 218.3 ± 121.1***,† 1.7 ± 0.2** 0.3 ± 0.2* 4.4 ± 1.9*** Note. Chemical composition of myelin isolated from brains of control and exposed pups at PND 22. The values are expressed as mean ± SEM, n = 5 litters. **p < 0.05, *p < 0.01, and ***p < 0.001 significantly different from control group (WN). †p < 0.001 significantly different from UN group. Open in new tab Ultrastructurally myelin sheath of undernourished pups (UN and UN70) showed a lower number of sheets with respect to WN pups. The ID was increased in UN70 (19%) with respect to WN pups. In addition, the MDLt was diminished in WN 70, UN70, and also in UN (see Table 2). TABLE 2 Morphometric Study at Ultrastructural Level of the Myelin around Corpus Callosum Transversally Sectioned Axons Number of sheets ID (nm) MDLt (nm) WN 7.90 ± 1.90 9.12 ± 0.39 2.70 ± 0.12 WN70 6.00 ± 1.50 8.01 ± 0.68 2.18 ± 0.30* UN 5.60 ± 2.00* 8.82 ± 1.00 2.30 ± 0.17* UN70 5.90 ± 1.50* 10.83 ± 0.96*,** 1.78 ± 0.34*,** Number of sheets ID (nm) MDLt (nm) WN 7.90 ± 1.90 9.12 ± 0.39 2.70 ± 0.12 WN70 6.00 ± 1.50 8.01 ± 0.68 2.18 ± 0.30* UN 5.60 ± 2.00* 8.82 ± 1.00 2.30 ± 0.17* UN70 5.90 ± 1.50* 10.83 ± 0.96*,** 1.78 ± 0.34*,** Note. From 12 electronmicrographs at 31,500× (primary magnification), the mean of the number of sheet in the myelin sheath of each axon transversally sectioned and the SEM are shown. From other 12 electronmicrographs at 100,000× (primary magnification), the mean of ID and MDLt with their SEM are shown. Besides, the mean of the number of myelin sheet for each axon transversally sectioned and the SEM are shown. *p < 0.01 relative to WN group. **p < 0.01 relative to UN pups. Open in new tab TABLE 2 Morphometric Study at Ultrastructural Level of the Myelin around Corpus Callosum Transversally Sectioned Axons Number of sheets ID (nm) MDLt (nm) WN 7.90 ± 1.90 9.12 ± 0.39 2.70 ± 0.12 WN70 6.00 ± 1.50 8.01 ± 0.68 2.18 ± 0.30* UN 5.60 ± 2.00* 8.82 ± 1.00 2.30 ± 0.17* UN70 5.90 ± 1.50* 10.83 ± 0.96*,** 1.78 ± 0.34*,** Number of sheets ID (nm) MDLt (nm) WN 7.90 ± 1.90 9.12 ± 0.39 2.70 ± 0.12 WN70 6.00 ± 1.50 8.01 ± 0.68 2.18 ± 0.30* UN 5.60 ± 2.00* 8.82 ± 1.00 2.30 ± 0.17* UN70 5.90 ± 1.50* 10.83 ± 0.96*,** 1.78 ± 0.34*,** Note. From 12 electronmicrographs at 31,500× (primary magnification), the mean of the number of sheet in the myelin sheath of each axon transversally sectioned and the SEM are shown. From other 12 electronmicrographs at 100,000× (primary magnification), the mean of ID and MDLt with their SEM are shown. Besides, the mean of the number of myelin sheet for each axon transversally sectioned and the SEM are shown. *p < 0.01 relative to WN group. **p < 0.01 relative to UN pups. Open in new tab Electronmicrograph studies also showed a compact myelin covering on central axons of corpus callosum from control, WN and UN pups, without disruption (see Figs. 6A, 7A, and 8A; and Figs. 6B, 7B, and 8B). Nevertheless, a lower number of myelin sheets in the UN pups (Figs. 6A and 6B) with respect to the sheets in the WN group (Figs. 7A and 7B), was observed. Although the number of sheets present in the cover of myelin of WN 70 group was similar to that of the WN pup group, the presence of and presence of a few disrupted myelin layers forming blebs and undulating major dense line (MDL) was observed (Fig. 7D). A reduced number of sheets but with presence of blebs, undulating MDL and disruptions of the ID were observed in UN70 pups (Fig. 6D). FIG. 6. Open in new tabDownload slide Electronmicrographs of myelinated axons from corpus callosum of UN (A and B) and UN70 (C and D) pups. Myelinated axons sectioned transversally (a) showed the cover of myelin (A and C). In UN (A and B) the myelin was compact but with scarce number of sheets, the inner mesaxon (IM) was present without disruption. In UN70 (C and D) the myelin was not compacted, there was disruption of the intraperiod line (asterisks). Calibration bars: 2 μm (A and C); 50 nm (B and D). FIG. 6. Open in new tabDownload slide Electronmicrographs of myelinated axons from corpus callosum of UN (A and B) and UN70 (C and D) pups. Myelinated axons sectioned transversally (a) showed the cover of myelin (A and C). In UN (A and B) the myelin was compact but with scarce number of sheets, the inner mesaxon (IM) was present without disruption. In UN70 (C and D) the myelin was not compacted, there was disruption of the intraperiod line (asterisks). Calibration bars: 2 μm (A and C); 50 nm (B and D). FIG. 7. Open in new tabDownload slide Electronmicrographs of myelinated axons from corpus callosum of WN (A and B) WN70 (C and D). The axons (A) showed the typical cover of compact myelin with approximately eight sheets (B). (C) A deficient number of sheets are present in the cover of myelin. It is possible to see the presence of blebs (b) in the myelin sheaths that is evident in (D) and the presence of disruption of the myelin sheet due to the disruption of intraperiod line (asterisks). Calibration bars: 2 μm (A and C); 50 nm (B and D). FIG. 7. Open in new tabDownload slide Electronmicrographs of myelinated axons from corpus callosum of WN (A and B) WN70 (C and D). The axons (A) showed the typical cover of compact myelin with approximately eight sheets (B). (C) A deficient number of sheets are present in the cover of myelin. It is possible to see the presence of blebs (b) in the myelin sheaths that is evident in (D) and the presence of disruption of the myelin sheet due to the disruption of intraperiod line (asterisks). Calibration bars: 2 μm (A and C); 50 nm (B and D). FIG. 8. Open in new tabDownload slide Electronmicrographs of myelinated axons from corpus callosum of control rats with saline. Note sections of control myelinated axons in (A) and the compact disposition of the myelin sheath in (B). Calibration bars: 2 μm (A) and 50 nm (B). FIG. 8. Open in new tabDownload slide Electronmicrographs of myelinated axons from corpus callosum of control rats with saline. Note sections of control myelinated axons in (A) and the compact disposition of the myelin sheath in (B). Calibration bars: 2 μm (A) and 50 nm (B). On the other hand, no effect of DMSO in the ultrastructural organization of the central myelin was detected (compare Fig. 8A with Fig. 7A and Fig. 8B with Fig. 7B). DISCUSSION Myelination has been regarded as one of the most useful morphological parameters to observe the development and maturation of the CNS. In the rat brain, most oligodendrocytes complete a proliferation phase between approximately 1-2 weeks after birth, which is followed by a period of a very rapid rate of myelination that increases to a maximum at about 20–22 days of age and then declines to a low level that appears to be maintained throughout the life of the rats (Miller, 1992). Myelin deposition could be affected by insults to the process of myelination during different stages of development. It is known, that myelination is sensitive to nutritional factors. Brains of UN rats from birth contain a lower amount of total lipids, cholesterol, phospholipids and a 50% deficit of cerebrosides (Bass et al., 1970; Benton et al., 1966). Morphological examination of UN animals from birth showed poorly stained myelin and the number of myelin lamellae per axon and the number of myelin lamellae for a given axon diameter were both lower (Krigman and Hogan, 1976). Similar results in our UN pups which showed lower levels in myelin components (total protein, cholesterol, galactolipid, MBP, and PLP) were found in this study. In addition, although myelin does not present compaction problems, a lower number of myelin sheets and a decrease in MDLt were observed. Our results also agree with Fuller et al. (1984) who described that the myelin deposition would decrease about 22–25% with a diminution of body weight of 25–30%. On the other hand, the elaboration of CNS myelin requires the large-scale synthesis of myelin-specific lipids and membrane-associated proteins (Campagnoni, 1995; Pfeiffer et al., 1993) presumed to play specific roles in spiral wrapping, membrane compaction and fine architecture of the multilayer myelin sheath. These abundant myelin components play a key role in the formation of structurally normal CNS myelin. Among them, galactolipids—the significant glycolipids of myelin—appear to facilitate the maturation of both, oligodendrocytes and myelinated fibers and, are necessary for the normal development of axo-glial interaction at nodes of Ranvier. Galactolipids from myelin of UN and all 2,4-D exposed pups were diminished with respect to WN pups. During myelination, specific lipid and protein components are assembled into the highly ordered plasma membrane of the tongue-like processes of oligodendrocytes. They address up to 100 axons and ensheath them in a wrapping process in which the cytoplasm is “squeezed” out, and the surfaces of the inner leaflets become closely apposed forming the MDL. Several studies have demonstrated that MBP is essential in formation of MDL (Readhead et al., 1987) and that there are interactions between MBP and sulfatides (Maggio and Yu, 1989). In addition, myelin PLP is the major intrinsic protein of CNS myelin. PLP forms stabilizing membrane junction after myelin compaction similar to a “zipper.” It is also known, that CNPase is not present in compact myelin but it is enriched at the cytoplasmic side of all other surface membrane of myelinating oligodendrocytes and participates in membrane expansion and migration (Yin et al., 1997). Alterations in some of these components lead to disruptions in myelin formation, compaction, conduction, etc. Individual immunostaining from the main myelin proteins showed that MBP and PLP were decreased in all 2,4-D exposed pups but, only PLP was decreased in UN pups. However, WB analyses showed lower contents of CNP and MBP in all treated animals (PLP WB was not done because the antibody does not work). A lower content of PLP and/or MBP, turns myelin physically labile and tends to delaminate and to present a clear disruption of the compaction of myelin sheaths as could be observed after 2,4-D exposure. The decreased quantity of the main myelin proteins and lipids could be explained by the action of the xenobiotic on gene expression, abnormal sorting and/or transport to the corresponding place. The ultrastructure of myelin is characterized by its periodicity between the MDL, representing the cytosolic cleft between the apposed inner surfaces of the plasma membrane and the intermediate line imaging the space between the apposed external membrane surface. A decreased MDLt in myelin of 2,4-D exposed pups (both WN and UN ones) and from UN pups was determined in this study. These morphometric results at electron microscopic level agree with an abnormal myelination processes. Both WN and UN groups of rats that received only DMSO and those rats that were not given DMSO showed a distance between two consecutive MDLs of 8 nm (Table 2). This distance was increased in UN70 pups, showing a clear disruption of the myelin sheath, with expanded intraperiod spaces, disruption of the ID, undulating MDL and also the presence of blebs. Although WN70 pups did not present a significant ID increase compared with the control group, its myelin sheath showed expanded intraperiod spaces and presence of disrupted myelin layers forming blebs, indicating myelin compaction alterations. Previous studies from our laboratory, demonstrated that the CNS is essentially involved in the 2,4-D toxicity, especially if the compound is present in the brain during CNS maturation (Duffard et al., 1987, 1996). This work found, (through a morphometric, immunohistological, and chemical study) hypomyelination in WN 2,4-D exposed rats, which have no body weight variation with respect to controls. These results indicate that undernourishment and 2,4-D exposure during myelination are two independent hypomyelinic factors. In conclusion, the results obtained in this study show that undernourishment hypomyelination was expressed by a diminution of the number of sheets and in the MDLt, whereas 2,4-D hypomyelination had a normal number of sheets but presented altered compaction of myelin sheaths, increased ID and the presence of a higher number of blebs. Taking into account that 2,4-D induces neuronal death of granular cerebellar cells in culture (De Moliner et al., 2002) and also a diminution of the axon length in those live neurons (Rosso et al., 2000a), it is possible to deduce that (1) there are less axons to be ensheathed or (2) a direct 2,4-D action on oligodendrocytes prevents their normal maturation and thus the number of the tongue-like processes of each oligodendrocyte diminishes or (3) both processes could also be present. 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Chemical and Ultrastructural Studies in Rats JF - Toxicological Sciences DO - 10.1093/toxsci/kfn085 DA - 2008-08-01 UR - https://www.deepdyve.com/lp/oxford-university-press/neonatal-hypomyelination-by-the-herbicide-2-4-dichlorophenoxyacetic-kvDBqX4VzZ SP - 332 EP - 340 VL - 104 IS - 2 DP - DeepDyve ER -