2019 Volume 44 Issue 11 Pages 799-809
In the present study, we investigated the effects of lead (Pb) and ascorbic acid co-administration on rat cerebellar development. Female rats were randomly divided into the following groups: control, Pb, and Pb plus ascorbic acid (PA) groups. From one week prior to mating, female rats were administered Pb (0.3% Pb acetate in drinking water) and ascorbic acid (100 mg/kg, oral intubation). The chemical administration was stopped on postnatal day 21 when the morphology of the offspring’s cerebellum is similar to that of the adult brain. The blood Pb level was significantly increased following long-term Pb exposure. Ascorbic acid reduced Pb levels in the dams and offspring. Nissl staining demonstrated that the number of Purkinje cells was significantly reduced following Pb exposure, while ascorbic acid ameliorated this effect in the cerebellum of the offspring. Calcium-binding proteins, such as calbindin, calretinin, and parvalbumin were commonly expressed in Purkinje cells, and Pb exposure and ascorbic acid treatment resulted in similar patterns of change, namely Pb-induced impairment and ascorbic acid-mediated amelioration. The gamma-aminobutyric acid transporter 1 (GABAT1) is expressed in the pinceau structure where the somata of Purkinje cells are entwined in inhibitory synapses. The number of GABAT1-immunoreactive synapses was reduced following Pb exposure, and ascorbic acid co-treatment prevented this effect in the cerebellar cortex. Therefore, it can be concluded that ascorbic acid supplementation to mothers during gestation and lactation may have potential preventive effects against Pb-induced impairments in the developing cerebellum via protection of inhibitory neurons and synapses.
Environmental and occupational exposure to lead (Pb) has been an important public health issue. The history of Pb uses dates to ancient Roman times when it was used in water pipes and the Middle Ages when it was used as a wine sweetener. The use of Pb increased markedly in the industrial age. Although strict restriction of Pb use has decreased its release into the environment, Pb that was previously used in petrol, paints, batteries, chemicals, solder, cable/tube sheeting, and water pipelines is still contaminating the environment (Castellino et al., 1994). Despite its usefulness in industry, Pb is a noxious non-essential heavy metal for human beings. Pb can be absorbed into our bodies by various routes, such as ingestion, inhalation, skin absorption, and placental/milk transmission (WHO, 2010). Generally, Pb is toxic to individuals of all ages, although there is high susceptibility during the developmental stage. Multiple organs are targets of Pb-induced toxicity, and the central nervous system is the primary target (WHO, 2010). Pb-induced neurotoxicity involves oxidative stress and excitotoxicity (Al-Quraishy et al., 2016). The developing brain is highly susceptible to the oxidative stress due to its high metabolic rates and high oxygen demand combined with its immature antioxidant systems (Ikonomidou and Kaindl, 2011; Shim and Kim, 2013). In our previous studies, we demonstrated that gestational low-dose Pb (0.2%) exposure led to neurotoxicity in the developing hippocampus and cerebellum (Chang et al., 2012; Nam et al., 2018a). The developmental period with the maximum level of neurogenesis in the cerebellum is the early postnatal period in rodents and the late period of gestation in humans (Stagni et al., 2015). Therefore, we exposed rats to Pb for a long period, from pre-gestation to lactation.
Ascorbic acid is an essential nutrient in humans and guinea pigs. We thus require ingestion of ascorbic acid from exogenous sources. In contrast, rats and mice can synthesize ascorbic acid in the liver (Du et al., 2012). Ascorbic acid is a cofactor of many enzymes and is involved in the synthesis of collagen, carnitine, and catecholamines (Iqbal et al., 2004). It is also an important reducing agent used for the regeneration of active enzymes from inactivated forms (Du et al., 2012). Ascorbic acid is involved in the development of several organ systems, such as bones, the brain, and the immune system (Kim et al., 2013, 2015a; NHS, 2015). Ascorbic acid levels are especially high in the brain, and its levels increase during the late phase of gestation in the rat brain (Kratzing et al., 1985). Ascorbic acid induces neuronal differentiation and synaptic maturation of the embryonic stem cells (Lee et al., 2003). It is also the primary antioxidant in the body, as its deficiency renders the developing brain vulnerable to oxidative stress and excitotoxicity (Tveden-Nyborg and Lykkesfeldt, 2009). Notably, ascorbic acid is useful as a supportive therapy for Pb intoxication by increasing its renal excretion (Lowry, 2010).
Purkinje cells and other gamma-aminobutyric acid (GABA)ergic cells in the developing cerebellar cortex are derived from the neuroepithelium of the ventricular zone. Glutamatergic granule cells are derived from rhombic lips and initially form an external granule cell layer. They then descend to shape the internal granule cell layer (Martinez et al., 2013). Appropriate cellular proliferation and migration are required for proper development of the cerebellum. Among the cellular components of the cerebellar cortex, Purkinje cells, stellate cells, basket cells, and Golgi cells are GABAergic cells (Schilling, 2018). Stellate cells and basket cells regulate the activities of Purkinje cells, which form the only output of the cerebellar cortex. The axons of Purkinje cells project to deep cerebellar nuclei and vestibular nuclei (Canto et al., 2016; Mordel et al., 2013). Axons of the stellate cells contact the dendritic trees of the Purkinje cells, while axons of basket cells wrap around the somata and initial axonal segments of the Purkinje cells, forming the pinceau structure (Laube et al., 1996; Manova et al., 1992). The proper development of neuronal cells and their maturation is associated with subsequent synapse formation, signal transmission, and normal functioning of the cerebellum (zur Nedden et al., 2018). The cerebellum is an important organ the controls the balance, motor coordination, and motor learning (Ito, 2000). Improper development and regulation of the cerebellar Purkinje cells is one of the causes of the development of ataxia (Hoxha et al., 2018).
Until now, Pb-induced changes in the inhibitory cellular components of the cerebellar cortex were not clearly understood. Additionally, previous studies including one from our group have investigated the effects of low-dose (0.1% or 0.2%) and high-dose (0.54% or 1%) Pb. However, few studies have investigated the effects of an intermediate dose (0.3%) of Pb (Hossain et al., 2016; Mousa et al., 2015; Nam et al., 2018a). Here, we investigated the effects of 0.3% Pb on inhibitory neuronal development in the immature cerebellum of rat offspring. In addition, we investigated the effects of ascorbic acid co-treatment on the development of the cerebellar cortex.
The experimental protocol of the present study was approved by the Institutional Animal Care and Use Committee of the Konkuk University (KU18133). Female (n = 12) and male (n = 6) Sprague Dawley rats (8 weeks old) purchased from Narabiotec Co., Ltd (Seoul, Korea) were housed under conditions of adequate temperature (23ºC) and humidity (60%), with a 12-hr light/12-hr dark cycle. The animals were used for experiments after one week of acclimation to a conventional environment at the animal facility in the College of Veterinary Medicine. Pregnancy was confirmed when vaginal plugs were present or when sperm was detected on vaginal smears. The day on which this confirmation took place was designated day 0. Male and female rats were separated again and pregnant females were single-housed until the end of the experiments. Animals were handled and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, which was issued by the Institute of Laboratory Animal Resources, USA, 1996.
Adult female Sprague Dawley rats were randomly divided into 3 groups: control group (n = 4), Pb group (n = 4), and Pb plus ascorbic acid (PA) group (n = 4). The doses of ascorbic acid were adopted from previous studies (Chang et al., 2012; Nam et al., 2018a, 2018b). Water-soluble Pb acetate (0.3%; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled water with glacial acetic acid (0.05%; Junsei Chemical Co., Tokyo, Japan) to prevent Pb precipitation (Dieter et al., 1993; Nam et al., 2018b). Ascorbic acid (100 mg/kg; Sigma-Aldrich) for oral administration was freshly prepared in saline daily. To adjust for the effects of stress during oral intubation, rats in the control and Pb groups were orally administered the same volume of saline. Pb and ascorbic acid treatments were started 1 week prior to the mating day and continued during gestation and delivery of offspring until the end of the experiment on a postnatal day (PND) 21. The body weights of offspring were measured and averaged per every week. To avoid the effects of litter size in the different experimental groups, 6 rat offspring per cage were randomly selected and the remaining offspring were sacrificed. Before sacrifice, the sex of every offspring was recorded. The researchers in the present study conducted experimental procedures carefully to minimize suffering and the number of animals used.
On PND21, 12 offspring per group and 3 dams per group were anesthetized with an intraperitoneal injection of 1.5 g/kg urethane (Sigma-Aldrich). Blood samples were collected from the heart for analysis of Pb levels.
As described in the previous study (Hwang et al., 2015), procedures for blood Pb analyses were performed in the Neodin Biovet Laboratory (Seoul, Korea), certified by the Korean Ministry of Health and Welfare. Blood Pb levels were analyzed by using the PinAAcle 500 flame atomic absorption spectrometer (Perkin Elmer Zeeman 5100; Norwalk, CT, USA) and an HGA-600 graphite furnace with Zeeman background correction. As reported in the previous report (Modesto et al., 2016), the samples were prepared by mixing 100 μL of whole blood (with anti-coagulant) with 900 μL matrix modifier. Matrix modifier was made by mixing 2 g of ammonium phosphate diabasic (Sigma Aldrich) and 2 mL of Triton X-100 (Sigma Aldrich) in distilled water 998 mL. On the other hand, the recovery samples were prepared by mixing 100 μL blood sample, 100 μL working standard solution and 800 μL matrix modifier. For internal quality assurance and control, commercial standard reference materials were obtained from Bio-Rad (Lyphocheck Whole Blood Metals Control; Hercules, CA, USA). The coefficients of variation were 0.68-4.11% upon analysis of reference samples. In terms of external quality control, the Neodin has passed the German External Quality Assessment Scheme operated by Friedrick Alexander University. The spiked Pb sample showed good percentage recoveries, the limit of detection was 0.070 μg/dL, and these values were similar to the previous study (Rawar and Rohman, 2016). The absorption wavelength was 283.3 nm and the r2 of the calibration curve exceeded 0.995. The weights of the brain and cerebellum of offspring were measured on PND21 (n = 12 per group).
For histological analysis, the other 12 offspring (n = 12 per group) were anesthetized using 1.5 g/kg urethane (Sigma-Aldrich) on PND21 and perfused transcardially with heparinized phosphate-buffered saline (PBS; 0.1 M, pH 7.4), followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The cerebella were removed and post-fixed in the same fixative overnight at 4ºC. Cerebellar tissues (n = 12 per group) were embedded in paraffin blocks and midsagittal sections of the vermis were cut into 5 μm slices at multiple levels. Three slides per offspring were selected, and a total of 36 paraffin sections per group were used in the histological analysis. Nissl staining was conducted using routine procedures. Immunohistochemistry for target marker proteins was conducted as follows. Deparaffinized sections were subjected to antigen retrieval using citrate buffer (pH 6.0). The sections were then sequentially treated with 0.3% hydrogen peroxide (H2O2) to quench endogenous peroxidase activity, and with 10% normal horse serum for blocking. Next, the sections were incubated overnight at 4°C in antibodies against calbindin-28kD (CB, 1:5000; Swant, Bellinzona, Switzerland), calretinin (CR, 1:2000; Swant), GABA transporter 1 (GABAT1, 1:500; Synaptic Systems, Göttingen, Germany), NeuN (1:500; Millipore, Billerica, MA, USA), or parvalbumin (PV, 1:5,000; Swant). Subsequently, sections were exposed to biotinylated immunoglobulin G (1:200; Vector, Burlingame, CA, USA) and streptavidin-peroxidase complex (1:200; Vector). They were then visualized by reaction with 3,3’-diaminobenzidine tetrachloride (Sigma) in 0.1 M Tris-HCl buffer (pH 7.2) and mounted on gelatin-coated slides. The sections were finally dehydrated and mounted in a toluene-based mounting medium (Richard-Allan Scientific, Thermo Scientific, Detroit, MI, USA).
All histopathological analyses described below were performed by an investigator blinded to the rat treatment. The quantification method of the present study was modified from the recent study (Kandeel et al., 2017). Specifically, the numbers of Purkinje cells in the cerebellum were counted in micrographs obtained at 100 × magnification per 5,000 μm length of Purkinje cell layer using arbitrary line probe of the DP2-BSW software (Olympus, Tokyo, Japan). The observations were carried out in the 2nd, 5th, and 8th lobules of the sagittal section of the cerebellar vermis (Nam et al., 2018b).
Data are expressed as means ± standard errors of the mean for each group, and the significance of the differences between these mean values was determined using one-way analysis of variance followed by Tukey’s test for multiple comparisons. All analyses were performed using GraphPad Prism 5.01 software (GraphPad Software, Inc.; La Jolla, CA, USA). P-values < 0.05 were considered significant.
We observed differences in body weight gain in the dams and offspring during the experiment. Pb exposure induced a decrease in the average body weight of the offspring, while ascorbic acid ameliorated the Pb-induced reduction. Additionally, Pb exposure during pregnancy and lactation affected cerebellar weight in offspring on PND21. The prominent Pb-induced reduction in cerebellar weight was ameliorated by ascorbic acid treatment in the offspring. Developmental processes in cerebellar morphogenesis, such as foliation, migration, and lamination were not affected by Pb exposure. Atomic absorption spectrometry demonstrated that blood Pb levels in dams and offspring were increased in the Pb group, and ascorbic acid co-treatment reduced the increase in Pb blood levels (Fig. 1).
Body weights of offspring (n = 12 per group) on a postnatal day (PND) 0, 7, 14, and 21 in control (CTL), lead (Pb), and lead plus ascorbic acid (PA) groups (A). Brain (B) and Cerebellar weights of offspring on PND21 (C). Blood lead levels in the dams and offspring on day 21 after delivery (D) (aP < 0.05, CTL group versus Pb group, bP < 0.05, Pb group versus PA group). The bars indicate means ± standard errors.
In the cerebellar cortex, we primarily evaluated the effects of Pb exposure and ascorbic acid treatment on the architecture of the cerebellum. On PND21, three layers of the cerebellar cortex were observed in all groups. After Nissl staining and CB immunohistochemistry, the number of the Purkinje cells was significantly reduced following Pb exposure in the cerebellum, while ascorbic acid ameliorated the Pb-induced reduction in the number of Purkinje cells in the cerebellum. Degenerating pyknotic cells were commonly detected in the Purkinje cell layer in rats in the Pb group. After ascorbic acid treatment, the frequency of detection of the pyknotic cells was reduced in the cerebellum. Images obtained at higher magnification revealed that the dendritic branches of CB-immunoreactive Purkinje cells were well-developed and intricately elaborate dendritic arbors were detected in the control group. Pb exposure impaired the development of dendrites of the Purkinje cells, and the remaining intact Purkinje cells developed dendrites to compensate for the degeneration of the Purkinje cells in their vicinity. However, ascorbic acid restored the numbers and dendritic branching of the Purkinje cells in the cerebellar cortex. Unlike cells in the Purkinje cell layer, those in the granule cell layer did not show any significant changes in different experimental groups (Fig. 2).
Nissl staining (A, B, and C) and immunohistochemistry for calbindin (CB) in the cerebellum (D, E, and F) in offspring from control (CTL), lead (Pb), and lead plus ascorbic acid (PA) groups. GCL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar = 25 μm. The numbers of intact Purkinje cells and CB-positive Purkinje cells in the cerebellar cortex (G) are expressed as percentages of the value in the CTL group (n = 12 per group; aP < 0.05, CTL group versus Pb group, bP < 0.05, Pb group versus PA group). The bars indicate the means ± standard errors of the mean.
CR was detected in the Purkinje cell layer and in some cells in the granule cell layer in the cerebellar cortex of the offspring. After long-term exposure to Pb, CR was weakly expressed in the Purkinje cells and the number of CR-positive Purkinje cells was prominently reduced. However, the co-administration of ascorbic acid attenuated the reduction in the number of CR-positive Purkinje cells (Fig. 3). Unlike CR-positive Purkinje cells, CR-positive cells in the granule cell layer were not significantly different among the three groups. A previous study reported that CR was expressed in the Golgi cells, other interneurons, and granule cells in the granular cell layer (Pibiri et al., 2017; Wierzba-Bobrowicz et al., 2011). We additionally evaluated the expression pattern of NeuN-positive granule cells to examine the differences among experimental groups. Similar to CR-expressing cells in the granule cell layer, NeuN-positive granule cells were not prominently affected by the Pb and ascorbic acid treatments (Fig. 3).
Immunohistochemistry (A, B, and C) for calretinin (CR) and NeuN (D, E, and F) in the cerebellum in offspring from control (CTL), lead (Pb), and lead plus ascorbic acid (PA) groups. GCL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar = 25 μm. The numbers of CR-positive Purkinje cells (G) in the cerebellar cortex are expressed as percentages of the value in the CTL group (n = 12 per group; aP < 0.05, CTL group versus Pb group, bP < 0.05, Pb group versus PA group). The bars indicate the means ± standard errors of the mean.
PV was detected mainly in the cells in the Purkinje cell layer and in some cells in the molecular and granule cell layers in the cerebellar cortex of the offspring. Compared to the control group, Pb treatment significantly reduced the number of PV-immunoreactive Purkinje cells. Additionally, the dendrites of PV-immunoreactive Purkinje cells degenerated following Pb exposure. Ascorbic acid treatment prevented the Pb-induced dendritic impairment and reduction in cell number in the PA group (Fig. 4).
Immunohistochemistry (A, B, and C) for parvalbumin (PV) and GABA transporter 1 (GABAT1) (D, E, and F) in the cerebellum in offspring from control (CTL), lead (Pb), and lead plus ascorbic acid (PA) groups. Note that the numbers of PV-positive Purkinje cells and GABAT1-positive pinceau structures near Purkinje cells were significantly reduced in the Pb group, and that ascorbic acid treatment ameliorated these reductions in the PA group. GCL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar = 25 μm. The numbers of PV-positive Purkinje cells (G) and GABAT1-positive pinceau structures (G) are expressed as percentages of the value in the CTL group in the cerebellum (n = 12 per group; aP < 0.05, CTL group versus Pb group, bP < 0.05, Pb group versus PA group). The bars indicate the means ± standard errors of the mean.
GABAT1 mediates the uptake of GABA, which is important for brain morphogenesis during development (Takayama and Inoue, 2005). In all groups, GABAT1 was mainly detected near the somata of the Purkinje cells, as scattered puncta in the molecular layer, and as puncta and ring-shaped structures in the granule cell layer of the cerebellar cortex. In the periphery of the Purkinje cells, GABAT1 immunoreactivity is due to basket cells, which wrap their somata and initial segments of the axon around Purkinje cells. The resulting specialized structure, called the pinceau, is important for regulating the cerebellar cortical output by inhibiting the axons of the Purkinje cells (Laube et al., 1996). In the present study, the pattern of the GABAT1 immunoreactivity in the pinceau structure was similar to the changes observed in Purkinje cells in the cerebellar cortex. The Pb exposure-induced reduction in the number of Purkinje cells was associated with the reduction in the number of GABAT-immunoreactive pinceau. Ascorbic acid ameliorated this Pb-mediated impairment by increasing the numbers of both Purkinje cells and pinceau structures. In other words, the synapses between the basket cells and Purkinje cells were susceptible to Pb-induced developmental defects, and ascorbic acid promoted the generation of such synapses (Fig. 4).
The process of neuronal cell generation, migration, maturation, and subsequent synapse formation in the developing brain is susceptible to exogenous materials that may affect the functionally immature glial system and fragile blood vessels in the developing brain (Saunders et al., 2012; Yang et al., 2013). Pb is a non-essential hazardous heavy metal that primarily targets the brain as it crosses the blood-brain barrier when transported through the blood. Pb is absorbed by ingestion or transferred from mother to offspring by crossing the placenta. Compared to those in the adult cerebellum, neurons in the developing cerebellum are more vulnerable to Pb-induced toxicity (Nam et al., 2018a, 2018b, 2019). Inhibitory Purkinje cells and excitatory granule cells are the main components of the cerebellum regulating balance and movement. These cells mature within the first 3 weeks after birth, a time when they are susceptible to the cellular insults (Stagni et al., 2015; Faustino and Ortiga-Carvalho, 2014). We thus investigated the effects of Pb exposure during gestation and lactation on cerebellar development in offspring.
First, in the present study, physiological parameters such as body weight gain, cerebellum weight, and blood Pb level were different among the control, Pb, and PA groups. Long-term Pb exposure showed a reduction in body weight and cerebellar weight while significantly increasing the blood Pb level in the offspring. Previous studies have also reported that low- and high-dose Pb treatments increase the blood and brain concentrations of Pb (Hossain et al., 2016; Kang et al., 2004). While ascorbic acid treatment was effective in reducing the blood Pb level in the mothers, indirect ingestion of ascorbic acid in the offspring resulted only in a slight reduction in the Pb level, which was not statistically significant. During the fetal period, Pb-exposed mothers transfer Pb to the offspring via the placenta. Postnatal rats are subsequently exposed to excreted Pb in their rearing environment via Pb-containing urine and feces. Secondarily, offspring are exposed to excreted Pb by suckling Pb-containing milk (Hossain et al., 2016). When compared to a previous study, exposure to an intermediate dose of Pb (0.3%) raised the blood Pb levels to higher values in both the mothers and offspring (Nam et al., 2018a). Similarly, ascorbic acid treatment was beneficial in reducing the blood level of Pb in both mothers and offspring, although the basal levels were higher given the present experimental conditions (Nam et al., 2018b; Sadeghi et al., 2013). In addition to enhancing the excretion of Pb, ascorbic acid reduces the absorption of Pb by chemically binding to Pb (Kim et al., 2015b).
Nissl staining revealed that gestational and lactational Pb exposure significantly reduced the number of Purkinje cells. In addition, degenerative changes, such as pyknosis and vacuolation were detected in the Purkinje cell layer. Ascorbic acid co-treatment with Pb efficiently protected against the Pb-induced impairment in the Purkinje cells. To clearly investigate changes in cerebellar development, we determined the expression levels of the calcium-binding proteins (CaBPs), CB, CR, and PV. CB immunostaining clearly revealed the morphologies of the Purkinje cells by showing dendritic branching and axonal projection. Long-term Pb exposure was detrimental to the dendritic branching development and axonal projections of Purkinje cells, while ascorbic acid ameliorated these changes in the cerebellar cortex. CR-positive Purkinje cells displayed a similar pattern of changes to those observed in CB-positive Purkinje cells, namely a Pb-induced reduction in the number and an ascorbic acid-mediated attenuation of the reduction in number. However, CR-positive interneurons, CR-positive granule cells, and NeuN-positive granule cells were not significantly affected by Pb exposure or ascorbic acid treatment. Other previous studies have reported similar results indicating that gestational Pb exposure significantly impaired the development of Purkinje cells in the cerebellar cortex without affecting the cells in the granule cell layer (Nam et al., 2018a, 2018b, 2019). Furthermore, high-dose Pb (1%) was reported to be highly lethal to the fetus and impair the postnatal development of the brain by causing a reduction in whole brain weight and poorly defined lamination of the cerebellum (Mousa et al., 2015). The protective effects of ascorbic acid treatment in Pb-exposed rats are supported by the previous demonstration of the role of ascorbic acid in the development of the cerebellum in a genetic model of the vitamin C deficiency, which displays atrophic changes in granule and Purkinje cells and subsequent defects in motor function (Kim et al., 2015a).
Next, we focused on the expression of PV in the cerebellum, as it is a relevant marker of inhibitory neurons in the brain. PV was detected in the Purkinje cells and some cells in the molecular and granule cell layers. Long-term treatment with Pb was injurious to the development of PV-immunoreactive Purkinje cells by reducing their numbers and impairing dendritic branching. Significant differences in other PV-immunoreactive cells in the molecular or granule cell layers were not detected in the Pb or PA groups when compared to the control group. Although the specific patterns of the expression of the three CaBPs (CB, CR, and PV) were different, all of them were expressed in the inhibitory Purkinje cells. Similar to the present results, rat offspring whose mothers were exposed to ethanol during pregnancy and lactation were shown to have reduced CB, CR, and PV expression in the cerebellar Purkinje cells on PND10 (Wierzba-Bobrowicz et al., 2011). The above CaBPs are important regulators of the concentration of the Ca2+ in cells (Caillard et al., 2000). Although we could not directly demonstrate that the Pb-induced reduction in the numbers of the Purkinje cells was mediated by Ca2+ dyshomeostasis, previous studies have reported that Pb competes with Ca2+ and acts as a Ca2+ mimetic. In addition, Pb treatment of chick embryos was reported to lead to reorganization of cytoskeletal proteins and apoptotic cell death (Choi et al., 2011; Scheuhammer, 1996). In addition to its calcium binding properties, CB has anti-apoptotic effects by inhibiting the activity of pro-apoptotic caspase-3 (Bellido et al., 2000). Van Den Bosch et al. (2002) have reported that PV has the protective effects against kainic acid-induced neuronal death. Importantly, PV is also importantly involved in the GABA release and signaling in the brain (Hu et al., 2014). Therefore, these results suggest that reductions in the levels of CaBPs are useful markers of Pb-mediated cerebellar mal-development and may comprise an underlying mechanism. The Pb-mediated changes were partially ameliorated by ascorbic acid co-administration, which increased the levels of CaBPs in the cerebellum.
In addition to the inhibitory Purkinje cells in the cerebellar cortex, we investigated the inhibitory synapses of the Purkinje cells in the pinceau structure, which regulates the output of the cerebellar cortex by entwining the cell bodies and axons of the Purkinje cells. GABAT1 was detected mainly in the pinceau structure and as dot-like structures in the molecular and granule cell layers. Ascorbic acid treatment was beneficial to the preservation of these structures near Purkinje cells in the face of Pb-induced impairment. We hypothesized that changes of the pinceau structure may reflect changes in Purkinje cells, as the pinceau structure is the inhibitory synapse between basket cells and Purkinje cells. In fact, a previous study by our group confirmed that gestational exposure to the low-dose Pb (0.2%) impaired c-kit-immunoreactive inhibitory synapses in the cerebellar cortex (Nam et al., 2018b). Long-term exposure to an intermediate level of Pb in the present study also negatively affected GABAT1-immunoreactive pinceau structures. However, we were unable to detect significant differences in other GABAT1-immunoreactive dot-like structures in the cerebellar cortex. Similarly, Pb exposure was reported to reduce the level of GABAT1 in the zebrafish embryos (Wirbisky et al., 2014). In addition, a low dose of Pb (996 ppm) was shown to be detrimental to the prefrontal cortex and hippocampus by significantly down-regulating the expression levels of the GABA receptor and the GABA synthesizing enzyme glutamic acid decarboxylase (Neuwirth et al., 2018). Based on these results, we hypothesize that the GABAergic system and GABAergic synapses in the developing brain are vulnerable to Pb exposure and that ascorbic acid can reduce the resulting Pb-induced toxicity.
In conclusion, the results presented here demonstrate that long-term exposure to an intermediate dose of Pb is detrimental to cerebellar development in rat offspring. Pb exposure significantly reduced the numbers of CB-, CR-, and PV-immunoreactive Purkinje cells and PV-immunoreactive inhibitory synapses of the Purkinje cells. Ascorbic acid was an effective remedy for Pb exposure, as it ameliorated the Pb-induced impairments and changes. Therefore, we suggest that ascorbic acid supplementation during gestation and lactation in mothers at high risk for Pb exposure is helpful in preventing potential developmental neurotoxicity due to Pb.
The authors would like to thank Mr. Tae-Hun Go for his technical support in the present study.
The authors declare that there is no conflict of interest.