Effects of Ascorbic Acid on Osteopontin Expression and Axonal Myelination in the Developing Cerebellum of Lead-Exposed Rat Pups

Abstract

Osteopontin (OPN) is a multi-functional protein that binds to integrin and calcium-binding phosphoprotein. OPN is required for normal neuronal development and its axonal myelination. We studied the combined effect of lead (Pb) and ascorbic acid treatment on OPN expression in the developing cerebellum. We randomly divided pregnant female rats into three groups: control, Pb (lead acetate, 0.3%, drinking water), and Pb plus ascorbic acid (PA; ascorbic acid, 100 mg/kg, oral intubation) groups. The blood level of Pb was significantly increased, while ascorbic acid reduced Pb levels in the dams and pups. At postnatal day (PND) 21, results from Nissl staining and OPN immunohistochemistry demonstrated that OPN was detected in the Purkinje cell layer in the cerebellum. Ascorbic acid treatment mitigated Pb exposure-induced reduction in the number of intact Purkinje cells and OPN immunoreactive Purkinje cells in the cerebellum of pups. In addition, Pb-induced reduction in the number of oligodendrocytes and myelin-associated glycoprotein is associated with the malformation of the myelin sheath. Ascorbic acid provided protection from Pb-induced impairments. Pb-induced structural deficits in the cerebellum resulted in functional deterioration observed during locomotive tests (bar holding test and wire mesh ascending test), while ascorbic acid ameliorated these harmful effects. Present results suggest that the change of OPN is associated with myelination in the developing cerebellum. The results also demonstrated that exposure to Pb is harmful, while ascorbic acid treatment is beneficial.

Keywords: ascorbic acid; cerebellum; lead (Pb) toxicity; locomotive test; oligodendrocyte; osteopontin.

Figure 1

Body weights of dams (n = 3 per group) during gestation and offspring (n = 12 per group) on postnatal days (PND) 0, 7, 14, and 21 in control (CTL), lead (Pb), and Pb+ ascorbic acid (PA) groups (A). Cerebellar weights of offspring on PND21 (B). Blood Pb levels in the dams and offspring on day 21 after delivery (C) (^a p < 0.05, indicating a significant difference compared with the control group, ^b p < 0.05, indicating a significant difference compared with the Pb group). The bars indicate means ± standard errors of mean.

Figure 2

Nissl staining of cerebellum (A–C) of offspring from CTL, Pb, and PA groups. GCL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar = 25 μm. (D) The numbers of intact Purkinje cells in the cerebellum are expressed as percentages of the value in the CTL group. The number of intact Purkinje cells in three sections per offspring in non-overlapping fields, 12 rats were included in each group (n = 12 offspring per group; ^a p < 0.05, indicating a significant difference compared with the control group, ^b p < 0.05, indicating a significant difference compared with the Pb group). The bars indicate the means ± standard errors of the mean.

Figure 3

Immunohistochemistry for osteopontin (OPN) in the cerebellum (A–C) of offspring from CTL, Pb, and PA groups. GCL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Bar = 25 μm. (D) The numbers of OPN-positive Purkinje cells in the cerebellar cortex are expressed as percentages of the value in the CTL group (n = 12 offspringper group; ^a p < 0.05, indicating a significant difference compared with the control group, ^b p < 0.05, indicating a significant difference compared with the Pb group). The bars indicate the means ± standard errors of the mean.

Figure 4

Immunohistochemistry for oligodendrocytes (brown) in the cerebellum (A–C) of offspring from CTL, Pb, and PA groups. Note that the numbers of Olig2-positive oligodendrocytes in the cerebellum are reduced in the Pb group, and ascorbic acid treatment ameliorated these reductions in the PA group. GM-GCL (blue), granule cell layer in gray matter; WM, white matter. Bar = 25 μm. (D) The numbers of OPN-positive Purkinje cells in the cerebellar cortex are expressed as percentages of the value in the CTL group (n = 12 offspring per group; ^a p < 0.05, indicating asignificant difference compared with the control group, ^b p < 0.05, indicating a significant difference compared with the Pb group). The bars indicate the means ± standard errors of the mean.

Figure 5

Immunohistochemistry for myelin-associated glycoprotein (MAG, brown) in the cerebellum (A–C) of offspring from CTL, Pb, and PA groups. Note that the MAG-immunoreactivity in the WM in the cerebellum are reduced in the Pb group, and ascorbic acid treatment ameliorated this reduction in the PA group. GM-GCL (blue), granule cell layer in gray matter. (D) The MAG-immunoreactivity in the WM in the cerebellum is expressed as percentages of the value in the CTL group (n = 12 offspring per group; ^a p < 0.05, indicating a significant difference compared with the control group, ^b p < 0.05, indicating a significant difference compared with the Pb group). The bars indicate the means ± standard errors of the mean.

Figure 6

Effect of Pb exposure and ascorbic acid treatment in the bar holding test (A) and wire mesh ascending test (B) among offspring from CTL, Pb, and PA groups. (A) The times during which the animal stayed on the bar in the bar holding test. (B) The amount of time the animal spent to reach the top of the wire mesh in the wire mesh ascending test (n = 12 offspring per group; ^a p < 0.05, indicating a significant difference compared with the control group, ^b p < 0.05, indicating a significant difference compared with the Pb group).