Effect of antenatal betamethasone treatment on microtubule-associated proteins MAP1B and MAP2 in fetal sheep

Abstract

Betamethasone has been used extensively to accelerate fetal lung maturation, yet little is known of its effects on neuronal morphogenesis in the developing fetus. Microtubule-associated proteins (MAPs) are a diverse family of cytoskeletal proteins that are important for brain development and the maintenance of neuroarchitecture. Vehicle (n = 7) or betamethasone (10 ug h-1, n = 7) was infused I.V. to fetal sheep over 48 h beginning at 0.87 of gestation (128 days of gestation), producing fetal plasma betamethasone concentrations resembling those to which the human fetus is exposed during antenatal glucocorticoid therapy. Paraffin sections of the left hemisphere were stained with monoclonal antibodies against MAP1B and the MAP2 isoforms MAP2a,b,c and MAP2a,b. The level of the juvenile isoform MAP2c was determined by comparison of the two MAP2 immunostainings. We were able to detect MAP1B and MAP2 immunoreactivity (IR) in the fetal sheep brain. MAP2c was the major MAP2, constituting 90.2 % of the total MAPBetamethasone exposure diminished MAP1B IR in the frontal cortex and caudate putamen (P < 0.05) but not in the hippocampus. A decrease of MAP2 IR was found in the frontal cortex, hippocampus and caudate putamen (P < 0.05). Loss of MAP2 IR was mainly due to the loss of MAP2c IR. Haematoxylin-eosin staining did not demonstrate irreversible neuronal damage. Regional cerebral blood flow determined using coloured microspheres was significantly decreased by 28 % in the frontal cortex and by 36 % in the caudate putamen but not in the hippocampus 24 h after the onset of betamethasone exposure (P < 0.05). The loss of MAP1B and MAP2a,b,c IR showed a significant correlation to the cerebral blood flow decrease only in the frontal cortex (P < 0.05). These data suggest that mechanisms other than metabolic insufficiency caused by the decreased cerebral blood flow may contribute to the loss of MAPs. The results suggest that clinical doses of betamethasone may have acute effects on cytoskeletal proteins in the fetal brain.

Figure 2. Photomicrographs of MAP2a,b,c immunostaining (brown precipitate) of the frontal cerebral cortex (top), caudate putamen (middle) and hippocampus (bottom), counterstained with haematoxylin (blue), of a vehicle- (A, C, E) and a betamethasone-treated fetus (B, D, F) at 130 dGA

MAP2a,b,c IR is evident in neuronal perikarya and processes. In the cerebral cortex (A), the anatomical orientation of the apical dendrites (arrowheads) of pyramidal cells (Py) is clearly visible. In the caudate putamen (C), neuronal populations are recognised by MAP2a,b,c antibody in contrast to white fibre bundles. In the CA1 region of the hippocampus (E) the pyramidal cell layer (PL), stratum oriens (SO) and stratum radiatum (SR) are densely labelled by MAP2a,b,c. Loss of MAP2 IR was found in the frontal cortex (B), caudate putamen (D) and hippocampus (F) 48 h after the onset of betamethasone treatment. Nuclei counterstained with haematoxylin do not show irreversible neuronal damage. Scale bar, 100 μm.

Figure 3. Photomicrographs of MAP2a,b,c (A) and MAP2a,b (B) immunostaining (brown precipitate) in the frontal cerebral cortex counterstained with haematoxylin (blue)

Note the small amount of immunoreactivity of MAP2a,b (B) in comparison to that of MAP2a,b,c (A). Scale bar, 100 μm.

Figure 5. Acute effects of antenatal betamethasone (BETA) treatment on MAP1B IR in fetal sheep at 130 dGA

Vehicle-treated fetuses, n = 7; betamethasone-treated fetuses, n = 7; means ±s.e.m., *P < 0.05.

Figure 6. Acute effects of antenatal betamethasone (BETA) treatment on MAP2a,b,c IR in fetal sheep at 130 dGA

Vehicle-treated fetuses, n = 7; betamethasone-treated fetuses, n = 7; means ±s.e.m.; *P < 0.05.

Figure 4. Comparison of the acute effects of antenatal betamethasone (BETA) treatment on MAP2a,b,c and MAP2a,b IR in fetal sheep at 130 dGA in the frontal cerebral cortex

Filled bars, MAP2a,b; hatched bars, MAP2a,b,c; vehicle-treated fetuses, n = 7; betamethasone (BETA)-treated fetuses, n = 7; *P < 0.05. Difference between MAP2a,b,c and MAP2a,b IR gives the amount of MAP2c. Note the loss of MAP2a,b,c IR (P < 0.05) in contrast to MAP2a,b IR after betamethasone treatment.

Figure 1. Photomicrographs of MAP1B immunostaining (brown precipitate) of the frontal cerebral cortex (top), caudate putamen (middle) and hippocampus (bottom), counterstained with haematoxylin (blue), of a vehicle- (A, C, E) and a betamethasone-treated fetus (B, D, F) at 130 dGA

MAP1B IR is evident in cell bodies and processes of neurons and the neuropil. In the middle cortical layers (A) apical cortical dendrites (arrowheads) show their typical parallel orientation. The most dense MAP1B immunostaining was found in the caudate putamen (C). Loss of MAP1B IR in the frontal cortex (B) and caudate putamen (D) of the betamethasone-treated fetuses is clearly visible. Note the loss of clear demarcation of the neuronal processes in the stratum radiatum (SR) and stratum oriens (SO) of the hippocampus after betamethasone treatment (F). PL, pyramidal cell layer of the CA1 region. Nuclei counterstained with haematoxylin do not show irreversible neuronal damage. Scale bar, 100 μm.

Figure 7. Correlation of the area of MAP1B IR and MAP2a,b,c IR to the relative CBF 24 h after the onset of betamethasone exposure

CBF is given relative to baseline. Vehicle-treated fetuses, n = 5; betamethasone-treated fetuses, n = 5. **P < 0.01, *P < 0.05, †P < 0.06.