Role of late maternal thyroid hormones in cerebral cortex development: an experimental model for human prematurity

Abstract

Hypothyroxinemia affects 35-50% of neonates born prematurely (12% of births) and increases their risk of suffering neurodevelopmental alterations. We have developed an animal model to study the role of maternal thyroid hormones (THs) at the end of gestation on offspring's cerebral maturation. Pregnant rats were surgically thyroidectomized at embryonic day (E) 16 and infused with calcitonin and parathormone (late maternal hypothyroidism [LMH] rats). After birth, pups were nursed by normal rats. Pups born to LMH dams, thyroxine treated from E17 to postnatal day (P) 0, were also studied. In developing LMH pups, the cortical lamination was abnormal. At P40, heterotopic neurons were found in the subcortical white matter and in the hippocampal stratum oriens and alveus. The Zn-positive area of the stratum oriens of hippocampal CA3 was decreased by 41.5% showing altered mossy fibers' organization. LMH pups showed delayed learning in parallel to decreased phosphorylated cAMP response element-binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression in the hippocampus. Thyroxine treatment of LMH dams reverted abnormalities. In conclusion, maternal THs are still essential for normal offspring's neurodevelopment even after onset of fetal thyroid function. Our data suggest that thyroxine treatment of premature neonates should be attempted to compensate for the interruption of the maternal supply.

Figure 1.

(A–C) Low-magnification photomicrographs of coronal sections of the parietal cortex and hippocampus showing BrdU-immunoreactive cells after E17–20 injections in control, LMH + T₄, and LMH pups at P40. (D–F) Details (boxes in A, B, and C) showing that both in layers I–VI and white matter (wm) the neocortex and the alveus (al) and stratum oriens (or) of the hippocampus, the radial distribution of BrdU-immunoreactive cells is more widespread in LMH pups than in control and LMH + T₄ pups. Note the increased number of heterotopic BrdU-immunoreactive cells in wm, al, and or in LMH pups compared with that of control and LMH + T₄ pups. The borders between layers are indicated by horizontal lines.

Figure 2.

(A–C) Plots of coronal sections of the parietal cortex and hippocampus showing the distribution of type 1 and 2 BrdU-immunoreactive cells after E17–20 injections in control, LMH + T₄, and LMH pups at P40. Note the increased number of heterotopic BrdU-immunoreactive cells in the wm of LMH pups compared with that of control and LMH + T₄ pups. (D) Histogram showing the percentage of BrdU-immunoreactive cells in different layers of the parietal cortex in control, LMH + T₄, and LMH pups (see also Supplementary Table 3). Error bars represent ± standard deviation across layers from the same group; n.s., no significant differences; *P <0.05 for LMH group compared with C or LMH + T₄ group (n = 12 for each group).

Figure 3.

Confocal photomicrographs of the primary somatosensory cortex (A–C, G–I, J) and the hippocampal CA1 (D–F) of control (J, K) and LMH (A–I, L) pups at P40. Single labeling to BrdU (A, D, G) and to NeuN (B, E, H) and double labeling (C, F, I, J, yellow) are shown. Note that almost all BrdU-positive cells are neurons (double labeled with NeuN; C, F, I, J). Panels G–I show heterotopic neurons in the subcortical white matter (wm) of the primary somatosensory cortex in LMH pups (for comparisons between experimental groups and percentages, see Figure 6 and Supplementary Table 4). Immunofluorescence labeling of glial cells (BrdU positive and NeuN negative; arrows) in the primary somatosensory cortex of control (J) and LMH (G, I) pups. Glial cells in the hippocampal CA1 of LMH (D, F) pups are also indicated (arrows). Light microscope photomicrographs are shown of primary somatosensory cortex of control (K) and LMH (L) pups. Double-labeled astrocytes (arrows in K and L) are shown. Note that BrdU-labeled astrocytes show partial staining limited to clumped chromatin within the nuclei corresponding to type 3 labeling of Takahashi et al. (1992).

Figure 4.

(A, D, G) Low-magnification photomicrographs of coronal sections of the parietal cortex and hippocampus showing NeuN-immunoreactive neurons in control (A–C), LMH + T₄ (D–F) and LMH (G–I) pups at P40. (B, C, E, F, H, I) Magnified boxes showing details of heterotopic NeuN-immunoreactive neurons in the subcortical white matter (wm; B, E, H) and in the alveus (al) and stratum oriens (or) of the hippocampus (C, F, I). Note the increased number of heterotopic neurons in LMH pups compared with control and LMH + T₄ pups.

Figure 5.

Photomicrographs of coronal sections of the parietal cortex showing NeuN-immunoreactive neurons in control (A, D, G, J), LMH + T₄ (B, E, H, K), and LMH (C, F, I, L) pups at P0, P8, P15, and P20. At P0, the subplate in LMH pups is thicker than in normal and LMH + T₄ pups (compare C with A and B). At P15, the border between the subplate and adjacent layer VI is more clear-cut in LMH pups (compare I with G and H).

Figure 6.

Plots of NeuN-immunoreactive neurons taken at similar rostrocaudal and anteroposterior of the parietal cortex in control (A, D, G, J, M), LMH + T₄ (B, E, H, K, N), and LMH (C, F, I, L, O) pups at P0, P8, P15, P2, and P40. At P15, the subplate is still present in LMH pups (compare I with G and H). (P–T) Histograms showing the percentage of NeuN-immunoreactive neurons in different layers of the parietal cortex in control, LMH + T₄, and LMH pups at different postnatal ages (see also Supplementary Table 4). Note the high percentage of cells in the subplate (SP) at P15 and in the white matter (wm) at all ages in LMH compared with control and LMH + T₄ pups. At P20 and P40, the percentage of NeuN-immunoreactive neurons decreased in layers II–III and IV and increased in layer VI. Error bars represent ± standard deviation across layers from the same group; n.s., no significant differences; *P < 0.1 and **P <0.05 for LMH group compared with control or LMH + T₄ group (n = 15 for each group); CP, cortical plate.

Figure 7.

(A, B) Low-magnification photomicrographs of coronal sections of the parietal cortex showing NeuN-immunoreactive neurons (A) and TUNEL-labeled cells (B) in LMH at P13 after ibotenic acid injections at P12. Note that the zone affected by the ibotenic acid injection comprises all the cortical layers including the subplate (arrow in A). Photomicrograph in (B) is an adjacent section to (A) showing TUNEL-labeled cells in LMH pups. Note that the TUNEL labeling in (B) matches the injured area in (A). This box is magnified in (C) showing details of the subplate (SP) and subcortical white matter (wm) in the injured area (left) and in the adjacent undamaged cortex (right).

Figure 8.

PLP/DM20 in situ hybridization showing the distribution of oligodendrocytes both in the parietal cortex (A, B) and hippocampus (C, D) in control and LMH pups at P40. (E) Histogram showing the percentages of PLP/DM20-labeled cells in control and LMH pups. No statistically significant differences (n.s.) were found between normal and LMH pups (n = 6 for each group) in the different layers and white matter (wm).

Figure 9.

(A–C) Low-magnification photomicrographs of coronal sections of the hippocampus showing Zn labeling in control (A, D), LMH + T₄ (B, E), and LMH (C, F) pups at P40. Note the heavier labeling in the hilus of CA4 (arrow) and in the strata radiatum (double arrows) and oriens (arrowhead) of CA3. (D–F) Plots showing the Zn labeling of the stratum oriens in control, LMH + T₄, and LMH pups.

Figure 10.

Photomicrographs of coronal sections of the hippocampus showing ZnT-3 labeling in control (A) and LMH (B) pups at P40. A decreased intensity of the immunolabeling can be observed in the stratum oriens (arrowhead) in LMH pups compared with controls. DG, dentate gyrus.

Figure 11.

Histogram showing step-down latencies in seconds at 1, 3, and 24 h after the initial footshock in control and LMH pups at P39. Pups from LMH dams show a 24.9% reduction in the step-down latency at 1 h after the footshock. Error bars represent ± standard deviation; n.s., no significant differences; *P <0.001 for LMH compared with control group (n = 23 for control and n = 8 for LMH group).

Figure 12.

Western blots obtained from the hippocampus of control and LMH pups at P40, immunolabeled for pATF1, pCREB, and CREB (A) and ERK2, pERK1, and pERK2 (C). Histograms showing that the pCREB/pATF1 and pCREB/CREB (B) and pERK1/ERK2 and pERK2/ERK2 (D) ratios are reduced by 59.1%, 66.7%, 44.4%, and 42.9%, respectively, in LMH compared with control pups. Error bars represent ± standard deviation; *P <0.001 for LMH compared with control group (n = 6 for each group).