Treatment with thyroxine restores myelination and clinical recovery after intraventricular hemorrhage

Abstract

Intraventricular hemorrhage (IVH) remains a major cause of white matter injury in preterm infants with no viable therapeutic strategy to restore myelination. Maturation of oligodendrocytes and myelination is influenced by thyroid hormone (TH) signaling, which is mediated by TH receptor α (TRα) and TRβ. In the brain, cellular levels of TH are regulated by deiodinases, with deiodinase-2 mediating TH activation and deiodinase-3 TH inactivation. Therefore, we hypothesized that IVH would decrease TH signaling via changes in the expression of deiodinases and/or TRs, and normalization of TH signaling would enhance maturation of oligodendrocytes and myelination in preterm infants with IVH. These hypotheses were tested using both autopsy materials from human preterm infants and a rabbit model of IVH. We found that deiodinase-2 levels were reduced, whereas deiodinase-3 levels were increased in brain samples of both humans and rabbits with IVH compared with controls without IVH. TRα expression was also increased in human infants with IVH. Importantly, treatment with TH accelerated the proliferation and maturation of oligodendrocytes, increased transcription of Olig2 and Sox10 genes, augmented myelination, and restored neurological function in pups with IVH. Consistent with these findings, the density of myelinating oligodendrocytes was almost doubled in TH-treated human preterm infants compared with controls. Thus, in infants with IVH the combined elevation in deiodinase-3 and reduction in deiodinase-2 decreases TH signaling that can be worsened by an increase in unliganded TRα. Given that TH promotes neurological recovery in IVH, TH treatment might improve the neurodevelopmental outcome of preterm infants with IVH.

Figure 1.

Thyroid hormone receptor α increased after IVH in preterm human infants. A, Representative immunofluorescence of cryosections from the germinal matrix of 23–27 gw infants with and without IVH, double labeled with TRα and MAP2/Olig2/GFAP-specific antibodies. Note that TRα expression colocalized extensively with MAP2⁺ cells, but occasionally with Olig2⁺ or GFAP⁺ cells. TRα immunoreactivity was stronger in infants with IVH compared with controls, shown in insets at high magnification. B, Representative cryosections from the germinal matrix were double labeled with TRβ and MAP2/Olig2/GFAP-specific antibodies. Note TRβ was expressed on MAP2⁺, Olig2⁺, and GFAP⁺ cells. C, Representative Western blot for TRα in infant autopsy samples from the cortex, white matter (WM), and germinal matrix (GM). Rat brain was used as the positive control. Bar graphs show the mean ± SEM (n = 9 each group). Protein concentration normalized to β-actin. Note the higher expression of TRα in infants with IVH compared with controls without IVH in the white matter and germinal matrix. D, Representative Western blot for TRβ performed on autopsy samples from preterm infants with and without IVH, as indicated in C. Rat brain was used as the positive control. Bar graphs show the mean ± SEM (n = 9 each group). Protein concentration normalized to β-actin. Note the lower expression of TRβ in the germinal matrix of infants with IVH compared with neonates without IVH. *p < 0.01 for the comparison between IVH and no IVH. ^#p ≤ 0.01 cortex versus GM. ^ψp ≤ 0.01 WM versus GM. Scale bar, 20 μm. Insets show images under high magnification. Ctrl, Control.

Figure 2.

D2 decreased and D3 increased after IVH in preterm human infants. A, Representative immunofluorescence from the germinal matrix of 23–27 gw infants with and without IVH, double labeled with D2 and MAP2/O4/GFAP-specific antibodies. Note that D2 is expressed in GFAP⁺ astrocytes and radial glia, but is almost absent in MAP2⁺ neurons and O4⁺ OLs. B, Representative cryosections from the germinal matrix double labeled with D3 and MAP2/Olig2/GFAP-specific antibodies. D3 is abundantly expressed on Olig2⁺ OLs, and weakly expressed on MAP2⁺ and GFAP⁺ cells. Note the higher D3 immunoreactivity in infants with IVH versus infants without IVH. C, Representative Western blot analysis of D2 in cortex, white matter (WM), and germinal matrix (GM) from infants with and without IVH. Rat brain was used as the positive control. Bar graphs show the mean ± SEM (n = 9 each group). Protein concentration was normalized to β-actin. Note the higher expression of D2 in the germinal matrix compared with cortex and white matter. D, Western blot analysis was performed for D3 on tissues samples from the three brain regions, as indicated in C. Rat brain was used as the positive control. Bar graphs show the mean ± SEM (n = 9 each group). Protein concentration was normalized to β-actin. Note the higher expression of D3 in the germinal matrix of infants with IVH compared with infants without IVH. *p < 0.05 for the comparison between IVH and no IVH. Scale bar, 20 μm.

Figure 3.

Deiodinase and TR levels in rabbit pups with and without IVH. A, Coronal section through the frontal lobe of E29 rabbit pups showing small hemorrhage in the ventricle (arrows; left), moderate hemorrhage in the ventricle (block arrows; middle), and severe hemorrhage resulting in fusion of the two ventricles (arrowheads; right). Scale bar, 1 cm. B, D2 and D3 mRNA expression assayed by qRT-PCR (n = 6 each group). D2 expression was comparable between pups with and without IVH at both 3 and 7 d. Note the higher D3 expression in pups with IVH than without IVH at 7 d, but not at 3 d. Thyroxine treatment significantly elevated D3 mRNA at 3 d and reduced it at 7 d. C, Representative Western blot analysis of D3 in rabbit pups with and without IVH. Rat brain was used as the positive control. Bar graphs show the mean ± SEM (n = 4 each group). Protein concentration was normalized to β-actin. Note the higher expression of D3 in pups with IVH than without IVH at 7 and 14 d, and thyroxine treatment of pups with IVH reduced D3 protein to control levels at 14 d. D, TRα and TRβ mRNA expression assayed by qRT-PCR (n = 6 each group). Note that thyroxine treatment elevated TRα expression at 3 d and reduced TRβ levels at 7 d relative to vehicle-treated pups with IVH. ^#p < 0.05, ^###p < 0.001 for pups with IVH versus without IVH. *p < 0.05, ***p < 0.001 for vehicle-treated versus thyroxine-treated pups with IVH. ^†p < 0.05 for pups with no IVH versus thyroxine-treated pups with IVH.

Figure 4.

Thyroxine treatment enhances myelination. A, Representative immunofluorescence of MBP in the corona radiata of 14 d pups. Error bars indicate the mean ± SEM (n = 5 each group). The volume fraction of MBP was elevated in the corpus callosum and corona radiata of thyroxine-treated pups compared with vehicle-treated pups with IVH. Scale bar, 100 μm. V, Ventricle. B, Representative Western blot analysis for MBP in the forebrain of three sets of premature rabbit pups as indicated at 14 d. Adult rat brain was used as the positive control. Each lane represents lysate from a whole coronal slice taken at the level of midseptal nucleus of one brain. The bar graph shows the mean ± SEM (n = 5 each group). MBP expression was higher in thyroxine-treated pups compared with vehicle-treated pups. C, Western blot analysis for MAG in the forebrain of three sets of pups as indicated at 14 d. Adult rat brain was used as the positive control. The bar graph shows the mean ± SEM (n = 5 each group). MAG expression was higher in thyroxine-treated pups compared with vehicle-treated pups. D, Typical electron micrograph from rabbit pups without and with IVH, and pups with IVH treated with thyroxine at 14 d. Note that myelinated axons were fewer in pups with IVH relative to controls without IVH, and that thyroxine treatment significantly increased the number of myelinated axons in pups with IVH. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, pups with versus without IVH. *p < 0.05, **p < 0.01, vehicle-treated versus thyroxine-treated pups with IVH.

Figure 5.

Thyroxine treatment does not affect gliosis. A, Typical appearance of GFAP labeling in the groups of pups indicated at 14 d. Bar graphs show the mean ± SEM. Note the increased volume fraction (load) of astrocytes and glial fibers in pups with IVH compared with controls, and no effect of thyroxine treatment in pups with IVH. B, Representative Western blot analysis for GFAP in the groups of pups indicated at 14 d. Bar graphs are the mean ± SEM (n = 5 each group). Protein concentration normalized to β-actin. Rat brain was used as the positive control. Levels of GFAP were elevated in pups with IVH compared with controls, and thyroxine treatment did not alter GFAP levels in pups with IVH. C, Gene expression of GFAP assayed by qRT-PCR at 3 and 7 d. Note that IVH elevates the transcription of GFAP at 3 d, and that thyroxine does not affect GFAP mRNA expression in pups with IVH. ^#p < 0.05, pups with versus without IVH.

Figure 6.

Thyroxine treatment enhances the proliferation and maturation of OLs. A, Representative cryosections from 3-d-old pups double labeled with Olig2 and Ki67 antibodies. Bar graphs show the mean ± SEM (n = 5 each group). Note the enhanced density after thyroxine treatment at 3 d, not at 7 d. B, Typical double labeling of PDGFRα and Ki67 in the corona radiata and corpus callosum of 3-d-old pups. Bar graphs show the mean ± SEM (n = 5 each group). Note that thyroxine treatment enhances the density of both total and cycling PDGFRα⁺ cells. C, Representative immunolabeling of the corona radiata of 7-d-old pups using O4- and APC-specific antibodies. Bar graphs show the mean ± SEM (n = 5 each group). O4⁺APC⁺ cells are higher in density in thyroxine-treated pups. D, Western blot analyses of CNPase in rabbit pups without IVH and vehicle- or thyroxine-treated pups with IVH. Bar graphs show the mean ± SEM (n = 5 each group). Protein concentration normalized to actin. Thyroxine treatment restores normal levels of CNPase in pups with IVH. Scale bar, 20 μm. ^#p < 0.05, ^###p < 0.001, pups with versus without IVH. *p < 0.05, **p < 0.01, ***p < 0.001, vehicle-treated versus thyroxine-treated pups with IVH. ^†p < 0.05, no IVH versus thyroxine-treated pups with IVH.

Figure 7.

Thyroxine treatment enhances the maturation of OLs in human preterm infants with IVH. A, Representative immunofluorescence of cryosections from the white matter of a 25 gw premature infant labeled with O4 and O1 antibodies. Note the higher density of myelinating OLs, double labeled with O4 and O1 (arrowheads), in thyroxine-treated infants compared with untreated controls. Arrows indicate O4⁺O1⁻ OLs. B, Quantification of myelinating (immature) OLs was performed in three thyroxine-treated cases and three matched untreated controls. Each bar represents one case. Data are the mean ± SEM. Note the higher percentage of immature OLs in thyroxine-treated cases compared with controls.

Figure 8.

Thyroxine treatment elevates Olig2 and Sox10 transcription factors. qRT-PCR was performed in pups without IVH as well as vehicle- and thyroxine-treated pups with IVH. Note the reduced expression of Olig2 and Sox10 mRNA in pups with IVH relative to controls without IVH, and the enhanced expression of Olig2 and Sox10 genes in thyroxine-treated compared with vehicle-treated pups with IVH. *p < 0.05, **p < 0.01, vehicle-treated versus thyroxine-treated pups with IVH. ^#p < 0.05, pups with versus without IVH.

Figure 9.

Thyroxine treatment does not affect apoptosis in rabbit pups. A, B, Representative Western blot analyses for caspase 3 in the forebrain of three sets of premature rabbit pups as indicated at 3 and 7 d. Bar graphs show the mean ± SEM (n = 5 each group). Protein concentration normalized to β-actin. Newborn rat brain used as the positive control. Note the elevated cleaved caspase in pups with IVH relative to controls without IVH; and thyroxine treatment does not affect the cleaved caspase level. C, Western blot analyses for vehicle-treated and thyroxine-treated healthy pups without IVH at 3 d. Bar graphs show the mean ± SEM (n = 5 each group). Protein concentration was normalized to actin. There is no difference in the cleaved caspase level between the two groups. D, Representative immunofluorescence of 7-d-old rabbit pups labeled with Olig2 and TUNEL. Left, Bar graph shows the mean ± SEM of the total TUNEL⁺ cells in the germinal matrix (GM) and corona radiata (CR) of 7-d-old rabbit pups. Right, Bar graph indicates the mean ± SEM of the percentage of Olig2⁺ cells that are TUNEL⁺ in the GM and CR. No difference was seen in the extent of apoptosis between vehicle-treated or thyroxine-treated pups with IVH.

Figure 10.

Thyroxine treatment reduces proliferation but does not affect the maturation of OLs or myelination in healthy rabbit pups. A, Coronal sections were stained using a combination of Olig2/Ki67, PDGFRα/Ki67, or O4/APC antibodies. Bar graphs show the mean ± SEM (n = 5 each group). Total and cycling Olig2 cells are similar in density between groups. However, proliferating PDGFRα⁺ cells are reduced in thyroxine-treated pups compared with untreated controls. Densities of O4⁺ and O4⁺/APC⁺ cells are comparable between groups. B, C, Representative Western blot analyses for CNPase and MBP in forebrain homogenates of thyroxine-treated and untreated pups without IVH at 14 d. Bar graphs show the mean ± SEM (n = 5 each group). Protein concentration was normalized to β-actin. Adult rat brain was used as the positive control. No difference was seen between groups.