Impact of thyroid hormone perturbations in adult mice: brain weight and blood vessel changes, gene expression variation, and neurobehavioral outcomes

Abstract

Mouse models of hyper- and hypothyroidism were used to examine the effects of thyroid hormone (TH) dyshomeostasis on the aging mammalian brain. 13-14 month-old mice were treated for 4months with either levothyroxine (hyperthyroid) or a propylthiouracil and methimazole combination (PTU/Met; hypothyroid). Hyperthyroid mice performed better on Morris Water Maze than control mice, while hypothyroid mice performed worse. Brain weight was increased in thyroxine-treated, and decreased in PTU/Met-treated animals. The brain weight change was strongly correlated with circulating and tissue T4. Quantitative measurements of microvessels were compared using digital neuropathologic methods. There was an increase in microvessel area in hyperthyroid mice. Hypothyroid mice showed a trend for elevated glial fibrillary acidic protein-immunoreactive astrocytes, indicating an increase in neuroinflammation. Gene expression alterations were associated with TH perturbation and astrocyte-expressed transcripts were particularly affected. For example, expression of Gli2 and Gli3, mediators in the Sonic Hedgehog signaling pathway, were strongly impacted by both treatments. We conclude that TH perturbations produce robust neurobehavioral, pathological, and brain gene expression changes in aging mouse models.

Keywords: Astrocytes; Collagen IV; Digital neuropathology; Hyperthyroidism; Hypothyroidism; ScanScope.

Figure 1.. Brain Weight Changes with TH Level.

Mice were treated with either levothyroxine or PTU/Met to induce hyper- or hypothyroidism, respectively. A. Following four months of treatment, the average weight of the hemibrains was increased in thyroxine treated mice, while decreased in PTU/Met-treated mice. Both serum (B) and brain (C) T4 were positively correlated with brain weight. Neither serum (D) nor brain (E) T3 significantly correlated with brain weight. N = 12–13 / treatment group. Orange = Control, Green = Thyroxine-treated, Blue = PTU/Met-treated.

Figure 2.. TH Perturbation Affects Spatial Learning and Memory.

Morris Water Maze (MWM) was performed at two and four months of treatment. A. Representative swim paths for each treatment group. Latency to platform (B), distance to platform (C), and velocity (D) were unchanged in both treatment groups at two months of treatment. E. At four months of treatment, thyroxine-treated mice had a shorter latency to platform on acquisition day four, while PTU/Met-treated mice had a longer latency to platform on day five, compared with control-treated mice. F. Similarly, thyroxine-treated mice had a shorter swim distance on day four, while PTU/Met-treated mice had a longer swim distance on day three. G. There was not a significant difference in swim speed in either treatment group. H. Several probe trials were performed during the two and four months MWM testing. There was no difference in the time spent in proximity to platform after five days of testing during the two month MWM. Another probe trial was performed prior to the four month MWM. There was no difference in platform proximity in either treatment group during this probe trial. I. The platform location was moved after initial probe trial at four months. Additional probe trials were performed after acquisition testing on day two, four, and five. Thyroxine-treated mice spent significantly more time in proximity to the platform area on both day two and five, compared with control-treated mice. N = 12–13 / treatment group. Orange = Control, Green = Thyroxine-treated, Blue = PTU/Met-treated.

Figure 3.. Representative H&E and GFAP Staining.

After four months of treatment, hemibrains were collected, fixed in formalin, and embedded in paraffin. Sections from each mouse were stained with hematoxylin and eosin (H & E) or GFAP immunoreactivity. Representative images for each treatment group are shown.

Figure 4.. Analysis of GFAP Immunoreactivity.

A. A representative section immunostained for GFAP. The top inset panel is a higher magnification view of the hippocampus. The lower inset panel is a magnified view of the image processed with the Positive Pixel Count algorithm in the ImageScope software. The red color denotes positive (NovaRed) staining, while the blue color denotes negative (hematoxylin) staining. In both the cortex (B) and hippocampus (C) the amount of GFAP-positive staining trended towards an increase in the PTU/Met-treated group, as compared with the thyroxine-treated. GFAP immunoreactivity weakly, negatively correlates with brain weight in both the cortex (C) and hippocampus (D). N = 10–12 / treatment group. Orange = Control, Green = Thyroxine-treated, Blue = PTU/Met-treated.

Figure 5.. Representative Collagen IV-Stained Brain Sections.

A. A representative Collagen-IV stained section. The top inset panel is a higher magnification view of the hippocampus. The lower inset panel is a magnified view of the image processed with the Microvessel Algorithm in ImageScope. Green denotes the identified vessels. B. Representative images for each treatment group.

Figure 6.. Analysis of Microvessel Parameters.

In the hippocampus, vessel area (A), vessel perimeter (B), lumen area (C), vascular area (D), and vessel wall thickness (E) trended towards an increase in thyroxine-treated animals. In the cortex, vessel area (F), vessel perimeter (G), and vascular area (I) trended towards an increase in thyroxine-treated animals. Lumen area (H) and vessel wall thickness (J) did not change in either treatment group. In the hippocampus, vessel area (K) and vessel perimeter (L) were positively correlated with brain weight. Wall thickness (M) was not correlated with brain weight. In the cortex, vessel area (N) and vessel perimeter (O) were positively correlated with brain weight. Cortical vessel wall thickness (P) was not correlated with brain weight. N = 8–12 / treatment group. Orange = Control, Green = Thyroxine-treated, Blue = PTU/Met-treated.

Figure 7.. TH Perturbation Alters Brain mRNA Transcript Levels.

Gene expression was measured in homogenized hemibrains after four months of treatment. Shown is a heat map of the fold change in gene expression relative to the control group (2^−ΔΔC_T). The transcripts tested are grouped by the cell type with which they are associated. Many transcripts are present in multiple cell types. Two transcripts (Gli2 and Dio3) were increased beyond the range depicted by the color key and are shown in bright green.