Spatial and temporal dynamics of HDACs class IIa following mild traumatic brain injury in adult rats

Abstract

The fundamental role of epigenetic regulatory mechanisms involved in neuroplasticity and adaptive responses to traumatic brain injury (TBI) is gaining increased recognition. TBI-induced neurodegeneration is associated with several changes in the expression-activity of various epigenetic regulatory enzymes, including histone deacetylases (HDACs). In this study, PET/CT with 6-([¹⁸F]trifluoroacetamido)-1- hexanoicanilide ([¹⁸F]TFAHA) to image spatial and temporal dynamics of HDACs class IIa expression-activity in brains of adult rats subjected to a weight drop model of diffuse, non-penetrating, mild traumatic brain injury (mTBI). The mTBI model was validated by histopathological and immunohistochemical analyses of brain tissue sections for localization and magnitude of expression of heat-shock protein-70 kDa (HSP70), amyloid precursor protein (APP), cannabinoid receptor-2 (CB2), ionized calcium-binding adapter protein-1 (IBA1), histone deacetylase-4 and -5 (HDAC4 and HDAC5). In comparison to baseline, the expression-activities of HDAC4 and HDAC5 were downregulated in the hippocampus, nucleus accumbens, peri-3rd ventricular part of the thalamus, and substantia nigra at 1-3 days post mTBI, and remained low at 7-8 days post mTBI. Reduced levels of HDAC4 and HDAC5 expression observed in neurons of these brain regions post mTBI were associated with the reduced nuclear and neuropil levels of HDAC4 and HDAC5 with the shift to perinuclear localization of these enzymes. These results support the rationale for the development of therapeutic strategies to upregulate expression-activity of HDACs class IIa post-TBI. PET/CT (MRI) with [¹⁸F]TFAHA can facilitate the development and clinical translation of unique therapeutic approaches to upregulate the expression and activity of HDACs class IIa enzymes in the brain after TBI.

Fig. 1. PET/CT images of the rat brain at 20–30 min post [¹⁸F]TFAHA administration obtained at baseline, 1–3 days, and 7–8 days post-TBI.

Axial images (top row) and coronal images (lower three rows) reflect [¹⁸F]TFAHA accumulation (Standard uptake values; SUV) in the following brain structures outlined by the red dots: n. accumbens (row 2), hippocampus and substantia nigra (row 3), and peri-3rd ventricular thalamic gray matter (row 4). The corresponding cross-sections of stereotactic Atlas of the Rat Brain are provided for reference in the left.

Fig. 2. Changes in [¹⁸F]TFAHA accumulation in different structures of the rat brain at baseline and different days post TBI in peri-3rd ventricular thalamic gray matter (PTG), n. accumbens, hippocampus, and substantia nigra.

A SUV and B volumes of distribution (VD). Data are mean ± SEM. Statistical significance was determined via one-way ANOVA for repeated measures: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. Expression of HDAC4 in the rat hippocampus at baseline, 1–3 days, and 7–8 days post-TBI, visualized by IHC.

Left column: stitched images (×10 magnification) with black rectangles indicating hippocampal CA1, CA2, dorsal dentate gyrus (DDG), and ventral dentate gyrus (VDG) areas shown in the corresponding ×40 magnification.

Fig. 4. Expression of HDAC4 and HDAC5 in the paraventricular thalamic nucleus in the rat brain.

IHC staining of the brain tissue sections for HDAC4 and HDAC5 was performed at baseline, 1–3 days, and 7–8 days post mTBI. Images are at ×40 magnification.

Fig. 5. Expression of HDAC5 in the rat hippocampus at baseline, 1–3 days, and 7–8 days post-TBI, visualized by IHC.

Left column: stitched images (×5 magnification) with black rectangles indicating hippocampal CA1, CA2, dorsal dentate gyrus (DDG), and ventral dentate gyrus (VDG) areas shown in the corresponding images at ×40 magnification.

Fig. 6. The ratio of cytoplasmic vs nuclear localization/expression levels of HDAC4 and HDAC5 in different structures of the rat brain.

Measurements were obtained by optical densitometry of immunohistochemically stained brain tissue sections from different animals at baseline, 1–3 days, and 7–8 days post-TBI for A HDAC4 and B HDAC5. Data are mean ± SEM. Statistical significance was determined via one-way ANOVA for repeated measures. *p < 0.05, **p < 0.01, ***p < 0.001.