Immunohistochemical analysis of histone H3 acetylation in the trigeminal root entry zone in an animal model of trigeminal neuralgia

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OBJECTIVE

The trigeminal root entry zone (TREZ) is a transitional zone between the central nervous system (CNS) and peripheral nervous system (PNS), adjacent to the brainstem. Microvascular compression of the TREZ has been considered to be the primary etiology in most cases of trigeminal neuralgia (TN), but whether epigenetic regulation is involved in the pathogenesis of TN is still unclear. Therefore, this study was designed to investigate the epigenetic regulation of histone H3 acetylation in the TREZ in an animal model of TN.

METHODS

An animal model of TN was established, and adult male Sprague-Dawley rats were randomly assigned to a TN group with trigeminal nerve root compression, sham operation group, TN+HDACi group (TN plus selective histone deacetylase inhibitor injection into the TREZ), or TN+Veh group (TN plus vehicle injection into the TREZ). To measure the length of the central portion of the TREZ from the junction of the trigeminal nerve root entering the pons to the interface of the dome-shaped CNS-PNS transitional zone, immunofluorescent staining of glia and glial nuclei was performed using glial fibrillary acidic protein (GFAP) antibody and DAPI, respectively. To investigate the acetylation of histone H3 within the TREZ in a TN animal model group and a sham operation group, localization of histone H3K9, H3K18, and H3K27 acetylation was examined via immunohistochemical staining methods.

RESULTS

Measurements of the CNS-PNS transitional zone in the TREZ revealed that the average length from the junction of the trigeminal nerve root connecting the pons to the glial fringe of the TREZ in the TN group was longer than that in the sham operation group (p < 0.05) and that the interface gradually migrated distally. Cells that stained positive for acetylated histone H3K9, H3K18, and H3K27 were distributed around both sides of the border of the CNS-PNS junction in the TREZ. The ratio of immunoreactive H3K9-, H3K18- and H3K27-positive cells in the TN group was obviously higher than that in the sham operation group on postoperative days 7, 14, 21, and 28 (p < 0.05).

CONCLUSIONS

These results suggested that chronic compression of the trigeminal nerve root may be involved in the pathogenesis of TN in an animal model by influencing the plasticity of the CNS-PNS transitional zone and the level of histone acetylation in the TREZ.

ABBREVIATIONS CNS = central nervous system; DMSO = dimethyl sulfoxide; GFAP = glial fibrillary acidic protein; HAT = histone acetyltransferase; HDACi = histone deacetylase inhibitor; mAb = monoclonal antibody; PBS = phosphate-buffered saline; PNS = peripheral nervous system; SAHA = suberoylanilide hydroxamic acid; TN = trigeminal neuralgia; TREZ = trigeminal root entry zone.

Article Information

Correspondence Daoshu Luo: Basic Medical College, Fujian Medical University, Fujian, PR China. luods2004@163.com.

INCLUDE WHEN CITING Published online November 2, 2018; DOI: 10.3171/2018.5.JNS172948.

R.L. and L.L. share first authorship and contributed equally to this study.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Histogram statistics (left) show the behavioral results of the orofacial mechanical stimulus threshold in two TN animal model groups after HDACi or vehicle administration. Asterisks indicate a significant difference (p < 0.05). There were significantly more acetylated cells in the TN+HDACi group (A1–F1) than in the TN+Veh group (A–F) on postoperative days 7 and 14. Bar = 100 μm. Figure is available in color online only.

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    Merged images illustrating the dynamic changes in the CNS-PNS interface in the TREZ. The glial fringe of the CNS-PNS transitional zone in the TREZ is distinguished by GFAP (red) and DAPI (blue) immunofluorescence staining. Dotted line indicates the junction of the trigeminal root and the pons. The central length of the CNS-PNS interface has no obvious change in the sham operation group (A–D), while it gradually extends distally after the trigeminal root mechanical compression operation in the TN animal group (E–H). Bar = 500 μm. Figure is available in color online only.

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    Measuring the length of the TREZ in the rat. A: Note the TREZ, where central glia appear in the CNS side (GFAP, red, proximal part of the root) and peripheral glia appear on the PNS side (DAPI, blue, distal part of the root). Dotted line indicates the junction of the trigeminal root and the pons; dashed box indicates sites for the quantification of acetylation of histone H3. The lateral axis (L-axis) and central axis (C-axis; bidirectional arrows) represent the lateral and central axis lengths, respectively, from the trigeminal root–pons junction to the glial fringe of the CNS-PNS transitional zone in the TREZ. Bar = 500 μm. B and C: Average L-axis and C-axis lengths of the TREZ measured on postoperation days in the sham group and TN group, respectively. Asterisks indicate a significant difference (p < 0.05). Figure is available in color online only.

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    Immunohistochemical detection of histone H3K9 acetylation in the CNS-PNS transitional zone of the TREZ. In the sham group (A–D), immunoreactive acetylated H3K9 cells (green) decreased from postoperative day 7 to day 21, and few were detected on day 28. In the TN group (E–H), acetylated H3K9 cells (green) gradually increased from postoperative day 7 to day 28. Merged images show the positive ratio of immunoreactive acetylated H3K9 cells (green) among the DAPI-positive cells (blue) around the CNS-PNS transitional zone of the TREZ (same area as in the white dashed box in Fig. 3A) in the sham group (A1–D1) and TN group (E1–H1). The ratio of H3K9 acetylated cells in the TN group was higher than that in the sham operation group (I). Asterisks indicate a significant difference (p < 0.05). Most of the oligodendrocytes (arrows, J) and astrocytes (triangles) were acetylated in the central part of the TREZ, and Schwann cells (double arrows, K) were acetylated in the peripheral part of the TREZ. Bar = 150 μm (A–H1) and 50 μm (J and K). Figure is available in color online only.

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    Immunohistochemical detection of histone H3K18 acetylation in the CNS-PNS transitional zone of the TREZ. In the sham group (A–D), immunoreactive acetylated H3K18 cells (green) decreased from postoperative day 7 to day 21, and few were detected on day 28. In the TN group (E–H), acetylated H3K18 cells (green) gradually increased from postoperative day 7 to day 28. Merged images show the positive ratio of immunoreactive acetylated H3K18 cells (green) among the DAPI-positive cells (blue) around the CNS-PNS transitional zone of the TREZ (same area as in the white dashed box in Fig. 3A) in the sham group (A1–D1) and the TN group (E1–H1). The ratio of H3K18 acetylated cells in the TN group was higher than that in the sham operation group (I). Asterisks indicate a significant difference (p < 0.05). Most of the oligodendrocytes (arrows, J), astrocytes (triangles), and Schwann cells (double arrows, K) were acetylated in the TREZ. Bar = 150 μm (A–H1) and 50 μm (J and K). Figure is available in color online only.

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    Immunofluorescence staining of histone H3K27 acetylation in the CNS-PNS transitional zone of the TREZ. The level of histone H3K27 acetylation cells (green) in the sham group (A–D) was relatively lower than that in the TN group after operation. In the TN group (E–H), the amount of immunoreactive acetylated H3K27 cells (green) decreased from postoperative day 7 to day 28. Merged images show the positive ratio of immunoreactive acetylated H3K27 cells (green) among the DAPI-positive cells (blue) around the CNS-PNS transitional zone of the TREZ (same area as in the white dashed box in Fig. 3A) in the sham group (A1–D1) and the TN group (E1–H1). The ratio of H3K27 acetylated cells in the TN group was higher than that in the sham operation group (I). Asterisks indicate a significant difference (p < 0.05). Oligodendrocytes (arrows, J), astrocytes (triangles), and Schwann cells (double arrows, K) were acetylated in the TREZ. Bar = 150 μm (A–H1) and 50 μm (J and K). Figure is available in color online only.

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