Perioperative Medicine  |   June 2017
Growth Arrest and DNA-damage–inducible Protein 45β-mediated DNA Demethylation of Voltage-dependent T-type Calcium Channel 3.2 Subunit Enhances Neuropathic Allodynia after Nerve Injury in Rats
Author Notes
  • From the Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan (C.-Y.L.); Department of Medicine, Mackay Medical College, New Taipei, Taiwan (C.-Y.L., M.-C.H., Y.-C.H., A.-S.L., H.-H.W., J.-K.C., Y.-P.C., H.-Y.P.); Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan (M.-C.H.); and Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan (J.-K.C.).
  • C.-Y.L. and M.-C.H. contributed equally to this study.
    C.-Y.L. and M.-C.H. contributed equally to this study.×
  • Submitted for publication July 11, 2016. Accepted for publication February 1, 2017.
    Submitted for publication July 11, 2016. Accepted for publication February 1, 2017.×
  • Address correspondence to Dr. Peng: Department of Medicine, Mackay Medical College, No.46, Sec. 3, Zhongzheng Rd, Sanzhi Dist, New Taipei 25245, Taiwan. hsien.yu@gmail.com. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’S articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Basic Science / Central and Peripheral Nervous Systems / Pain Medicine
Perioperative Medicine   |   June 2017
Growth Arrest and DNA-damage–inducible Protein 45β-mediated DNA Demethylation of Voltage-dependent T-type Calcium Channel 3.2 Subunit Enhances Neuropathic Allodynia after Nerve Injury in Rats
Anesthesiology 6 2017, Vol.126, 1077-1095. doi:10.1097/ALN.0000000000001610
Anesthesiology 6 2017, Vol.126, 1077-1095. doi:10.1097/ALN.0000000000001610
Abstract

Background: Growth arrest and DNA-damage–inducible protein 45β reactivates methylation-silenced neural plasticity-associated genes through DNA demethylation. However, growth arrest and DNA-damage–inducible protein 45β–dependent demethylation contributes to neuropathic allodynia-associated spinal plasticity remains unclear.

Methods: Adult male Sprague–Dawley rats (654 out of 659) received a spinal nerve ligation or a sham operation with or without intrathecal application of one of the following: growth arrest and DNA-damage–inducible protein 45β messenger RNA–targeted small interfering RNA, lentiviral vector expressing growth arrest and DNA-damage–inducible protein 45β, Ro 25–6981 (an NR2B-bearing N-methyl-d-aspartate receptor antagonist), or KN-93 (a calmodulin-dependent protein kinase II antagonist) were used for behavioral measurements, Western blotting, immunofluorescence, dot blots, detection of unmodified cytosine enrichment at cytosine-phosphate-guanine site, chromatin immunoprecipitation quantitative polymerase chain reaction analysis, and slice recordings.

Results: Nerve ligation-enhanced growth arrest and DNA-damage–inducible protein 45β expression (n = 6) in ipsilateral dorsal horn neurons accompanied with behavioral allodynia (n = 7). Focal knockdown of growth arrest and DNA-damage–inducible protein 45β expression attenuated ligation-induced allodynia (n = 7) by reducing the binding of growth arrest and DNA-damage–inducible protein 45β to the voltage-dependent T-type calcium channel 3.2 subunit promoter (n = 6) that decreased expression of and current mediated by the voltage-dependent T-type calcium channel 3.2 subunit (both n = 6). In addition, NR2B-bearing N-methyl-d-aspartate receptors and calmodulin-dependent protein kinase II act in an upstream cascade to increase growth arrest and DNA-damage–inducible protein 45β expression, hence enhancing demethylation at the voltage-dependent T-type calcium channel 3.2 subunit promoter and up-regulating voltage-dependent T-type calcium channel 3.2 subunit expression. Intrathecal administration of Ro 25–6981, KN-93, or a growth arrest and DNA-damage–inducible protein 45β–targeting small interfering RNA (n = 6) reversed the ligation-induced enrichment of unmodified cytosine at the voltage-dependent T-type calcium channel 3.2 subunit promoter by increasing the associated 5-formylcytosine and 5-carboxylcytosine levels.

Conclusions: By converting 5-formylcytosine or 5-carboxylcytosine to unmodified cytosine, the NR2B-bearing N-methyl-d-aspartate receptor, calmodulin-dependent protein kinase II, or growth arrest and DNA-damage–inducible protein 45β pathway facilitates voltage-dependent T-type calcium channel 3.2 subunit gene demethylation to mediate neuropathic allodynia.