Fig. 4.
Spinal nerve ligation (SNL) increases growth arrest and DNA-damage–inducible protein 45β (Gadd45β) binding to the voltage-dependent T-type calcium channel 3.2 subunit (CaV3.2) gene promoter to enhance CaV3.2 expression in the dorsal horn. (A, B) Intrathecal administration with l-ascorbic acid (a selective CaV3.2 antagonist; day 7 after spinal nerve ligation [SNL 7D] + l-ascorbic acid; 10, 30, and 100 μM, 10 μl), but not the vehicle solution (SNL 7D + Veh, 10 μl), increased the withdrawal threshold of the ipsilateral hind paw on SNL 7D. Nevertheless, both these treatments failed to affect the contralateral hind paw at 2 h after injection (SNL 7D + l-ascorbic acid; 100 μM, 10 μl; von Frey test). **P < 0.01 versus SNL 7D. n = 7. (C) Intrathecal injections of lentiviral vector expressing Gadd45β into naive rat (Gadd45β LV; 2.4 × 107 U/ml, 15 μl) resulted in a decreased withdrawal threshold on day 14 after treatment. Intrathecal injections of control vector into naive rat (Control LV; 2.2 × 107 U/ml, 15 μl). **P < 0.01 versus Naive. n = 7. (D) Representative Western blot and statistical analyses (normalized to glyceraldehyde 3-phosphate dehydrogenase protein [GAPDH]) showing that, on day 14 after treatment, intrathecal injections of lentiviral vector expressing Gadd45β (Gadd45β LV; 2.4 × 107 U/ml, 15 μl), not control vector (Control LV; 2.2 × 107 U/ml, 15 μl), into naive rat (Gadd45β LV; 2.4 × 107 U/ml, 15 μl) increased Gadd45β and CaV3.2 protein levels in ipsilateral dorsal horn. **P < 0.01 versus Naive. n = 6. (E–J) Representative reverse-transcription polymerase chain reaction, Western blot, and statistical analysis (normalized to GAPDH) revealing that, compared with sham operation (Sham 7D), spinal nerve ligation (SNL 7D) increased CaV3.2 transcription or expression in ipsilateral dorsal horn of the spinal cord (SC) and dorsal root ganglion (DRG) samples harvested on day 7 postoperation, which was reversed by intrathecal application of Gadd45β mRNA–specific antisense siRNA (SNL 7D + Gadd45β RNAi; 5 μg, 10 μl; once daily on days 3 to 6 after SNL). Missense siRNA (MS RNAi; 5 μg, 10 μl). **P <0.01 versus Sham 7D. #P < 0.05. ##P < 0.01 versus SNL 7D. n = 6. The SNL-enhanced CaV3.2 transcription or expression in the DRG was markedly lesser than that in the dorsal horn (SC; normalized to Sham 7D). **P < 0.01 versus SC. n = 6. (K) Chromatin immunoprecipitation (ChIP) quantitative reverse-transcription polymerase chain reaction analysis showed that intrathecal application of Gadd45β mRNA–specific antisense siRNA (SNL 7D + Gadd45β RNAi; 5 μg, 10 μl; once daily on days 3 to 6 after SNL) reversed the SNL-enhanced binding of Gadd45β to the CaV3.2 promoter region in the ipsilateral dorsal horn on day 7 postoperation. **P < 0.01 versus Sham 7D. ##P < 0.01 versus SNL 7D. n = 6. Data represent mean ± SD. IB = immunoblotting.

Spinal nerve ligation (SNL) increases growth arrest and DNA-damage–inducible protein 45β (Gadd45β) binding to the voltage-dependent T-type calcium channel 3.2 subunit (CaV3.2) gene promoter to enhance CaV3.2 expression in the dorsal horn. (A, B) Intrathecal administration with l-ascorbic acid (a selective CaV3.2 antagonist; day 7 after spinal nerve ligation [SNL 7D] + l-ascorbic acid; 10, 30, and 100 μM, 10 μl), but not the vehicle solution (SNL 7D + Veh, 10 μl), increased the withdrawal threshold of the ipsilateral hind paw on SNL 7D. Nevertheless, both these treatments failed to affect the contralateral hind paw at 2 h after injection (SNL 7D + l-ascorbic acid; 100 μM, 10 μl; von Frey test). **P < 0.01 versus SNL 7D. n = 7. (C) Intrathecal injections of lentiviral vector expressing Gadd45β into naive rat (Gadd45β LV; 2.4 × 107 U/ml, 15 μl) resulted in a decreased withdrawal threshold on day 14 after treatment. Intrathecal injections of control vector into naive rat (Control LV; 2.2 × 107 U/ml, 15 μl). **P < 0.01 versus Naive. n = 7. (D) Representative Western blot and statistical analyses (normalized to glyceraldehyde 3-phosphate dehydrogenase protein [GAPDH]) showing that, on day 14 after treatment, intrathecal injections of lentiviral vector expressing Gadd45β (Gadd45β LV; 2.4 × 107 U/ml, 15 μl), not control vector (Control LV; 2.2 × 107 U/ml, 15 μl), into naive rat (Gadd45β LV; 2.4 × 107 U/ml, 15 μl) increased Gadd45β and CaV3.2 protein levels in ipsilateral dorsal horn. **P < 0.01 versus Naive. n = 6. (E–J) Representative reverse-transcription polymerase chain reaction, Western blot, and statistical analysis (normalized to GAPDH) revealing that, compared with sham operation (Sham 7D), spinal nerve ligation (SNL 7D) increased CaV3.2 transcription or expression in ipsilateral dorsal horn of the spinal cord (SC) and dorsal root ganglion (DRG) samples harvested on day 7 postoperation, which was reversed by intrathecal application of Gadd45β mRNA–specific antisense siRNA (SNL 7D + Gadd45β RNAi; 5 μg, 10 μl; once daily on days 3 to 6 after SNL). Missense siRNA (MS RNAi; 5 μg, 10 μl). **P <0.01 versus Sham 7D. #P < 0.05. ##P < 0.01 versus SNL 7D. n = 6. The SNL-enhanced CaV3.2 transcription or expression in the DRG was markedly lesser than that in the dorsal horn (SC; normalized to Sham 7D). **P < 0.01 versus SC. n = 6. (K) Chromatin immunoprecipitation (ChIP) quantitative reverse-transcription polymerase chain reaction analysis showed that intrathecal application of Gadd45β mRNA–specific antisense siRNA (SNL 7D + Gadd45β RNAi; 5 μg, 10 μl; once daily on days 3 to 6 after SNL) reversed the SNL-enhanced binding of Gadd45β to the CaV3.2 promoter region in the ipsilateral dorsal horn on day 7 postoperation. **P < 0.01 versus Sham 7D. ##P < 0.01 versus SNL 7D. n = 6. Data represent mean ± SD. IB = immunoblotting.

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