CCI-induced HDAC mRNA expression in the dorsal lumbar spinal cord
Time course changes in Hdac1–11 mRNA expression after CCI are shown in Figure 1. Hdac1–11 expression in the ipsilateral lumbar SCDH of sham-operated rats and RT-qPCR was examined on day 3, 7, 14, and 28 d after CCI. hdac1 (A), hdac2 (B), hdac3 (C), hdac4 (D), hdac5 (E), hdac6 (F), hdac7 (G), hdac8 (H), hdac9 (I), hdac10 (J), and hdac11 (K) mRNA were quantitative results in the ipsilateral and contralateral of SCDH. Compared with the sham group and the contralateral side in each group, ipsilateral Hdac3 and Hdac6 mRNA increased significantly on 7, 14, and 28 d after CCI (Fig. 1C and 1D). Ipsilateral Hdac4 mRNA levels were significantly higher than in the contralateral and sham ipsilateral lumbar SCDH at 7, 14, and 28 d after CCI surgery (Fig. 1F). Ipsilateral Hdac1 and Hdac7 were significantly higher than the contralateral side 7 d after CCI (Fig. 1A and 1G). Ipsilateral Hdac10 was significantly upregulated 3 d after CCI (Fig. 1J). Therefore, spinal Hdac3, Hdac4, and Hdac6 may play vital roles in NP.
CCI-induced upregulation of HDAC3, HDAC4, and HDAC6 protein expression in the spinal cord
To verify whether CCI impacted HDAC3, HDAC4, and HDAC6 protein upregulation, we performed western blot of the ipsilateral lumbar SCDH. Compared with the sham group, CCI significantly increased HDAC3 expression on days 14 and 28. HDAC4 expression was significantly upregulated on 3 and 7 d after CCI. HDAC6 expression was significantly upregulated on 7, 14, and 28 d after CCI (Fig. 2A and 2B). Ipsilateral lumbar SCDH tissue lysates were subjected to HDAC activity analysis with fluorometric ELISA kits. CCI-induced HDAC activity on the ipsilateral side was significantly higher than that in the sham group and the contralateral side on days 7 and 14 (Fig. 2C) after CCI. Thus, CCI upregulated HDAC3, HDAC4, and HDAC6 protein expression and activity in the rat spinal cord.
CCI attenuated spinal H3K9 expression in the spinal cord
H3K9 acetylation plays a dominant role in histone deposition and chromatin assembly. We assessed whether CCI-induced HDAC upregulation attenuated H3K9 expression. Fig. 3 shows western blot (Fig. 3A) and immunofluorescence staining (Fig. 3C) of H3K9 expression in the ipsilateral lumbar SCDH on 3, 7, 14, and 28 d after CCI. H3K9 protein levels were quantified and normalized against b-actin level and are expressed versus the sham group. H3K9 expression decreased significantly from days 3 to 28 after CCI compared with the sham group (Fig. 3B). Similar to the western blot results, immunofluorescence staining revealed significant CCI-induced downregulation of H3K9 in lamina I–III of the SCDH (Fig. 3D). Hence, nociception downregulates H3K9 expression in the SCDH.
CCI upregulated HDAC3, HDAC4, and HDAC6 expression in the ipsilateral gray matter of the lumbar SCDH after CCI
Fig. 4 shows the topographic ipsilateral distribution of HDAC3, HDAC4, and HDAC6 (Fig. 4A, 4C, and 4E) immunofluorescence (red) in the rat lumbar SCDH of sham and CCI rats at postoperative days 3, 7, 14, 21, and 28. HDAC immunoreactive quantification of the fluorescence intensity was determined with ImageJ software (Fig. 4B, 4D, and 4F). Compared with the sham group and the contralateral side, HDAC3 increased significantly 7 and 14 d after CCI (Fig. 4A and 4B). Compared with the sham group and the contralateral side, HDAC4 was rapidly and persistently upregulated by CCI on the ipsilateral side, starting at day 3, peaking at day 14, and maintained until day 28 after CCI (Fig. 4C and 4D). Additionally, HDAC6 was significantly higher than the sham group and the contralateral side on days 14 and 28 after CCI (Fig. 4E and 4F). Based on the results in Figs. 2 and 4, CCI-induced nociceptive sensitization upregulates HDAC3, HDAC4, and HDAC6 expression in the ipsilateral SCDH at the gene and protein levels.
CCI upregulates HDAC3, HDAC4, and HDAC6 expression in neuronal cells
Fig. 5 demonstrates double immunofluorescence staining of HDAC3, HDAC4, and HDAC6 (red) with three specific cell markers (green), NeuN (a neuronal-specific marker), GFAP (an astrocyte-specific marker), and OX-42 (a microglial cell-specific marker) in ipsilateral lumbar SCDH after CCI. HDAC3, HDAC4, and HDAC6 co-localized with neurons and were less visible in astrocytes and microglia from day 3 to 28 after CCI and in the sham group. CCI-induced HDAC3, HDAC4, and HDAC6 co-localized with neurons and were relatively higher expressed 14 d after CCI (Fig. 5A–5C; yellow).
The analgesic effect of the HDAC inhibitor panobinostat in CCI rats
As shown in Fig. 6, rats were randomly separated into three groups: sham, CCI, and CCI + panobinostat. Nociceptive behavior, thermal hyperalgesia (paw withdrawal latency) (Fig. 6A), and mechanical allodynia (paw withdrawal threshold) (Fig. 6D) were assessed every two days after CCI. Thermal hyperalgesia and mechanical allodynia were significantly induced in rats for 28 d after CCI surgery. Compared with the CCI group, daily intrathecal (i.t.) administration of 1mg panobinostat, an HDACi, significantly improved CCI-induced thermal hyperalgesia from day 7 to 28 and mechanical allodynia from day 5 to 28.
Panobinostat attenuates CCI-induced HDAC activity
In Fig. 7, tissue lysates from the ipsilateral lumbar SCDH 28 d after CCI showed that HDAC activity was significantly higher than that in the sham group. To verify whether HDACi downregulates HDAC under neuropathy conditions, we used panobinostat, a non-specific HDACi. In the CCI + panobinostat group, HDAC activity rather than expression was significantly attenuated in the SCDH.
Intrathecal panobinostat restores GAD65 and GABA after CCI
GAD65, regulated by HDAC, participates in transforming glutamate into GABA. Therefore, we utilized immunofluorescence staining to verify whether GAD65 (red) and GABA (red) expression are mediated by HDACs in the SCDH (Fig. 8A and 8C). GAD65 and GABA expression was significantly attenuated in the CCI group and dramatically enhanced in the CCI+ panobinostat group (Fig. 8B and 8D). Thus, panobinostat, a non-specific HDACi, may increase GAD65 expression by ameliorating HDAC activity, resulting in enhanced GABA production in the SCDH under neuropathy.
The role of spinal HDAC on neuropathy-induced nociceptive sensitization
As is shown in Fig. 9, CCI-induced neuronal HDAC3, HDAC4, and HDAC6 upregulation attenuates H3K9 expression in the ipsilateral SCDH. The activation of HDACs represses Gad2 by attenuating H3K9 expression, resulting in GAD65 downregulation, which decreases the production of the inhibitory neurotransmitter GABA but increases the excitatory neurotransmitter glutamate. Both contribute to nociceptive sensitization. Panobinostat administration inhibited HDAC activity and attenuated CCI-induced H3K9, Gad2, and GAD65 downregulation. The restoration of GAD65 could increase GABA synthesis and decrease the amount of glutamate. Thus, HDAC inhibition may have an antinociceptive effect on NP.