Table1_Downregulation of nuclear STAT2 protein in the spinal dorsal horn is involved in neuropathic pain following chronic constriction injury of the rat sciatic nerve.XLSX

Regulation of gene transcription in the spinal dorsal horn (SDH) plays a critical role in the pathophysiology of neuropathic pain. In this study, we investigated whether the transcription factor STAT2 affects neuropathic pain and evaluated its possible mechanisms. A proteomic analysis showed that the nuclear fraction of STAT2 protein in the SDH was downregulated after chronic constriction injury of the rat sciatic nerve, which was associated with the development of neuropathic pain. Similarly, siRNA-induced downregulation of STAT2 in the SDH of naïve rats also resulted in pain hypersensitivity. Using RNA-sequencing analysis, we showed that reduction of nuclear STAT2 after chronic constriction injury was associated with increased expression of microglial activation markers, including the class II transactivator and major histocompatibility complex class II proteins. In addition, siRNA-induced downregulation of STAT2 promoted microglial activation and pro-inflammatory cytokine expression in the SDH. Taken together, these results showed that chronic constriction injury caused downregulation of nuclear STAT2 in the SDH, which may result in microglial activation and development of neuropathic pain. Our findings indicate that restoration of nuclear expression of STAT2 could be a potential pathway for the treatment of neuropathic pain.</p

Double immunofluorescence staining of p300/CBP and cyclooxygenase-2 (Cox-2) in the ipsilateral spinal dorsal horn (laminae I–IV) of vehicle-treated CCI rats.

<p>Cox-2 is localized in the cytoplasm and cell membranes surrounding immunoreactive P300 or CBP nuclei. Arrows indicate the co-localization of Cox-2 and p300 or CBP. Scale bar = 45 µm.</p

Binding of p300/CBP and H3K9ac/H4K5ac to the promoter of BDNF and Cox-2 gene.

<p>The chromatin immunoprecipitation assay was performed with antibodies against p300, CBP, H3K9ac, H4K5ac or non-immune rabbit IgG, after 7 days of treatment with DMSO or curcumin at 20, 40, and 60 mg/kg body weight. Binding of p300/CBP and H3K9ac/H4K5ac to the BDNF promoter (A). Binding of p300/CBP and H3K9ac/H4K5ac to the Cox-2 promoter (B). *p<0.05, CCI versus sham; **p<0.01, CCI versus sham; ^#p<0.05 curcumin versus vehicle; ^##p<0.01 curcumin versus vehicle (n = 10 per group). cur20: curcumin 20 mg/kg; cur40: curcumin 40 mg/kg; cur60: curcumin 60 mg/kg.</p

Protein expression of BDNF and Cox-2 protein.

<p>Western immunoblotting analysis was performed 7/kg body curcumin. β-actin was used as an internal control. Representative images of BDNF expression (A). Relative amount of BDNF protein (B). Representative images of Cox-2 expression (C). Relative amount of Cox-2 protein (D). *p<0.05, CCI versus sham; **p<0.01, CCI versus sham; ^#p<0.05 curcumin versus vehicle (n = 10 per group). Sham: vehicle-treated sham rats; vehicle: vehicle-treated CCI rats; cur20: curcumin 20 mg/kg-treated CCI rats; cur40: curcumin 40 mg/kg-treated CCI rats; cur60: curcumin 60 mg/kg-treated CCI rats.</p

mRNA expression of BDNF and Cox-2.

<p>Quantitative real time polymerase chain reaction was performed 7/kg body weight curcumin. Relative amount of BDNF gene (A). Relative amount of Cox-2 gene (B). Data were normalized to the β-actin gene. **p<0.01, CCI versus sham; ^#p<0.05 curcumin versus vehicle; ^##p<0.01 curcumin versus vehicle (n = 10 per group). sham: vehicle-treated sham rats; vehicle: vehicle-treated CCI rats; cur20: curcumin 20 mg/kg-treated CCI rats; cur40: curcumin 40 mg/kg-treated CCI rats; cur60: curcumin 60 mg/kg-treated CCI rats.</p

Double immunofluorescence staining of p300/CREB binding protein (CBP) and brain derived neurotrophic factor (BDNF) in the ipsilateral spinal dorsal horn (laminae I–IV) of vehicle-treated CCI rats.

<p>Immunoreactivity of BDNF is localized in the cytoplasm and cell membranes surrounding immunoreactive p300 or CBP nuclei. Arrows indicate the co-localization of BDNF and p300 or CBP. Scale bar = 45 µm.</p

Intracerebral gene expression of inflammatory cytokines in young and old animals after TBI.

<p>Differential mRNA expression of interleukin 1β (IL1β; <b>B</b>), tumor-necrosis-factor α (TNFα; <b>C</b> and <b>D</b>) and interleukin 6 (IL6; <b>E</b> and <b>F</b>) were determined in young (2 months, light grey) and old (21 months, dark grey) C57/Bl6 mice at 15 minutes ( = 15 m, n = 6), 24 hours ( = 24 h, n = 7) and 72 hours ( = 72 h, n = 7) after CCI (data are normalized to the housekeeping gene cyclophilin A and are presented as mean±SD in % of age matched naïve animals; p-values were adjusted for multiple comparison by Bonferroni).</p

Quantification of blood differential cell count.

<p>Hemoglobin concentration and platelets were not significantly different at any time point between age groups (young: 2 months = 2 mo, old: 21 months = 21 mo). Number of white blood cells (WBC) was by trend higher 15 minutes after TBI. In old animals 24 hours after TBI WBC were reduced indicating a systemic CNS injury-induced immunodepression (CIDS; <b>^§</b>p<0.05 in old animals (21 months): 15 min vs. 24 h). In young animals reduction of WBC did not reach level of significance (p = 0.067). Lymphocytes were significantly lower whereas neutrophil granulocytes levels were relatively increased in old animals compared to young animals after 24 hours (*p<0.05, 24 h: 2 mo vs. 21 mo). Basophil, eosinophil granulocytes and monocytes were not regulated differently in old and young mice at any time point (Data are presented as mean±SD).</p

Specific primer and probes and optimized temperature conditions for real-time PCR.

<p>Specific primer and probes and optimized temperature conditions for real-time PCR.</p

Contusion Volume, neurological function, and brain edema formation after TBI in young and old mice.

<p><b>A and B:</b> Contusion volumes were assessed in Nissl-stained brain sections in young (2 months, light grey) and old (21 months, dark grey) C57/Bl6 mice at 15 minutes ( = 15 m, n = 6), 24 hours ( = 24 h, n = 7) and 72 hours ( = 72 h, n = 7) after CCI and is presented as absolute values (<b>A</b>; mm³) and in percentage of the contralateral hemisphere (<b>B</b>; %). 15 min-groups represent the primary lesion group. 24 hours after CCI contusion volume increased significantly in both groups. After 72 hours there was an additional increase of brain damage volume in both groups without difference between age groups. <b>C:</b> To determine the neurological function in these animals a Neurological Severity Score (NSS) was performed before CCI (baseline values; additionally data of naïves included), and 24 hours and 72 hours after CCI. 24 hours after CCI NSS increased in both groups without significant difference between young and old animals. 72 hours after TBI neurological impairment improved in young animals, while motor function deteriorated in old animals. <b>D:</b> Body weight loss (in percent of initial weight) was determined as an additional marker for the well being of the animals and showed similar results to the neurological data. After 24 hours weight loss was present in both age groups. However, 72 hours after insult young, but not old animals started to regain weight. <b>E:</b> Brain water content was assessed 24 hours after CCI in young (2 months, light grey, n = 7) and old (21 months, dark grey, n = 7) and is presented as difference to the contralateral hemisphere. Increase in brain water content was significantly higher in aged animals, but (<b>F</b>) nonlinear regression analysis failed to show a correlation between neurological function and brain water content (r = 0.08).</p