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. 2024 May;61(5):2672-2685.
doi: 10.1007/s12035-023-03717-w. Epub 2023 Nov 3.

Sigma-1 Receptor Inhibition Reduces Mechanical Allodynia and Modulate Neuroinflammation in Chronic Neuropathic Pain

Simona Denaro  1 , Lorella Pasquinucci  2 , Rita Turnaturi  2 , Cristiana Alberghina  1 , Lucia Longhitano  3 , Sebastiano Giallongo  3 , Giuliana Costanzo  2 , Salvatore Spoto  4 , Margherita Grasso  5 , Agata Zappalà  1 , Giovanni Li Volti  3 , Daniele Tibullo  3 , Nunzio Vicario  6 , Rosalba Parenti  7 , Carmela Parenti  4
Affiliations

Sigma-1 Receptor Inhibition Reduces Mechanical Allodynia and Modulate Neuroinflammation in Chronic Neuropathic Pain

Simona Denaro et al. Mol Neurobiol. 2024 May.

Abstract

Neuropathic pain is one of the most debilitating forms of chronic pain, resulting from an injury or disease of the somatosensory nervous system, which induces abnormal painful sensations including allodynia and hyperalgesia. Available treatments are limited by severe side-effects and reduced efficacy in the chronic phase of the disease. Sigma-1 receptor (σ1R) has been identified as a chaperone protein, which modulate opioid receptors activities and the functioning of several ion channels, exerting a role in pain transmission. As such, it represents a druggable target to treat neuropathic pain. This study aims at investigating the therapeutic potential of the novel compound (+)-2R/S-LP2, a σ1R antagonist, in reducing painful behaviour and modulating the neuroinflammatory environment. We showed that repeated administration of the compound significantly inhibited mechanical allodynia in neuropathic rats, increasing the withdrawal threshold as compared to CCI-vehicle rats. Moreover, we found that (+)-2R/S-LP2-mediated effects resolve the neuroinflammatory microenvironment by reducing central gliosis and pro-inflammatory cytokines expression levels. This effect was coupled with a significant reduction of connexin 43 (Cx43) expression levels and gap junctions/hemichannels mediated microglia-to-astrocyte communication. These results suggest that inhibition of σ1R significantly attenuates neuropathic pain chronicization, thus representing a viable effective strategy.

Keywords: Astrocyte; Connexin 43; Gap junction; Microglia; Neuropathic pain; Sigma-1.

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Conflict of interest statement

The authors have no relevant financial or non-financial interests to disclose.

Figures

Fig. 1
Fig. 1
Behavioural analysis of mechanical allodynia in Sham-Vehicle and CCI-treated rats at 0, 9, 11, 13 and 16 dpl. a Schematic representation of experimental procedure. b Repeated measures of withdrawal threshold following Von Frey filaments stimulation during the time-course of CCI; data are shown as mean (dots) ± SD of n = 4 rats per group; black ***p-value < 0.001 and black **p-value < 0.01 versus sham vehicle group; orange ***p-value < 0.001 versus CCI vehicle group; two-way ANOVA and Holm-Sidak’s post-hoc test. c Aligned dot plots and frequency distribution curves of Von Frey filaments test in sham vehicle, CCI vehicle and CCI (+)-2R/S-LP2 rats; data are shown as dots plot ± SD and frequency distribution curves are reported for each group. ***p-value < 0.001, **p-value < 0.01 between groups; two-way ANOVA and Holm-Sidak’s post-hoc test. CCI, chronic constriction injury; dpl, days post-ligatures; SD, standard deviation
Fig. 2
Fig. 2
CCI rats show a pro-apoptotic signal on spinal cord resident cell populations. Confocal-assisted representative images. a–b Representative pictures (a) and quantification (b) of Cl Casp 3 and NeuN double positive cells per mm2; c–d representative pictures (c) and quantification (d) of Cl Casp 3 and Gfap double positive cells per mm2; e–f representative pictures (e) and quantification (f) of Cl Casp 3 and Iba1 double positive cells per mm2. Analysis was performed in the ipsi-lateral dorsal horn. Arrowheads in a, c and e indicate Cl Casp3 positive cells. Data are shown as aligned dot plot and mean ± SD of n = 4 replicates per group. **p-value < 0.01 between groups; one-way ANOVA and Holm-Sidak’s post-hoc test. CCI: chronic constriction injury; Cl Casp3: cleaved caspase 3; SD: standard deviation. Scale bar: 10 µm
Fig. 3
Fig. 3
CCI induces a robust astrogliosis in spinal cord dorsal horns, reversed by sigma-1 antagonist (+)-2R/S-LP2. a Representative pictures of Gfap positive cells on ipsi-lateral dorsal horns of the spinal cord indicating laminae 1–4 and representative ROI. b Quantification of the number of Gfap positive cells over laminae 1–4 of the lumbar region of the spinal cord. c Representative pictures of Iba1 positive cells on ipsi-lateral dorsal horns of the spinal cord indicating laminae 1–4 and representative ROI. d Quantification of the number of Iba1 positive cells over laminae 1–4 of the lumbar region of the spinal cord. Data are shown as aligned dot plots and mean FC ± SD of n = 4 samples per group. ***p-value < 0.001, **p-value < 0.01 between groups; One-way ANOVA and Holm-Sidak’s post-hoc test. CCI: chronic constriction injury; FC: fold change; HTX: hematoxylin; ROI: region of interest; SD: standard deviation
Fig. 4
Fig. 4
Sigma-1 receptor targeting ameliorates CCI-induced pro-inflammatory milieu. mRNA expression levels of (a) Il6, b Tnf and (c) Gja1 in the spinal cord of Sham vehicle, CCI vehicle and CCI (+)-2R/S-LP2 rats. Data are shown as dot plots and mean FC ± SD of n ≥ 3 samples per group. ***p-value < 0.001, **p-value < 0.01, *p-value < 0.05 between groups; one-way ANOVA and Holm-Sidak’s post-hoc test. CCI: chronic constriction injury; FC: fold change; SD: standard deviation
Fig. 5
Fig. 5
( +)-2R/S-LP2 disrupts astrocytes-microglia signalling via Cx43 down-regulation. a Representative pictures of Gfap, Iba1 and Cx43 immunostaining and (b) quantification of the MFI of Cx43 in Sham vehicle, CCI vehicle and CCI (+)-2R/S-LP2 dorsal horns spinal cords. Data are shown as dot plots and mean FC ± SD of n = 4 rats per group (c) Profile plot of Gfap, Iba1 and Cx43 MFI expressed as arbitrary unit (a.u.) and (d–e) peak-distance profile analysis of Cx43/Gfap (d) and Cx43/Iba1 (e) clusters. Data are shown as dot plots and mean ± SD of n ≥ 9 peak-distance per groups. f Inverted immunofluorescence pictures displaying Cx43 (black dots) localization analysis on Gfap and Iba1 skeletons, bordered in black. Data are shown as mean ± SD. ***p-value < 0.001, *p-value < 0.05 between groups; One-way ANOVA and Holm-Sidak’s post-hoc test. a.u.: arbitrary unit; CCI: chronic constriction injury; FC: fold change; MFI: mean fluorescence intensity; SD: standard deviation. Scale bar = 25 µm
Fig. 6
Fig. 6
Sigma-1 receptor antagonism modulates heterocellular cell coupling Cx43-mediated. a 3D confocal-assisted representative immunofluorescence pictures of spinal cord section of Sham, CCI vehicle and CCI (+)-2R/S-LP2 treated rats; arrowheads indicate Cx43 positive area between Iba1 and Gfap positive cells. b Quantification of Cx43 MFI over area of Gfap- and Iba1-positive cells. Data are shown as dot plot and mean ± SD of n ≥ 16 cells per group and frequency distribution curves are reported for each group. ***p-value < 0.001, *p-value < 0.05 between groups; One-way ANOVA and Holm-Sidak’s post-hoc test. CCI: chronic constriction injury; MFI: mean fluorescence intensity; SD: standard deviation. Scale bar = 10 µm
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