Sigma-1 receptor expression in sensory neurons and the effect of painful peripheral nerve injury

We first used immunohistochemical (IHC) methods to identify if σ1R protein is present in the DRG. Fluorescent images of σ1R staining in DRG sections revealed the presence of σ1R in neuronal somata in a punctate distribution in the cytoplasm (Figure 1A,B), consistent with its location in the ER[1, 43]. Double staining for σ1R combined with NeuN, a neuron-specific marker, revealed expression of σ1R in all neurons (Figure 1A). Using glutamine synthetase[44, 45] as a marker for astrocyte-like SGCs[46], sections also demonstrated expression of σ1R in SGCs (Figure 1B). This finding is consistent with prior reports of the presence of σ1R in glial cells such as astrocytes and Schwann cells[3, 47]. When classified into different neuronal subpopulations by size, large neurons comparatively higher levels of σ1R expression (Figure 1C). This suggests that there may be neuron subtype-specific expression of σ1R. Accordingly, we compared σ1R expression in neuronal groups characterized by expression of CGRP, a marker of small unmyelinated peptidergic primary sensory neurons, by binding of IB4, a marker of small unmyelinated non-peptidergic sensory neurons, and by expression of NF200, a marker of large, myelinated neurons[48, 49]. This analysis confirmed comparatively higher σ1R expression in the NF200 group (Figure 1D, E), consistent with their larger size.

Transcripts for σ1R have previously been found in neuronal tissues[50, 51], but their presence has not been examined in peripheral sensory neurons. We identified σ1R at the transcript level in DRG lysates (Figure 2A), confirming the IHC findings of its expression there. As an initial examination of the effect of injury on sensory neuron σ1R expression, we also examined lysates from animals that developed painful neuropathy following SNL. Examination of transcript level (Figure 2A) did not show any significant changes. However, Western immunoblotting of lysates using a commercially available antibody (Invitogen) demonstrated significantly down-regulated σ1R protein expression in SNL L5 DRGs compared to Control, while neighboring SNL L4 DRGs showed an intermediate level of σ1R protein expression (Figure 2B,C). These Western findings were duplicated using another σ1R antibody (gift from Drs. T. P. Su and T. Hayashi at NIDA; data not shown).

To further evaluate the effect of injury on σ1R expression, we quantified IHC images from DRG sections following SNL (Figure 3A). Immunofluorescent intensity of σ1R was attenuated in both axotomized SNL L5 DRGs and, to a lesser extent, in the neighboring SNL L4 DRG (Figure 3B). The finding is consistent with our Western blotting result (Figure 2), in which σ1R protein expression decreased more in SNL L5 than in SNL L4 DRGs. This pattern was found in small and medium size groups (Figure 3C), although large neurons failed to show an effect on the SNL L5 group, which were few in number.

For the neuronal subpopulation labeled with CGRP (Figure 4A), we found that σ1R expression decreased not only in SNL L5 but also in neighboring uninjured L4 DRG neurons, compared to Control (Figure 4B). IB4 showed plasma membrane and perinuclear staining (Figure 5A), consistent with a previous description[52]. Double staining for σ1R with IB4 revealed that expression of σ1R decreased in SNL L5 DRGs but not in SNL L4 neurons (Figure 5B). Double staining for σ1R with NF200 (Figure 6A) showed decreased σ1R expression in both L4 and L5 DRGs after SNL, compared to Control (Figure 6B).

Using immunostaining of glutamine synthetase to detect satellite glial cells, we observed diminished σ1R immunofluorescent intensity within the satellite glial cells of the L5 DRG following SNL (Figure 7A,B).

Rats weighing 150 to 180 g were subjected to SNL modified from the original technique[73]. Specifically, rats were anesthetized with 2% isoflurane in oxygen and the right paravertebral region was exposed. The L6 transverse process was removed, after which the L5 and L6 spinal nerves were ligated with 6-0 silk suture and transected distal to the ligature. To minimize non-neural injury, no muscle was removed, muscles and intertransverse fascia were incised only at the site of the two ligations, and articular processes were not removed. The muscular fascia was closed with 4-0 resorbable polyglactin sutures and the skin closed with staples. Control animals received skin incision and closure only. After surgery, rats were returned to their cages and kept under normal housing conditions with access to pellet food and water ad lib.

We measured the incidence of a pattern of hyperalgesic behavior that we have previously documented to be associated with conditioned place avoidance[74, 75]. Briefly, on day 10, 12, and 17 after surgery, right plantar skin was touched (10 stimuli/session) with a 22G spinal needle with adequate pressure to indent but not penetrate the skin. Control animals respond with only a brief reflexive withdrawal, whereas rats subjected to SNL may display a complex hyperalgesia response that includes licking, chewing, grooming and sustained elevation of the paw, which peaks in frequency by day 17. Because of day-to-day variability in individual rat responses, the average frequency of hyperalgesia responses over the 3 testing days was tabulated for each rat. After SNL, only rats that displayed a hyperalgesia-type response after at least 20% of stimuli were used further in this study.

Total RNA was isolated from the homogenized L4 and L5 DRG of control animals (n = 6), and separately from the L4 and L5 DRGs of SNL rats (n = 6) harvested 21 days after surgery, following the manufacturer’s (Invitrogen, Carlsbad, CA) instructions using Trizol reagent (from aqueous phase). After DNase treatment, cDNA was synthesized from amounts of RNA that were standardized for each experiment ranging from 152 to 232 ng for different experiments, using random hexamer primers (Superscript III first strand synthesis kit; Invitrogen, Grand Islands, NY). Quantitative real time PCR (qPCR) analysis was carried out using IQ Syber Green supermix (Biorad Laboratories, Hercules, CA) and specific primers to quantify the cDNA levels of the σ1R genes (forward primer (FP), 5’-CCTGCTGGCATTCGGGGCTC-3’; reverse primer (RP), 5’-TCACAGCTGCAGGCGAACGG-3’; NCBI reference sequence, NM_030996.1). A preliminary gel showed that PCR products had the expected molecular weight and a melting curve was run in each sample to confirm single product in every run (data not shown). Normalization was carried out using the geometric mean of two reference genes, glyceraldehyde 3-phohphate dehydrogenase (GAPDH; FP, 5′-AGACAGCCGCATCTTCTTGT-3′; RP, 5′-TGATGGCAACAATGTCCACT-3′) and mitogen-activated protein kinase 6 (MAPK6; FP, 5′-TAAAGCCATTGACATGTGGG-3′; RP, 5′-TCGTGCACAACAGGGATAGA-3′), which were chosen for their stability in the context of SNL injury[76]. For each sample, two inter-run determinations were averaged and the fold differences in expression (SNL L4, and SNL L5 DRG) were compared to that of the Control samples using the comparative C_T method. Statistical analysis of ∆∆C_T values used for σ1R, normalized with MAPK6/GAPDH, was performed using Kruskal-Wallis test (p = 0.073) with Post hoc by Dunn’s test. Graphs were plotted using 2^-ΔΔCT values for representation of σ1R gene expressions in different groups.

Total protein was isolated from individual L4 and L5 DRGs of Control animals, and separately from L4 and L5 DRGs of SNL animals. Harvested ganglia were homogenized in 150 μl of RIPA lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (Thermo Scientific, Rockford, IL), and incubated on ice for 30 minutes. Lysates were spun down at 14000 rpm for 10 min at 4°C. The supernatant was used for protein estimation using Pierce bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL). Equal amounts of protein (30 μg) were separated on 4-15% sodium dodecyl sulphate polyacrylamide gel electrophoresis gel (SDS-PAGE; Bio-Rad, Hercules, CA) and transferred onto a polyvinylidene fluoride membrane. After blocking with 5% milk in TBS-T (Tris-buffered saline plus 0.1% Tween 20), blots were sequentially probed overnight at 4°C with one of two anti-σ1R rabbit polyclonal antibodies, either a commercial product (1:500, Invitrogen, Camarillo, CA, catalog number 42-3300; Lot No. 797152A, polyclonal affinity-purified antibody raised against a synthetic peptide derived from the C-terminus region (amino acid 139-155) of the rat σ1R), or another that was a kind gift from Drs. Su and Hayashi of NIDA (1:1000, Lot No. 6405, polyclonal affinity-purified antibody raised against amino acid 52-69 from the C-terminus of rat σ1R)[6, 77]. Loading control was used anti-β-tubulin I mouse monoclonal antibody (1:5000, Sigma-Aldrich, St. Louis, MO, catalog number T7816, Lot No. 088 K4795, monoclonal antibody raised against a synthetic peptide corresponding to the C-terminus sequence of β-tubulin isotype I, conjugated to BSA)[45]. Three 15 min washes in TBS-T followed before probing with secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000, Pierce, Rockford, IL). Enhanced chemiluminescence (ECL plus; Amersham, Piscataway, NJ) was used to detect protein bands. Western blot restore stripping buffer (Thermo Scientific, Rockford, IL) was used to strip antibodies from the membrane. The bands obtained were quantified using ImageJ (U.S. National Institutes of Health, Bethesda, MD) program, and β-tubulin I was used to normalize the protein loading. The normalized values (to β-tubulin I) of Control, SNL L4 and SNL L5 isoform were used for the statistical analysis. Although the antibody from NIDA showed a strong expression band at the expected molecular weight (25 kDa), the blot also showed non-specific bands as well (data not shown). The more selective Invitrogen antibody was used for immunohistochemistry.

Twenty-one days post-surgery, the Control, SNL L4 and SNL L5 DRGs were harvested and cryoprotected in 4% paraformaldehyde with 15% sucrose in 0.1 M PBS for 1 h, followed by incubation in 30% sucrose 0.1 M PBS overnight[49]. Tissues embedded in Tissue-Tek optimal cutting temperature compound (Ted Pella, Inc., Redding, CA) were sectioned (10 μm) with a Leica cryostat (Jung CM 1800; Vienna, Austria), plated onto subbed slides (Superfrost Plus Gold; Fisher Scientific, Pittsburgh, PA), and post-fixed in 4% paraformaldehyde with 4% sucrose for 10 min. After blocking with 10% normal goat serum for 1 h at room temperature, the tissue was incubated overnight with anti-σ1R rabbit polyclonal antibody (1:100, Invitrogen, same antibody used Western)[77]. After four washes with PBST, sections were incubated with Alexa Fluor 568 Goat anti-rabbit antibody (1:500; Invitrogen, Camarillo, CA) for 2 h. To determine co-localization of σ1R with the neuron-specific nuclear protein (NeuN), neurofilament 200 (NF-200), CGRP, and glutamine synthetase, the sections were washed four times with PBST followed by incubated with anti-NeuN mouse monoclonal antibody (1:100, Millipore, Billerica, MA, catalog number MAB377, Lot No. LV1616015, monoclonal antibody raised against purified cell nuclei from mouse brain)[45], anti-NF-200 mouse monoclonal antibody (1:1000, Abcam, Cambridge, UK, catalog number ab28029, monoclonal antibody raised against a non phosphorylated epitope from 200kD Neurofilament Heavy of most mammalian species)[49], anti-CGRP mouse polyclonal antibody (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, catalog number SC-28920, Lot No. H1407, epitope corresponding to amino acid 81-128 mapping at the C-terminus of CGRP of human origin)[49], or anti-glutamine synthetase rabbit polyclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, catalog number SC-6640, Lot No. H1407, polyclonal affinity purified antibody raised against a peptide mapping at the C-terminus of glutamine synthetase of human origin)[45] for 2 h. Following four washes with PBST, sections were incubated with Alexa Fluor 488 goat anti-mouse antibody or Alexa Fluor 488 goat anti-rabbit antibody (1:1000; Molecular Probes, Camarillo, CA) for 1 h. To determine co-localization of σ1R with IB4, sections were incubated with Alexa Fluor 488 conjugate IB₄ (1:50, Invitrogen, Camarillo, CA, catalog number I21411, Lot No. 743633,)[78] after four washes with PBST. Sections were washed four times with PBST and covered with Prolong Gold Antifade mounting medium (Invitrogen, Camarillo, CA). Sections were examined by confocal microscopy. The expression level of σ1R protein was represented by the image intensity that was captured using standardized camera parameters (Metamorph, Downingtown, PA), and cell area was determined by outlining the neuronal profile by excluding its nucleus. A neuron was considered positive when intensity in cells of interest was two-fold greater than in background in sections incubated without targeting primary antibody. At least 3 sections from each DRG were randomly chosen for fluorescence intensity measurement, except in a case of SNL L5 DRG with σ1R/CGRP double staining, for which 2 sections were evaluated. The fluorescence intensity of all cells in the section was quantified. The individual who measured the fluorescence intensity was not completely blinded to the treatment due to obvious markers of axotomy such as eccentric location of the nucleus. Average fluorescence intensity of neurons was measured in traced cytoplasmic areas of interest after subtracting background fluorescence[49, 79]. Satellite glial cell σ1R average intensity was derived by creating a mask from a thresholded image of glutamine synthetase immunofluorescence to isolate areas of interest in the image of σ1R immunofluorescence using Photoshop (Adobe Systems Inc., New York City, NY).

Prism (version 6.1, GraphPad Software, Inc., San Diego, CA) was used to perform paired or unpaired Student’s t-test or one-way ANOVA. Data were derived from at least 3 DRGs for every group. Non-parametric Kruskal-Wallis test with Post hoc by Dunn’s test was used for analyzing the influence of injury on σ1R in qPCR and Western immunoblot experiments. In IHC experiments, main effects identified by ANOVA were further analyzed by Tukey’s test (for comparisons between all groups) to compare relevant means. Data are reported as median with interquartile range for qPCR and Western immunoblot experiments, and as mean ± SEM for IHC experiments. A P value less than 0.05 was considered significant.