Upregulation of the voltage-gated sodium channel beta2 subunit in neuropathic pain models: characterization of expression in injured and non-injured primary sensory neurons

The development of abnormal primary sensory neuron excitability and neuropathic pain symptoms after peripheral nerve injury is associated with altered expression of voltage-gated sodium channels (VGSCs) and a modification of sodium currents. To investigate whether the beta2 subunit of VGSCs participates in the generation of neuropathic pain, we used the spared nerve injury (SNI) model in rats to examine beta2 subunit expression in selectively injured (tibial and common peroneal nerves) and uninjured (sural nerve) afferents. Three days after SNI, immunohistochemistry and Western blot analysis reveal an increase in the beta2 subunit in both the cell body and peripheral axons of injured neurons. The increase persists for >4 weeks, although beta2 subunit mRNA measured by real-time reverse transcription-PCR and in situ hybridization remains unchanged. Although injured neurons show the most marked upregulation,beta2 subunit expression is also increased in neighboring non-injured neurons and a similar pattern of changes appears in the spinal nerve ligation model of neuropathic pain. That increased beta2 subunit expression in sensory neurons after nerve injury is functionally significant, as demonstrated by our finding that the development of mechanical allodynia-like behavior in the SNI model is attenuated in beta2 subunit null mutant mice. Through its role in regulating the density of mature VGSC complexes in the plasma membrane and modulating channel gating, the beta2 subunit may play a key role in the development of ectopic activity in injured and non-injured sensory afferents and, thereby, neuropathic pain

Calpain-cleaved collapsing response mediator protein-3 induces neuronal death after glutamate toxicity and cerebral ischemia.: J.of Neuroscience

NRC publication: Ye

Mu opioid receptors on primary afferent nav1.8 neurons contribute to opiate-induced analgesia: insight from conditional knockout mice.

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund's Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain

Generation of mu opioid receptor conditional knockout mice.

<p>(<b>A</b>) The conditional <i>Oprm1</i> (<i>Oprm1</i>^fl) allele was created by homologous recombination. The scheme shows the wild-type <i>Oprm1</i> allele, the targeting vector, targeted allele and conditional allele obtained after excision of Hygro^r by a Cre recombinase treatment of ES cells. The <i>Oprm1</i>^fl conditional allele - or “floxed” allele - harbors two <i>loxP</i> sites flanking the <i>Oprm1</i> exon 2 and 3. Black boxes, exons; Mf, Mfe1; Sa, SalI, Sp, Spe1 restriction sites; white triangles, <i>loxP</i> sites; Hygro box, floxed hygromycin-resistance cassette, grey box, probe for Southern blot analysis. Dash lines indicate expected labeled DNA fragments in Southern blot analysis. (<b>B</b>) Southern blot analysis of wild-type and targeted alleles in ES cells. Genomic DNA was digested using Mfe1 and hybridized to a 3’ external probe, shown in 1A. The expected bands at 8.5 and 15.7 kb were obtained. (<b>C</b>) Conditional mutant mice. Right part shows the <i>Oprm1</i>^fl conditional allele and excised allele (deletion of exons 2 & 3) after intercrossing <i>Oprm1</i>^fl/fl mice with Nav1.8-Cre mice. A and B indicate PCR primers used to detect gene excision, and C & D PCR primers for the floxed allele. PCR shows exon 2-3 deletion in DRGs but not brain of mu-cKO mice. In DRGs, the two bands result from gene excision in Nav1.8⁺ neurons but not in other Nav1.8-negative cells. Mu-KO mice show full deletion in both DRGs and brain. (<b>D</b>) Conditional knockout of mu opioid receptor gene in DRGs but not brain. Quantitative RT-PCR was used to measure <i>Oprm1</i> mRNA levels from mu^fl, mu-cKO and mu-KO mice. <i>Oprm1</i> mRNA expression was normalized to mu^fl control samples, and is decreased in mu-cKO animals. Oprm1 transcripts were undetectable in DRG and brain from mu-KO animals. ★★ <i>P</i><0.01, mu-cKO <i>vs</i> mu^fl controls. (<b>E</b>) Conditional KO of the mu opioid receptor gene occurs in small/medium DRG cells. Left, representative <i>in </i><i>situ</i> hybridization on DRG sections from mu^fl, mu-cKO and mu-KO mice. Thin, medium and large arrows point to small, medium and large cells, respectively. Scale bar = 100 µm. Right, cell size distribution of <i>Oprm1</i>-positive neurons in DRGs. The % of <i>Oprm1</i>-positive neurons in control and mu-cKO DRGs are shown in white and black, respectively. The % of <i>Oprm1</i>-positive cells is significantly reduced in small and medium, but not large diameter (>700 µm²) neurons from mu-cKO mice. ★★★ <i>P</i><0.001 mu-cKO <i>vs</i> mu^fl controls, Student t-test.</p

Spontaneous guarding pain behavior after paw-CFA and CFA-inflammatory pain at day 9 in conditional mu-cKO mice.

<p>(<b>A</b>) The effect the conditional mutation on ongoing pain behavior was evaluated by quantifying the duration of guarding behavior over 6 min in mu^fl, mu-cKO and mu-KO mice before CFA-induced inflammation and at days 1 and 2 post-CFA. All mouse lines showed the same behavior (mu^fl, n=14; mu-cKO, n=6; mu-KO, n=12). Results are expressed as means ± sem. ★ <i>P</i><0.05, ★★ <i>P</i><0.01, ★★★ <i>P</i><0.001 post-CFA <i>vs</i> naïve. (B) Following CFA injection into tail, mu-cKO and mu^fl mice showed similar heat hyperalgesia at days 2, 6 and 9. The dashed line represents baseline (pre-CFA) sensitivity in the tail immersion tests at 48°C. Morphine (i.p.) produced anti-hyperalgesia in both genotypes, and that was reduced in mu-cKO mice as compared to controls. n=19/genotype., two-way ANOVA (genotype x treatment F(1,71) = 48.812, <i>P</i> <0.001 for treatment; F(1,71) = 5.999, <i>P</i> <0.05 for genotype. Post-hoc Bonferroni test, ✰✰✰ <i>P</i><0.001, morphine <i>vs</i> saline; ★ <i>P</i><0.05, cKO vs flox controls.</p

Acute pain responses are unchanged in conditional mu-cKO mice.

<p>(A) Acute thermal responses were similar in mu-cKO mice and mu^fl controls in the tail immersion test at 48, 50 and 54°C (n=15/genotype), tail flick test (n=10/genotype), Hargreaves test at three different intensities (n=10/genotype), hot plate test (48, 50 and 54°C, n=15 /genotype) and cold tail immersion test at 5°C (n=19/genotype). (B) In the experimental conditions of (A), mu receptor total knockout mice displayed higher sensitivity in the hot plate assay only (conventional KO vs WT, n =11-14/genotype, ★ <i>P</i><0.05) whereas they behaved as controls for heat tail immersion (n=11-14/genotype), tail flick (n=12/genotype), Hargreaves (n=20/genotype) and cold (5°C) tail immersion (n=12-13/genotype) tests. (C) Nociceptive responses to mechanical and chemical stimuli were unchanged in the conditional mutant mice when assessed in the tail pressure (n = 8/genotype) or von Frey filaments (n = 10-13/genotype) test, nocifensive responses to capsaicin (n=10/genotype) and abdominal writhing induced by acetic acid (n=13-14/genotype). (D) In the same experimental conditions as in (C), mu receptor total knockout mice were more sensitive than control mice in the von Frey filaments test for touch perception (conventional KO vs WT, n = 33-57/genotype, ★★ <i>P</i><0.01, Student t-test). Total knockout mice behaved as controls in the tail pressure test (n = 8/genotype), nocifensive responses to capsaicin (n=6/genotype) and abdominal writhing induced by acetic acid (n=4/genotype).</p

Morphine-induced constipation is maintained in conditional mu-cKO mice.

<p>Left: Morphine-induced inhibition of small intestinal transit. Mice were treated with saline (0 mg/kg morphine bars) or morphine (10 mg/kg) and 20 min. later given a charcoal gavage. For determination of small intestine transit, the distance travelled by charcoal was measured relative to the total length of the small intestine. White bars, mu^fl mice; black bars, mu-cKO mice; grey bars, mu-KO mice, n=5/group, two-way ANOVA (genotype X treatment F(2,24) = 18.206, <i>P</i> <0.001 for treatment; <i>P</i> = 0.184 for genotype), post-hoc Fisher test, ✰✰✰ <i>P</i> <0.001, morphine <i>vs</i> saline. Right: Morphine-induced inhibition of fecal boli accumulation. Mice were administered morphine or saline and fecal boli were collected after 4 hrs. n=10-12/group, two-way ANOVA (genotype X treatment F(2,62) = 21.138, <i>P</i> <0.001 for treatment; <i>P</i> = 0.934 for genotype), post-hoc Fisher test, ✰✰ <i>P</i> <0.01, morphine <i>vs</i> saline.</p