Electroacupuncture Promotes Nerve Regeneration and Functional Recovery Through Regulating lncRNA GAS5 Targeting miR-21 After Sciatic Nerve Injury.

Although the benefits of electroacupuncture (EA) for peripheral nerve injury (PNI) are well accepted in clinical practice, the underlying mechanism remains incompletely elucidated. In our study, we observed that EA intervention led to a reduction in the expression of the long non-coding RNA growth-arrest-specific transcript 5 (GAS5) and an increased in miR-21 levels within the injured nerve, effectively promoting functional recovery and nerve regeneration following sciatic nerve injury (SNI). In contrast, administration of adeno-associated virus expressing GAS5 (AAV-GAS5) weakened the therapeutic effect of EA. On the other hand, both silencing GAS5 and introducing a miR-21 mimic prominently enhanced the proliferation activity and migration ability of Schwann cells (SCs), while also inhibiting SCs apoptosis. On the contrary, inhibition of SCs apoptosis was found to be mediated by miR-21. Additionally, overexpression of GAS5 counteracted the effects of the miR-21 mimic on SCs. Moreover, SCs that transfected with the miR-21 mimic promoted neurite growth in hypoxia/reoxygenation-induced neurons, which might be prevented by overexpressing GAS5. Furthermore, GAS5 was found to be widely distributed in the cytoplasm and was negatively regulated by miR-21. Consequently, the targeting of GAS5 by miR-21 represents a potential mechanism through which EA enhances reinnervation and functional restoration following SNI. Mechanistically, the GAS5/miR-21 axis can modulate the proliferation, migration, and apoptosis of SCs while potentially influencing the neurite growth of neurons.

Therapeutic effects of cinnamon bark oil on sciatic nerve injury in rats.

OBJECTIVE: The aim of this study was to investigate the effects of cinnamon bark essential oil (CBO) on analgesia, motor activity, balance, and coordination in rats with sciatic nerve damage. MATERIALS AND METHODS: Rats were divided into three groups as simply randomized. The right sciatic nerve (RSN) of the Sham group was explored. Only vehicle solution was applied for 28 days. The RSN of the sciatic nerve injury (SNI) group was explored. Damage was created by unilateral clamping, and vehicle solution was applied for 28 days. The RSN of the sciatic nerve injury+cinnamon bark essential oil (SNI+CBO) group was explored. SNI was created by unilateral clamping and CBO was applied for 28 days. In the experiment study, motor activity, balance, and coordination measurements were made with rotarod and accelerod tests. A hot plate test was performed for analgesia measurements. Histopathology studies were carried out with the sciatic nerve tissues. RESULTS: In the rotarod test, there was a statistically significant difference between the SNI group and the SNI+CBO group (p<0.05). According to the accelerod test findings, there was a statistically significant difference between the SNI group with the Sham and SNI+CBO groups. In the hot plate test, there was a statistically significant difference between the SNI group with the Sham and SNI+CBO groups (p<0.05). In comparison to the Sham group and the SNI group, the SNI+CBO group was shown to have the greatest expression level of vimentin. CONCLUSIONS: We have concluded that CBO can be used as an adjuvant treatment in cases of SNI, increased pain, nociception, impaired balance, motor activity, and coordination. Our results will be supported by further studies.

Lentinan alleviates sciatic nerve injury by promoting autophagy to remove myelin fragments.

Lentinan, a natural drug with wide-ranging pharmacological activities, can regulate autophagy-the process through which Schwann cells (SCs) eliminate myelin fragments after peripheral nerve injury (PNI). However, the effect of lentinan after PNI and the role of accelerated myelin debris removal via autophagy in this process are unclear. This study examined the effect of lentinan on rat sciatic nerve repair following crush injury and the underlying mechanisms. After the successful establishment of the sciatic nerve compression injury model, group-specific treatments were performed. The treatment group received 20 mg/kg lentinan via intraperitoneal injection, while the model group was treated with normal saline. The recovery in each group was then evaluated. Further, a rat SC line (RSC96) was cultured in medium with/without lentinan after supplementation with homogenous myelin fractions to evaluate the removal of myelin particles. Our results showed that lentinan promotes autophagic flux in vivo via the AMPK/mTOR signaling pathway, accelerates the clearance of myelin debris by SCs, and inhibits neuronal apoptosis, thereby promoting neurological recovery. Similarly, in vitro experiments showed that lentinan promotes the phagocytosis of myelin debris by SCs. In conclusion, our results suggest that lentinan primarily promotes nerve regeneration by accelerating the autophagic clearance of myelin debris in SCs, and this process is likely regulated by the AMPK/mTOR signaling pathway. Therefore, this study provides compelling evidence that lentinan may be a cost-effective and natural treatment agent for PNI.

Electroacupuncture relieves neuropathic pain by inhibiting degradation of the ecto-nucleotidase PAP in the dorsal root ganglions of CCI mice.

BACKGROUND: Although electroacupuncture is widely used in chronic pain management, it is quite controversial due to its unclear mechanism. We hypothesised that EA alleviates pain by inhibiting degradation of the ecto-nucleotidase prostatic acid phosphatase (PAP) and facilitating ATP dephosphorylation in dorsal root ganglions (DRGs). METHODS: We applied EA in male C57 mice subjected to chronic constriction injury (CCI) and assessed extracellular ATP and 5'-nucleotidease expression in DRGs. Specifically, we used a luminescence assay, quantitative reverse transcriptase-polymerase chain reaction, Western blotting, immunohistochemistry and nociceptive-related behavioural changes to gather data, and we tested for effects after PAP expression was inhibited with an adeno-associated virus (AAV). Moreover, membrane PAP degradation was investigated in cultured DRG neurons and the inhibitory effects of EA on this degradation were assessed using immunoprecipitation. RESULTS: EA treatment alleviated CCI surgery-induced mechanical pain hypersensitivity. Furthermore, extracellular ATP decreased significantly in both the DRGs and dorsal horn of EA-treated mice. PAP protein but not mRNA increased in L4-L5 DRGs, and inhibition of PAP expression via AAV microinjection reversed the analgesic effect of EA. Membrane PAP degradation occurred through a clathrin-mediated endocytosis pathway in cultured DRG neurons; EA treatment inhibited the phosphorylation of adaptor protein complex 2, which subsequently reduced the endocytosis of membrane PAP. CONCLUSIONS: EA treatment alleviated peripheral nerve injury-induced mechanical pain hypersensitivity in mice by inhibiting membrane PAP degradation via reduced endocytosis and subsequently promote ATP dephosphorylation in DRGs. SIGNIFICANCE: In a mouse model of chronic pain, electroacupuncture treatment increased levels of prostatic acid phosphatase (PAP: an ecto-nucleotidase known to relieve pain hypersensitivity) by inhibiting PAP degradation in dorsal root ganglions. This promoted extracellular ATP dephosphorylation, inhibited glia activation and eventually alleviated peripheral nerve injury-induced mechanical pain hypersensitivity in mice. Our findings represent an important step forward in clarifying the mechanisms of pain relief afforded by acupuncture treatment.

Local vibration therapy promotes the recovery of nerve function in rats with sciatic nerve injury.

OBJECTIVE: It has been reported that local vibration therapy can benefit recovery after peripheral nerve injury, but the optimized parameters and effective mechanism were unclear. In the present study, we investigated the effect of local vibration therapy of different amplitudes on the recovery of nerve function in rats with sciatic nerve injury (SNI). METHODS: Adult male Sprague-Dawley rats were subjected to SNI and then randomly divided into 5 groups: sham group, SNI group, SNI + A-1 mm group, SNI + A-2 mm group, and SNI + A-4 mm group (A refers to the amplitude; n = 10 per group). Starting on the 7th day after model initiation, local vibration therapy was given for 21 consecutive days with a frequency of 10 Hz and an amplitude of 1, 2 or 4 mm for 5 min. The sciatic function index (SFI) was assessed before surgery and on the 7th, 14th, 21st and 28th days after surgery. Tissues were harvested on the 28th day after surgery for morphological, immunofluorescence and Western blot analysis. RESULTS: Compared with the SNI group, on the 28th day after surgery, the SFIs of the treatment groups were increased; the difference in the SNI + A-2 mm group was the most obvious (95% confidence interval [CI]: [5.86, 27.09], P < 0.001), and the cross-sectional areas of myocytes in all of the treatment groups were improved. The G-ratios in the SNI + A-1 mm group and SNI + A-2 mm group were reduced significantly (95% CI: [-0.12, -0.02], P = 0.007; 95% CI: [-0.15, -0.06], P < 0.001). In addition, the expressions of S100 and nerve growth factor proteins in the treatment groups were increased; the phosphorylation expressions of ERK1/2 protein in the SNI + A-2 mm group and SNI + A-4 mm group were upregulated (95% CI: [0.03, 0.96], P = 0.038; 95% CI: [0.01, 0.94], P = 0.047, respectively), and the phosphorylation expression of Akt in the SNI + A-1 mm group was upregulated (95% CI: [0.11, 2.07], P = 0.031). CONCLUSION: Local vibration therapy, especially with medium amplitude, was able to promote the recovery of nerve function in rats with SNI; this result was linked to the proliferation of Schwann cells and the activation of the ERK1/2 and Akt signaling pathways.

Effect of herbal extracts on peripheral nerve regeneration after microsurgery of the sciatic nerve in rats.

BACKGROUND: Recent experimental studies using herbal extracts have shown the possibility of peripheral nerve regeneration. This study aimed to investigate the effects of herbal extracts on peripheral nerve regeneration in a rat sciatic nerve injury model. METHODS: A total of 53 rats were randomly assigned to a control group or one of four experimental groups. In all rats, the sciatic nerve was completely severed and microscopic epineural end-to-end neurorrhaphy was performed. Normal saline (2 mL) was topically applied to the site of nerve repair in the control group, whereas four different herbal extracts - 2 mL each of Astragalus mongholicus Bunge, Coptis japonica (Thunb.) Makino, Aconitum carmichaelii Debeaux, or Paeonia lactiflora Pall. - were topically applied to the site of nerve repair in each experimental group. Nerve conduction studies were performed at an average of 11.9 weeks after the operation, and conduction velocity and proximal and distal amplitudes were measured. Biopsies were performed at an average of 13.2 weeks after the initial neurorrhaphy. The quality of nerve anastomosis and perineural adhesion to the surrounding soft tissues was macroscopically evaluated. The neuroma size at the site of the neurorrhaphy was microscopically measured, whereas the size of the scar tissue was evaluated relative to the diameter of the repaired nerve. RESULTS: The nerve conduction study results showed the highest nerve conduction velocity in the experimental group that used the Coptis japonica (Thunb.) Makino extract and the highest proximal and distal amplitudes in the experimental group that used the Aconitum carmichaelii Debeaux extract. Macroscopic evaluations after the second operation showed that grade 2 perineural adhesion was found in 70.8% of rats. The mean neuroma size in the Coptis japonica (Thunb.) Makino, Aconitum carmichaelii Debeaux, and Paeonia lactiflora Pall. groups showed statistically significant decreases relative to the control group. The mean scar tissue formation index in the Paeonia lactiflora Pall. group showed a statistically significant decrease relative to the control group. CONCLUSIONS: The peripheral nerve regeneration effect of the herbal extracts was confirmed through decreased neuroma and scar tissue formation.

Brainstem local microglia induce whisker map plasticity in the thalamus after peripheral nerve injury.

Whisker deafferentation in mice disrupts topographic connectivity from the brainstem to the thalamic ventral posteromedial nucleus (VPM), which represents whisker map, by recruiting "ectopic" axons carrying non-whisker information in VPM. However, mechanisms inducing this plasticity remain largely unknown. Here, we show the role of region-specific microglia in the brainstem principal trigeminal nucleus (Pr5), a whisker sensory-recipient region, in VPM whisker map plasticity. Systemic or local manipulation of microglial activity reveals that microglia in Pr5, but not in VPM, are necessary and sufficient for recruiting ectopic axons in VPM. Deafferentation causes membrane hyperexcitability of Pr5 neurons dependent on microglia. Inactivation of Pr5 neurons abolishes this somatotopic reorganization in VPM. Additionally, microglial depletion prevents deafferentation-induced ectopic mechanical hypersensitivity. Our results indicate that local microglia in the brainstem induce peripheral nerve injury-induced plasticity of map organization in the thalamus and suggest that microglia are potential therapeutic targets for peripheral nerve injury-induced mechanical hypersensitivity.

Electroacupuncture ameliorates mechanical hypersensitivity by down-regulating spinal Janus kinase 2/signal transducer and activation of transcription 3 and interleukin 6 in rats with spared nerve injury.

BACKGROUND: Evidence shows that the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway participates in the pathogenesis of neuropathic pain. Our previous study revealed that electroacupuncture (EA) attenuated neuropathic pain via activation of alpha-7 nicotinic acetylcholine receptor (α7nAChR) in the spinal cord. However, whether 2 Hz EA alleviates neuropathic pain by regulating the downstream molecules JAK2/STAT3 has not been fully clarified. METHODS: Paw withdrawal threshold (PWT) was used as a marker of mechanical allodynia in rats with spared nerve injury (SNI). After applying 2 Hz EA on day 3, 7, 14 and 21 post-surgery, spinal expression of JAK2, STAT3 and pro-inflammatory cytokine interleukin (IL)-6 was examined using quantitative reverse transcription and real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Intrathecal injection of the α7nAChR antagonist alpha-bungarotoxin (α-Bgtx) was used to further explore the mechanism underlying the effects of 2 Hz EA on expression of JAK2/STAT3 in SNI rats. RESULTS: It was found that levels of spinal STAT3 and IL-6 mRNA, as well as levels of phosphorylated (p)-JAK2, p-STAT3 and IL-6 protein, were markedly increased in SNI rats. 2 Hz EA attenuated the SNI-induced up-regulation of p-JAK2, p-STAT3 and IL-6 expression in the spinal cord. Furthermore, intrathecal injection of α-Bgtx (1.0 µg/kg) not only inhibited the effect of 2 Hz EA on mechanical hypersensitivity but also ameliorated the down-regulation of p-JAK2, p-STAT3 and IL-6 expression induced by 2 Hz EA. CONCLUSION: This study revealed that 2 Hz EA attenuated SNI-induced mechanical hypersensitivity and the concomitant up-regulation of spinal JAK2, STAT3 and IL-6 in SNI rats, suggesting that suppression of the JAK2/STAT3 signaling pathway might be the mechanism underlying the therapeutic effect of 2 Hz EA on neuropathic pain.

Neutralizing antibody to proNGF rescues erectile function by regulating the expression of neurotrophic and angiogenic factors in a mouse model of cavernous nerve injury.

BACKGROUND: Radical prostatectomy induces some degree of cavernous nerve injury (CNI) and causes denervation-induced pathologic changes in cavernous vasculature, regardless of the advances in surgical techniques and robotic procedures. The precursor for nerve growth factor (proNGF) is known to be involved in neuronal cell apoptosis and microvascular dysfunction through its receptor p75NTR . OBJECTIVES: To determine the expression of proNGF/p75NTR and the efficacy of proNGF neutralizing antibody (anti-proNGF-Ab) in a mouse model of ED induced by CNI. MATERIALS AND METHODS: Age-matched 12-week-old C57BL/6 mice were distributed into three groups: sham group and bilateral CNI group treated with intracavernous injections of PBS (20 µL) or of anti-proNGF-Ab (20 µg in 20 µL of PBS) on days -3 and 0. Two weeks after treatment, erectile function was measured by electrical stimulation of cavernous nerve. Penis tissues from a separate group of animals were harvested for further analysis. We also determined the efficacy of anti-proNGF-Ab on neural preservation in major pelvic ganglion (MPG) ex vivo. RESULTS: We observed increased penile expression of proNGF and p75NTR after CNI. Intracavernous administration of anti-proNGF-Ab increased nNOS and neurofilament expression probably by enhancing the production of neurotrophic factors, such as neurotrophin-3, NGF, and brain-derived neurotrophic factor. Anti-proNGF-Ab preserved the integrity of cavernous sinusoids, such as pericytes, endothelial cells, and endothelial cell-to-cell junctions, possibly by controlling angiogenic factors (angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor) and induced endogenous eNOS phosphorylation in CNI mice. And finally, treatment with anti-proNGF-Ab rescued erectile function in CNI mice. Anti-proNGF-Ab also enhanced neurite sprouting from MPG exposed to lipopolysaccharide. DISCUSSION AND CONCLUSION: The preservation of damaged cavernous neurovasculature through inhibition of the proNGF/p75NTR pathway may be a novel strategy to treat radical prostatectomy-induced erectile dysfunction.

Effects of low- and high-frequency electroacupuncture on protein expression and distribution of TRPV1 and P2X3 in rats with peripheral nerve injury.

BACKGROUND: Whether electroacupuncture (EA) stimulation at different frequencies has a similar effect on spared nerve injury (SNI) as other neuropathic pain models, and how EA at different frequencies causes distinct analgesic effects on neuropathic pain is still not clear. METHODS: Adult male Sprague-Dawley rats were randomly divided into sham SNI, SNI, 2 Hz, 100 Hz and sham EA groups. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured. EA was performed once a day on days 1 to 14 after SNI. The expressions of transient receptor potential cation subfamily V member 1 (TRPV1) and peripheral purinergic P2X receptor 3 (P2X3) were determined by western blotting and immunofluorescence. TRPV1 siRNA and P2X3 siRNA were administered by intrathecal injection. TRPV1 or P2X3 agonists were combined with EA. RESULTS: There were significant decreases in PWT, but no changes in PWL in the 14 days after SNI. EA using 2- or 100-Hz stimulation similarly increased PWT at every time point. The cytosol protein expression of P2X3 in the L4-L6 dorsal root ganglia (DRG) increased, but the expression of TRPV1 decreased in the SNI model. Both these effects were ameliorated by EA, with 2-Hz stimulation having a stronger effect than 100-Hz stimulation. Blocking either TRPV1 or P2X3 specific siRNAs attenuated the decreased PWT induced by SNI. Administration of either a TRPV1 or P2X3 agonist inhibited EA analgesia. CONCLUSION: 2- and 100-Hz EA similarly induced analgesic effects in SNI. This effect was related to up-regulation and down-regulation, respectively, of cytosol protein expression of P2X3 and TRPV1 in L4-L6 DRG, with 2 Hz having a better effect than 100 Hz.

Positive Regulatory Domain I-binding Factor 1 Mediates Peripheral Nerve Injury-induced Nociception in Mice by Repressing Kv4.3 Channel Expression.

BACKGROUND: The transcriptional repressor positive regulatory domain I-binding factor 1 (PRDM1) is expressed in adult mouse dorsal root ganglion and regulates the formation and function of peripheral sensory neurons. The authors hypothesized that PRDM1 in the dorsal root ganglion may contribute to peripheral nerve injury-induced nociception regulation and that its mechanism may involve Kv4.3 channel transcriptional repression. METHODS: Nociception was induced in C57BL/6 mice by applying chronic constriction injury, complete Freund's adjuvant, or capsaicin plantar injection. Nociceptive response was evaluated by mechanical allodynia, thermal hyperalgesia, cold hyperalgesia, or gait analysis. The role of PRDM1 was evaluated by injection of Prdm1 knockdown and overexpression adeno-associated viruses. The interaction of PRDM1 at the Kv4.3 (Kcnd3) promoter was evaluated by chromatin immunoprecipitation assay. Excitability of dorsal root ganglion neurons was evaluated by whole cell patch clamp recordings, and calcium signaling in spinal dorsal horn neurons was evaluated by in vivo two-photon imaging. RESULTS: Peripheral nerve injury increased PRDM1 expression in the dorsal root ganglion, which reduced the activity of the Kv4.3 promoter and repressed Kv4.3 channel expression (injured vs. uninjured; all P < 0.001). Knockdown of PRDM1 rescued Kv4.3 expression, reduced the high excitability of injured dorsal root ganglion neurons, and alleviated peripheral nerve injury-induced nociception (short hairpin RNA vs. Scram; all P < 0.05). In contrast, PRDM1 overexpression in naive mouse dorsal root ganglion neurons diminished Kv4.3 channel expression and induced hyperalgesia (PRDM1 overexpression vs. control, mean ± SD; n = 13; all P < 0.0001) as evaluated by mechanical allodynia (0.6 ± 0.3 vs. 1.2 ± 0.2 g), thermal hyperalgesia (5.2 ± 1.3 vs. 9.8 ± 1.7 s), and cold hyperalgesia (3.4 ± 0.5 vs. 5.3 ± 0.6 s). Finally, PRDM1 downregulation in naive mice reduced the calcium signaling response of spinal dorsal horn neurons to thermal stimulation. CONCLUSIONS: PRDM1 contributes to peripheral nerve injury-induced nociception by repressing Kv4.3 channel expression in injured dorsal root ganglion neurons.

Electroacupuncture Treatment Suppresses Transcription Factor IRF8 in Spinal Cord of Rats with Spared Nerve Injury.

Objective: Neuropathic pain with complex mechanisms has become a major public health problem that greatly impacts patients' quality of life. Therefore, novel and more effective strategies against neuropathic pain need further investigation. Electroacupuncture (EA) has an ameliorating effect on neuropathic pain following spared nerve injury (SNI), but the underlying mechanism remains to be fully clarified. Interferon regulatory factor 8 (IRF8), a critical transcription factor, was reported to be involved in the modulation of neuropathic pain. Here, we focused on exploring whether 2 Hz EA stimulation exerts an inhibitory action on spinal IRF8 in SNI rats. Methods: In this study, SNI rats were treated with 2 Hz EA once every other day for 21 days. Paw withdrawal threshold (PWT) was applied to determine the analgesic effect of 2 Hz EA on SNI rats. The spinal IRF8 and CX3CRl expressions were detected with qRT-PCR and western blot, and immunofluorescence staining was used to evaluate colocation of IRF8 or CX3CRl with microglial activation marker CD11b in the spinal cord. Results: It was found that SNI induced significant elevation of spinal IRF8 and CX3CRl mRNA and protein expression. Additionally, immunofluorescence results showed that SNI elicited the coexpression of IRF8 with CD11b, as well as CX3CRl with CD11b in the spinal cord. Meanwhile, 2 Hz EA treatment of SNI rats not only reduced IRF8 and CX3CRl mRNA and protein expression, but also reversed the coexpression of IRF8 or CX3CRl with CD11b in the spinal cord, along with an attenuation of SNI-evoked mechanical hypersensitivity. Conclusion: This experiment highlighted that 2 Hz EA can inhibit IRF8 expression and microglial activation in the spinal cord of SNI rats. Hence, targeting IRF8 may be a promising therapeutic strategy for 2 Hz EA treatment of neuropathic pain.

Flexible Multielectrode Array for Skeletal Muscle Conditioning, Acetylcholine Receptor Stabilization and Epimysial Recording After Critical Peripheral Nerve Injury.

Complete re-innervation after a traumatic injury severing a muscle's peripheral nerve may take years. During this time, the denervated muscle atrophies and loses acetylcholine receptors, a vital component of the neuromuscular junction, limiting functional recovery. One common clinical treatment for atrophy is electrical stimulation; however, epimysial electrodes currently used are bulky and often fail due to an excessive inflammatory response. Additionally, there remains a need for a device providing in vivo monitoring of neuromuscular regeneration and the maintenance of acetylcholine receptors. Here, an implantable, flexible microelectrode array (MEA) was developed that provides surface neuromuscular stimulation and recording during long-term denervation. Methods: The MEA uses a flexible polyimide elastomer and an array of gold-based microelectrodes featuring Peano curve motifs, which together maintain electrode flexibility. The devices were implanted along the denervated gastrocnemius muscles of 5 rats. These rats underwent therapeutic stimulation using the MEA daily beginning on post-operative day 2. Another 5 rats underwent tibial nerve resection without implantation of MEA. Tissues were harvested on post-operative day 14 and evaluated for quantification of acetylcholine receptors and muscle fiber area using immunofluorescence and histological staining. Results: The Young's modulus was 1.67 GPa, which is comparable to native tendon and muscle. The devices successfully recorded electromyogram data when implanted in rats. When compared to untreated denervated muscles, MEA therapy attenuated atrophy by maintaining larger muscle fiber cross-sectional areas (p < 0.05). Furthermore, the acetylcholine receptor areas were markedly larger with MEA treatment (p < 0.05). Conclusions: This proof-of-concept work successfully demonstrates the ability to combine conformability, tensile strength-enhancing metal micropatterning, electrical stimulation and recording into a functional implant for both epimysial stimulation and recording.

Attenuating Effect of Portulaca oleracea Extract on Chronic Constriction Injury Induced Neuropathic Pain in Rats: An Evidence of Anti-oxidative and Anti-inflammatory Effects.

BACKGROUND: Neuropathic pain responds poorly to drug treatments. The present study investigated the therapeutic effect of Portulaca oleracea, in chronic constriction injury (CCI)-induced neuropathic pain in rats. OBJECTIVE & METHODS: Neuropathic pain was performed by putting four loose ligatures around the sciatic nerve. CCI resulted in the development of heat hyperalgesia, mechanical allodynia and cold allodynia accompanied by an increase in the contents of TNF-α, IL1ß, malondialdehyde, with a reduction in total thiol content. RESULTS: Administration of Portulaca oleracea (100 and 200 mg/kg intraperitoneal) for 14 days in CCI rats significantly alleviated pain-related behaviors, oxidative damage and inflammatory cytokines in a dose-dependent manner. CONCLUSION: In conclusion, it is suggested that the antinociceptive effects of Portulaca oleracea might be due to antioxidant and anti-inflammatory properties.

Chronic electrical stimulation reduces hyperalgesia and associated spinal changes induced by peripheral nerve injury.

OBJECTIVES: We aimed to investigate if different protocols of electrical stimulation following nerve injury might improve neuropathic pain outcomes and modify associated plastic changes at the spinal cord level. MATERIALS AND METHODS: Adult rats were subjected to sciatic nerve transection and repair, and distributed in four groups: untreated (SNTR, n = 12), repeated acute electrical stimulation (rAES, 50 Hz, one hour, n = 12), chronic electrical stimulation (CES, 50 Hz, one hour, n = 12), and increasing-frequency chronic electrical stimulation (iCES, one hour, n = 12) delivered during two weeks following the lesion. The threshold of nociceptive withdrawal to mechanical stimuli was evaluated by means of a Von Frey algesimeter during three weeks postlesion. Spinal cord samples were processed by immunohistochemistry for labeling glial cells, adrenergic receptors, K+ -Cl- cotransporter 2 (KCC2) and GABA. RESULTS: Acute electrical stimulation (50 Hz, one hour) delivered at 3, 7, and 14 days induced an immediate increase of mechanical pain threshold that disappeared after a few days. Chronic electrical stimulation given daily reduced mechanical hyperalgesia until the end of follow-up, being more sustained with the iCES than with constant 50 Hz stimulation (CES). Chronic stimulation protocols restored the expression of ß2 adrenergic receptor and of KCC2 in the dorsal horn, which were significantly reduced by nerve injury. These treatments decreased also the activation of microglia and astrocytes in the dorsal horn. CONCLUSION: Daily electrical stimulation, especially if frequency-patterned, was effective in ameliorating hyperalgesia after nerve injury, and partially preventing the proinflammatory and hyperalgesic changes in the dorsal horn associated to neuropathic pain.

Down-Regulated Expression of Magnesium Transporter Genes Following a High Magnesium Diet Attenuates Sciatic Nerve Crush Injury.

BACKGROUND: Magnesium supplementation has potential for use in nerve regeneration. The expression of some magnesium transporter genes is reflective of the intracellular magnesium levels. OBJECTIVE: To assess the expression of various magnesium transporter genes as they relate to neurological alterations in a sciatic nerve injury model. METHODS: Sciatic nerve injury was induced in rats, which were then fed either basal or high magnesium diets. Magnesium concentrations and 5 magnesium transporter genes (SLC41A1, MAGT1, CNNM2, TRPM6, and TRPM7) were measured in the tissue samples. RESULTS: The high magnesium diet attenuated cytoskeletal loss in a dose-dependent manner in isolated nerve explants. The high magnesium diet augmented nerve regeneration and led to the restoration of nerve structure, increased S-100, and neurofilaments. This increased regeneration was consistent with the improvement of neurobehavioral and electrophysiological assessment. The denervated muscle morphology was restored with the high magnesium diet, and that was also highly correlated with the increased expression of desmin and acetylcholine receptors in denervated muscle. The plasma magnesium levels were significantly elevated after the animals consumed a high magnesium diet and were reciprocally related to the down-regulation of CNNM2, MagT1, and SCL41A1 in the blood monocytes, nerves, and muscle tissues of the nerve crush injury model. CONCLUSION: The increased plasma magnesium levels after consuming a high magnesium diet were highly correlated with the down-regulation of magnesium transporter genes in monocytes, nerves, and muscle tissues after sciatic nerve crush injury. The study findings suggest that there are beneficial effects of administering magnesium after a nerve injury.

Electrical stimulation of the pudendal nerve promotes neuroregeneration and functional recovery from stress urinary incontinence in a rat model.

The pudendal nerve can be injured during vaginal delivery of children, and slowed pudendal nerve regeneration has been correlated with development of stress urinary incontinence (SUI). Simultaneous injury to the pudendal nerve and its target muscle, the external urethral sphincter (EUS), during delivery likely leads to slowed neuroregeneration. The goal of this study was to determine if repeat electrical stimulation of the pudendal nerve improves SUI recovery and promotes neuroregeneration in a dual muscle and nerve injury rat model of SUI. Rats received electrical stimulation or sham stimulation of the pudendal nerve twice weekly for up to 2 wk after injury. A separate cohort of rats received sham injury and sham stimulation. Expression of brain-derived neurotrophic factor (BDNF) and ßII-tubulin expression in Onuf's nucleus were measured 2, 7, and 14 days after injury. Urodynamics, leak point pressure (LPP), and EUS electromyography (EMG) were recorded 14 days after injury. Electrical stimulation significantly increased expression of BDNF at all time points and ßII-tubulin 1 and 2 wk after injury. Two weeks after injury, LPP and EUS EMG during voiding and LPP testing were significantly decreased compared with sham-injured animals. Electrical stimulation significantly increased EUS activity during voiding, although LPP did not fully recover. Repeat pudendal nerve stimulation promotes neuromuscular continence mechanism recovery possibly via a neuroregenerative response through BDNF upregulation in the pudendal motoneurons in this model of SUI. Electrical stimulation of the pudendal nerve may therefore improve recovery after childbirth and ameliorate symptoms of SUI by promoting neuroregeneration after injury.

Different effects of dexmedetomidine and midazolam on the expression of NR2B and GABAA-α1 following peripheral nerve injury in rats.

Neuropathic pain is a complex, chronic pain condition and the treatment is a major clinical challenge. Recent studies have shown that two FDA approved drugs dexmedetomidine (DEX) and midazolam (MZL), may be useful in treating neuropathic pain, but the mechanism is not fully dementated. Here, we investigated the effects and mechanisms of DEX and MZL treatment in the peripheral nerve injury model. Intramuscular injection with DEX and MZL attenuated the development of mechanical allodynia and thermal hyperalgesia in rats with chronic constriction injury (CCI). Concurrently, the expression of NMDA receptor subunit 2B (NR2B), GABA (A) receptor subunit alpha1 (GABAA-α1), and Sonic Hedgehog (SHH) displayed different temporal patterns in the thalamus and the ipsilateral dorsal horn of the spinal cord after CCI. Such that (1) NR2B expression was decreased on day 1 and 14, whereas GABAA-α1 expression was increased on day 1 in the thalamus, and NR2B expression was decreased on day 1, whereas GABAA-α1 expression was increased on day 1 and day 30 in the ipsilateral spinal cord dorsal horn after DEX treatment. (2) NR2B expression was increased on day 1, then decreased on day 14 and returned to baseline on day30, whereas GABAA-α1 expression was no significant changes on day 1, 14, 30 in the thalamus, and NR2B expression was decreased on day 14 and 30, whereas GABAA-α1 expression was no changes on day 1 and 14 but increased on day 30 after MZL treatment. Furthermore, the mechanical allodynia was significantly attenuated after PUR administration. Meanwhile the expression of NR2B was significantly decreased, and the expression of GABAA-α1 was significantly increased, in the thalamus and in the ipsilateral spinal cord dorsal horn when detected on postoperative day 1, 7, and 14. Our findings indicate that DEX and MZL have different mechanisms in CCI rats, suggesting different strategies could be considered in managing neuropathic pain in different individuals. © 2018 IUBMB Life, 70(2):143-152, 2018.

Spot light on skeletal muscles: optogenetic stimulation to understand and restore skeletal muscle function.

Damage of peripheral nerves results in paralysis of skeletal muscle. Currently, the only treatment option to restore proper function is electrical stimulation of the innervating nerve or of the skeletal muscles directly. However this approach has low spatial and temporal precision leading to co-activation of antagonistic muscles and lacks cell-type selectivity resulting in pain or discomfort by stimulation of sensible nerves. In contrast to electrical stimulation, optogenetic methods enable spatially confined and cell-type selective stimulation of cells expressing the light sensitive channel Channelrhodopsin-2 with precise temporal control over the membrane potential. Herein we summarize the current knowledge about the use of this technology to control skeletal muscle function with the focus on the direct, non-neuronal stimulation of muscle fibers. The high temporal flexibility of using light pulses allows new stimulation patterns to investigate skeletal muscle physiology. Furthermore, the high spatial precision of focused illumination was shown to be beneficial for selective stimulation of distinct nearby muscle groups. Finally, the cell-type specific expression of the light-sensitive effector proteins in muscle fibers will allow pain-free stimulation and open new options for clinical treatments. Therefore, we believe that direct optogenetic stimulation of skeletal muscles is a very potent method for basic scientists that also harbors several distinct advantages over electrical stimulation to be considered for clinical use in the future.

Loss of synaptic zinc transport in progranulin deficient mice may contribute to progranulin-associated psychopathology and chronic pain.

Affective and cognitive processing of nociception contributes to the development of chronic pain and vice versa, pain may precipitate psychopathologic symptoms. We hypothesized a higher risk for the latter with immanent neurologic diseases and studied this potential interrelationship in progranulin-deficient mice, which are a model for frontotemporal dementia, a disease dominated by behavioral abnormalities in humans. Young naïve progranulin deficient mice behaved normal in tests of short-term memory, anxiety, depression and nociception, but after peripheral nerve injury, they showed attention-deficit and depression-like behavior, over-activity, loss of shelter-seeking, reduced impulse control and compulsive feeding behavior, which did not occur in equally injured controls. Hence, only the interaction of 'pain x progranulin deficiency' resulted in the complex phenotype at young age, but neither pain nor progranulin deficiency alone. A deep proteome analysis of the prefrontal cortex and olfactory bulb revealed progranulin-dependent alterations of proteins involved in synaptic transport, including neurotransmitter transporters of the solute carrier superfamily. In particular, progranulin deficiency was associated with a deficiency of nuclear and synaptic zinc transporters (ZnT9/Slc30a9; ZnT3/Slc30a3) with low plasma zinc. Dietary zinc supplementation partly normalized the attention deficit of progranulin-deficient mice, which was in part reminiscent of autism-like and compulsive behavior of synaptic zinc transporter Znt3-knockout mice. Hence, the molecular studies point to defective zinc transport possibly contributing to progranulin-deficiency-associated psychopathology. Translated to humans, our data suggest that neuropathic pain may precipitate cognitive and psychopathological symptoms of an inherent, still silent neurodegenerative disease.