Spinal Ganglion Neurons Research Articles

Morphofunctional Characteristics of Dorsal Root Ganglion Neurons in the Dynamics of Healing of a Purulent Wound

Abstract. between the morphological and functional state of spinal ganglia neurons and the dynamics of healing of a purulent wound in an experimental animal during its natural course and complex therapy with hydropulse sanitation (HIS) and the addition of platelet-rich blood plasma (PRP). Material and methods. In an experiment on 100 white outbred rats, a purulent wound on the lateral surface of the thigh was modeled by introducing a daily culture of Staphyllococcus aureus. Two groups were formed: natural healing and healing after therapy with hydropulse sanitation of the wound with an isotonic NaCl solution followed by the addition of platelet-enriched autoplasma. The material for the study was taken on the 1st, 3rd, 5th, 7th, 14th, and 21st days. The area of the wound and the dynamics of its healing were assessed using the planimetric method. For the study, lumbar spinal ganglia (SG) of segments LIII–LV. were taken. At the light-optical level, after staining with cresyl violet according to Nissl and azure B according to the S. Shea method, the area of nerve cells was measured, photometry of RNA content was carried out, and the number of satellite gliocytes was counted. The obtained data were processed using statistical analysis methods. Results. The healing process of a purulent skin wound ended with the formation of a scar on days 7–14 for both experimental groups. The use of GIS and PRP demonstrated better dynamics of defect closure - a reduction in wound area by 59.35±3.4% (p=0.018) in the period from 3 to 5 days, compared with 40.05±2.9% (p=0.009) with natural healing. The response of SG neurons during healing was characterized by pronounced polymorphism. The maximum number of cells with reactive changes during natural healing was observed on the 5th day of the experiment (17.2±3.2% for A-neurons and 36.7±4.3% for B-neurons). Complex therapy led to a decrease in the proportion of both reactively altered and destructive neurons. The cell area, RNA content, and the number of satellite glia increased on average by the 7th day of the experiment. Separate close correlations were identified between the dynamics of changes in the wound area and the studied quantitative characteristics. Conclusion. A purulent wound causes a complex of nonspecific reactions in the neurons of the SG. In the dynamics of wound healing, neurons with reactive changes more often demonstrated biphasic dynamics, more pronounced for B-type cells with a primary reaction on days 1–3 and a further transition to regenerative hypertrophy or destruction. Regenerative changes in SG neurons had a close correlation with the processes of regeneration of the wound defect.

Vincamine as an agonist of G-protein-coupled receptor 40 effectively ameliorates diabetic peripheral neuropathy in mice.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes, which has yet no curable medication. Neuroinflammation and mitochondrial dysfunction are tightly linked to DPN pathology. G-protein-coupled receptor 40 (GPR40) is predominantly expressed in pancreatic β-cells, but also in spinal dorsal horn and dorsal root ganglion (DRG) neurons, regulating neuropathic pain. We previously have reported that vincamine (Vin), a monoterpenoid indole alkaloid extracted from Madagascar periwinkle, is a GPR40 agonist. In this study, we evaluated the therapeutic potential of Vin in ameliorating the DPN-like pathology in diabetic mice. Both STZ-induced type 1 (T1DM) and db/db type 2 diabetic (T2DM) mice were used to establish late-stage DPN model (DPN mice), which were administered Vin (30 mg·kg-1·d-1, i.p.) for 4 weeks. We showed that Vin administration did not lower blood glucose levels, but significantly ameliorated neurological dysfunctions in DPN mice. Vin administration improved the blood flow velocities and blood perfusion areas of foot pads and sciatic nerve tissues in DPN mice. We demonstrated that Vin administration protected against sciatic nerve myelin sheath injury and ameliorated foot skin intraepidermal nerve fiber (IENF) density impairment in DPN mice. Moreover, Vin suppressed NLRP3 inflammasome activation through either β-Arrestin2 or β-Arrestin2/IκBα/NF-κB signaling, improved mitochondrial dysfunction through CaMKKβ/AMPK/SIRT1/PGC-1α signaling and alleviated oxidative stress through Nrf2 signaling in the sciatic nerve tissues of DPN mice and LPS/ATP-treated RSC96 cells. All the above-mentioned beneficial effects of Vin were abolished by GPR40-specific knockdown in dorsal root ganglia and sciatic nerve tissues. Together, these results support that pharmacological activation of GPR40 as a promising therapeutic strategy for DPN and highlight the potential of Vin in the treatment of this disease.

Characteristics of responsiveness of cochlear nerve to electrical stimulation in patients with cochlear nerve deficiency

Objectives: This study aimed to evaluate the responsiveness of cochlear nerve to electrical stimulation in patients with cochlear nerve deficiency(CND), to compare their results with those measured in implanted children with normal-sized cochlear nerves, and to investigate the characteristics of the cochlear nerve injury of children with CND. Methods: Participants were children who underwent cochlear implantation at Shandong Provincial ENT Hospital from January 2012 to January 2020, including CND group and control group. The CND group included 51 subjects (male:20; female: 31) who were diagnosed with CND and had normal cochlea. For the CND group, four children had been bilaterally implanted, the mean implantation age was (2.7±1.5) years old. The control group included 21 subjects (male:10; femal:11) who had normal-sized cochlear nerve and normal cochlea. For the control group, all children had been unilaterally implanted except one, and the mean implantation age was (3.0±1.9)years old. Three subjects in the CND group used CI422 electrode arrays, and all the other subjects used CI24RECA/CI512 electrode arrays. The electrically evoked compound action potentials (ECAP) had been tried to record for each electrode using Custom Sound EP software (v. 4.3, Cochlear Ltd.) at least six months post first activation. Furthermore, ECAP amplitude growth functions (AGF) were measured at multiple electrode locations across the electrode array. Generalized linear mixed effect models with the subject group and electrode location as the fixed effects and subjects as the random effect were used to compare results of ECAP measurements. Results: In the control group, ECAP could been recorded at all electrodes (100%), but it could only be recorded in 71% (859/1 210) electrodes in the CND group. Additionally, the percentage of electrodes with measurable ECAP decreased from electrode 1 to electrode 22 in the CND group. Compared to the control group, the ECAP thresholds significantly increased, the ECAP amplitudes and AGF slopes significantly decreased, and the ECAP latency significantly increased in the CND group (P<0.01). GLMM showed that the stimulating site had a significant effect on the ECAP threshold, maximum amplitude, and AGF slope (P<0.01), but had no significant effect on the ECAP latency (P>0.05) in the CND group. However, the stimulating site had no significant effects on the ECAP measurements in the control group. Furthermore, the functional status of cochlear nerve varied greatly among CND group. From electrode 1 to electrode 22, the ECAP thresholds gradually increased, the ECAP maximum amplitudes and AGF slopes gradually decreased in the CND group. Conclusion: Compared with patients with normal-sized cochlear nerve, not only the number of residual spinal ganglion neurons reduce,but also the function of spinal ganglion neurons damages in CND patients. The degree of cochlea nerve deterioration varies greatly among CND patients. Generally, the deterioration of cochlear nerve tends to increase from the basal to the apical site of the cochlea.

Resensitization of TRPV1 channels after the P2 receptor activation in sensory neurons of spinal ganglia in rats.

TRPV1 channels are responsible for detecting noxious stimuli such as heat (>43°C), acid, and capsaicin. P2 receptors are involved in numerous functions of the nervous system, including its modulation and specific response to the application of ATP. In our experiments, we investigated the dynamics of calcium transients in DRG neurons associated with TRPV1 channel desensitization and the effect of activation of P2 receptors on this process. We used DRG neurons from rats P7-8 after 1-2 days of culture to measure calcium transients by microfluorescence calcimetry using the fluorescent dye Fura-2 AM. We have shown that DRG neurons of small (d < 22 μm) and medium (d = 24-35 μm) sizes differ in TRPV1 expression. Thus, TRPV1 channels are mainly present in small nociceptive neurons (59% of the studied neurons). Short-term sequential application of the TRPV1 channel agonist capsaicin (100nM) leads to the desensitization of TRPV1 channels by the type of tachyphylaxis. We identified three types of sensory neurons based on responses to capsaicin: (1) desensitized 37.5%, (2) non-desensitized 34.4%, and (3) insensitive 23.4% to capsaicin. It has also been shown that P2 receptors are present in all types of neurons according to their size. So, the responses to ATP were different in different-sized neurons. Applying ATP (0.1 mM) to the intact cell membrane after the onset of tachyphylaxis caused recovery of calcium transients in response to the addition of capsaicin in these neurons. The amplitude of the capsaicin response after reconstitution with ATP was 161% of the previous minimal calcium transient in response to capsaicin. Significantly, the restoration of the amplitude of calcium transients under the ATP application is not associated with changes in the cytoplasmic pool of ATP because this molecule does not cross the intact cell membrane, thus, our results show the interaction between TRPV1 channels and P2 receptors. It is important to note that the restoration of the amplitude of calcium transients through TRPV1 channels after application of ATP was observed mainly in cells of 1-2 days of cultivation. Thus, the resensitization of capsaicin transients following P2 receptor activation may be associated with the regulation of the sensitivity of sensory neurons.

The Neuroanatomical Basis of Unfamiliar Presentations of Herpes Zoster: A Review and A Case Report

Herpes zoster (HZ, shingles) is caused by reactivated varicella zoster virus, that travels along the peripheral branches from its dormant location in neurons of spinal and cranial sensory ganglia. HZ is well known for its skin vesicular eruptions and severe sensory neuropathy. Over the years, reports have described the occurrence of segmental paresis of skeletal muscles and the involvement of the large and small intestine, clinically resembling intestinal obstruction.

Effects of Photodynamic Therapy on Nav1.7 Expression in Spinal Dorsal Root Ganglion Neurons.

The aim of this study was to examine the effects of photodynamic therapy (PDT) on the expression of Nav1.7 in spinal dorsal root ganglion (DRG) neurons. The primary DRG neurons from newborn SD rats were cultured. The cells were identified by neuron-specific enolase immunofluorescence staining. DRG neurons were divided into four groups: control group, photosensitizer group, laser group, and PDT group. The cell viability was detected by a cell counting kit-8 (CCK8) assay. qRT-PCR and Western blotting were used to determine the mRNA and protein expression levels of Nav1.7 in DRG neurons. The purity of the cultured primary DRG neurons was greater than 90%. Compared with the control group, no significant change was found in the cell viability of the photosensitizer group, while the viability in the laser group and the PDT group was significantly reduced. The mRNA and protein expression levels of Nav1.7 were significantly greater in the laser group and the PDT group than in the control group. At the same time, the mRNA and protein expression levels of Nav1.7 were greater in the laser group than in the PDT group. Both laser and PDT could upregulate the expression of Nav1.7 in DRG neurons, and the promoting effect might be related to the pain induced by clinical treatment. This study provides a research basis for the use of laser and PDT to treat pain. A better understanding of the relationship between Nav1.7 and PDT can help clinicians better manage PDT-related pain.

Emerging Roles of Type-I Interferons in Neuroinflammation, Neurological Diseases, and Long-Haul COVID.

Interferons (IFNs) are pleiotropic cytokines originally identified for their antiviral activity. IFN-α and IFN-β are both type I IFNs that have been used to treat neurological diseases such as multiple sclerosis. Microglia, astrocytes, as well as neurons in the central and peripheral nervous systems, including spinal cord neurons and dorsal root ganglion neurons, express type I IFN receptors (IFNARs). Type I IFNs play an active role in regulating cognition, aging, depression, and neurodegenerative diseases. Notably, by suppressing neuronal activity and synaptic transmission, IFN-α and IFN-β produced potent analgesia. In this article, we discuss the role of type I IFNs in cognition, neurodegenerative diseases, and pain with a focus on neuroinflammation and neuro-glial interactions and their effects on cognition, neurodegenerative diseases, and pain. The role of type I IFNs in long-haul COVID-associated neurological disorders is also discussed. Insights into type I IFN signaling in neurons and non-neuronal cells will improve our treatments of neurological disorders in various disease conditions.

Selective optogenetic activation of NaV1.7-expressing afferents in NaV1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli.

In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as NaV1.7, NaV1.8, and NaV1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite NaV1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of NaV1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic NaV1.7-iCre knock-in mice, which express iCre recombinase under the endogenous NaV1.7 gene promoter without disrupting NaV1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice. Compared with wild-type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous NaV1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in NaV1.7iCre/iCre;Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic NaV1.7-ChR2 mice.

Activation of α 6 -containing GABA A receptors induces antinociception under physiological and pathological conditions.

The loss of GABAergic inhibition is a mechanism that underlies neuropathic pain. Therefore, rescuing the GABAergic inhibitory tone through the activation of GABA A receptors is a strategy to reduce neuropathic pain. This study was designed to elucidate the function of the spinal α 6 -containing GABA A receptor in physiological conditions and neuropathic pain in female and male rats. Results show that α 6 -containing GABA A receptor blockade or transient α 6 -containing GABA A receptor knockdown induces evoked hypersensitivity and spontaneous pain in naive female rats. The α 6 subunit is expressed in IB4 + and CGRP + primary afferent neurons in the rat spinal dorsal horn and dorsal root ganglia but not astrocytes. Nerve injury reduces α 6 subunit protein expression in the central terminals of the primary afferent neurons and dorsal root ganglia, whereas intrathecal administration of positive allosteric modulators of the α 6 -containing GABA A receptor reduces tactile allodynia and spontaneous nociceptive behaviors in female, but not male, neuropathic rats and mice. Overexpression of the spinal α 6 subunit reduces tactile allodynia and restores α 6 subunit expression in neuropathic rats. Positive allosteric modulators of the α 6 -containing GABA A receptor induces a greater antiallodynic effect in female rats and mice compared with male rats and mice. Finally, α 6 subunit is expressed in humans. This receptor is found in CGRP + and P2X3 + primary afferent fibers but not astrocytes in the human spinal dorsal horn. Our results suggest that the spinal α 6 -containing GABA A receptor has a sex-specific antinociceptive role in neuropathic pain, suggesting that this receptor may represent an interesting target to develop a novel treatment for neuropathic pain.

Degeneration Of Spinal Ganglion And Segmental Apparatus Of The Spinal Neurons In Sciatic Nerve Injury: An Experimental Study

Objective — To investigate the extent of degenerative changes in neurons of spinal ganglion and segmental apparatus in various injuries to sciatic nerve in the experiment on white rats. Material and Methods — The research involved 40 white non-pedigree male rats distributed among four groups. The animals of Group 1 (n=10) underwent the compression of nerve trunks with Mosquito clamp forceps for 15 minutes. In Group 2 (n=10), the animals had their nerve trunks ligated; and in Group 3, they had their nerves completely transected in their middle thirds. The separate group of control animals (n=10) suffered no damage to their sciatic nerves. Spinal cords and spinal ganglia at L4-L6 level were the material for histopathological examination. We calculated the number (percent) of degenerated neurons in spinal cords and spinal ganglia at the affected sides on Day 30, and compared them to those at the intact sides. Results — The number (percent) of degenerated neurons in spinal cord and spinal ganglion, expressed as Me (Q1; Q2), constituted 2.52% (1.92; 2.74) and 3.75% (2.37; 4.74) in Group 1, 9.27% (9.03; 9.94) and 16.74% (16.01; 18.22) in Group 2, 25.59% (24.36; 26.29) and 31.94% (31.44; 33.03) in Group 3, respectively. Depending on the number (percent) of degenerated neurons, we classified three grades of change manifestation: mild (Group 1), medium (Group 2), and severe (Group 3). No degenerated neurons were found in the control animals. Conclusion — The compression, ischemic exposure on the sciatic nerve, and complete anatomical transection of its trunk resulted in Wallerian degeneration, as well as degeneration of segmental apparatus in spinal cord neurons.

Can Hybrid Synapse be Formed between Rat Spinal Motor Neurons and Major Pelvic Ganglion Neurons in vitro?

The purpose of this study is to determine whether synapses can be formed between spinal motor neurons (SMNs) and major pelvic ganglion (MPG) neurons of a rat in vitro. The green fluorescent protein (GFP)-labelled MPG cells were cultured together with SMNs in a specific medium. The synaptic-like contacts established between SMNs and MPG neurons were studied in co-cultures using morphologic and immunocytochemistry approaches. Phase-contrast observation of co-cultures showed apparent SMNs-MPG neurons contacts as early as three or four days in vitro. We demonstrate some evidence of synaptic contacts between SMNs and MPG neurons in vitro by immunostaining with antibody directed against postsynaptic density protein 95 (PSD-95). We describe the development process of a defined SMNs-MPG neurons co-culture system. The results suggest that the hybrid synapse formation that may occur between SMNs and MPG neurons in vitro played an essential role in the mechanisms of a regenerated bladder with an artificial somatic-autonomic reflex arc.

High-Mobility Group Box 1 Protein Signaling in Painful Diabetic Neuropathy.

Diabetes is a global epidemic and more than 50% diabetic patients are also diagnosed with neuropathy, which greatly affects the quality of life of the patients. Available treatments are not always successful due to the limited efficacy and complications, such as addiction and dependency. Studies have implicated that high mobility group box1 (HMGB1) protein plays a crucial role in neuroinflammation and the development of neuropathic conditions. HMGB1 is a proinflammatory cytokine that can be released from necrotic cells in passive form or in response to inflammatory signals as an active form. HMGB1 is the ligand for the receptor for advanced glycation end products (RAGE), and toll-like receptors, (TLR)-2 and TLR4, which also indirectly activates C-X-C chemokine receptor type 4 (CXCR4). We investigated whether blocking of HMGB1 can reduce pain and inflammation in diabetic neuropathic animals to further understand the role of HMGB1 in diabetic neuropathy. Type 2 diabetic rats and mice were treated with natural inhibitor of HMGB1, glycyrrhizin (GLC) for five days/week for four weeks at a dose of 50 mg/kg per day by intraperitoneal injection. The animals were divided into three categories: naïve control, diabetic alone, diabetic with GLC treatment. All of the behavioral analyses were conducted before and after the treatment. The expression of inflammatory markers and changes in histone acetylation in the peripheral nervous system were measured by immunohistochemistry and Western blot analysis after the completion of the treatment. Our study revealed that TLR4, HMGB1, CXCR4, and Nod-like receptor protein 3 (NLRP3) levels were increased in the spinal and dorsal root ganglia (DRG) neurons of Type 2 diabetic mice and rats with painful neuropathy. GLC treatment inhibited the increases in TLR4, NLRP3, and CXCR4 expressions and improved the mechanical and thermal pain threshold in these animals. Immunohistochemical studies revealed that hyperglycemia mediated inflammation influenced HMGB1 acetylation and its release from the neurons. It also altered histone 3 acetylation in the microglial cells. The inhibition of HMGB1 by GLC prevented the release of HMGB1 as well as H3K9 acetylation. These findings indicate that the interruption of HMGB1 mediated inflammation could ameliorate diabetic neuropathy and might exhibit a unique target for the treatment.

Regenerative Potential of Spinal Ganglion Neurons During Topical Administration of Platelet-Enriched Blood Plasma Into the Skin Wound.

We studied the reactions of spinal ganglion neurons accompanying regeneration of the skin wound complicated by S. aureus sp. infection. The appearance of neuron groups with different morphofunctional changes was noted: reversible dystrophic and compensatory reactions and irreversible degenerative changes. The proportion of small B neurons was higher than the proportion of large type A neurons at all terms of the experiment. Application of plateletrich plasma to the wound after preliminary hydroimpulsive treatment considerably reduced cell destruction and stimulated regenerative effects in neurons starting from day 7 of the experiment. Application of platelet-rich plasma without hydroimpulsive treatment decelerated wound regeneration and increased the number of destructively changed neurons even in comparison with spontaneous wound healing. Close correlations between protein synthesis markers and stages of the wound process and its characteristics were revealed.

Heteromerization of \u03bc-opioid receptor and cholecystokinin B receptor through the third transmembrane domain of the \u03bc-opioid receptor contributes to the anti-opioid effects of cholecystokinin octapeptide

Activation of the cholecystokinin type B receptor (CCKBR) by cholecystokinin octapeptide (CCK-8) inhibits opioid analgesia. Chronic opiate treatment leads to an increase in the CCK-8 concentration and thus enhances the antagonism of CCK-8 against opioid analgesia; the underlying molecular mechanisms remain of great interest. In the present study, we validated the colocalization of the μ-opioid receptor (MOR) and CCKBR in pain signal transmission-related spinal cord dorsal horn and dorsal root ganglion neurons of rats. Co-immunoprecipitation (Co-IP) and fluorescence lifetime-imaging-microscopy-based fluorescence resonance energy transfer (FLIM-FRET) assays showed that MOR heteromerized with CCKBR directly in transfected HEK293 cells. Combined with MOR mutant construction, the third transmembrane domain of MOR (TM3MOR) was demonstrated to participate in heteromerization with CCKBR. Receptor ligand binding, ERK phosphorylation and cAMP assays showed that MOR heteromerization with CCKBR weakened the activity of MOR. A cell-penetrating interfering peptide consisting of TM3MOR and TAT (a transactivator of HIV-1) sequences from the N terminal to the C terminal disrupted the MOR–CCKBR interaction and restored the activity of MOR in transfected HEK293 cells. Furthermore, intrathecal application of the TM3MOR-TAT peptide alleviated CCK-8-injection-induced antagonism to morphine analgesia in rats. These results suggest a new molecular mechanism for CCK-8 antagonism to opioid analgesia in terms of G-protein-coupled receptor (GPCR) interaction through direct heteromerization. Our study may provide a potential strategy for pain management with opioid analgesics.

Allogenic Adipose Derived Stem Cells Transplantation Improved Sciatic Nerve Regeneration in Rats: Autologous Nerve Graft Model.

We examined the effect of transplantation of allogenic adipose-derived stem cells (ADSCs) with properties of mesenchymal stem cells (MSCs) on posttraumatic sciatic nerve regeneration in rats. We suggested an approach to rat sciatic nerve reconstruction using the nerve from the other leg as a graft. The comparison was that of a critical 10 mm nerve defect repaired by means of autologous nerve grafting versus an identical lesion on the contralateral side. In this experimental model, the same animal acts simultaneously as a test model, and control. Regeneration of the left nerve was enhanced by the use of ADSCs, whereas the right nerve healed under natural conditions. Thus the effects of individual differences were excluded and a result closer to clinical practice obtained. We observed significant destructive changes in the sciatic nerve tissue after surgery which resulted in the formation of combined contractures in knee and ankle joints of both limbs and neurotrophic ulcers only on the right limb. The stimulation of regeneration by ADSCs increased the survival of spinal L5 ganglia neurons by 26.4%, improved sciatic nerve vascularization by 35.68% and increased the number of myelin fibers in the distal nerve by 41.87%. Moreover, we have demonstrated that S100, PMP2, and PMP22 gene expression levels are suppressed in response to trauma as compared to intact animals. We have shown that ADSC-based therapy contributes to significant improvement in the regeneration.

Cordycepin rescues lidocaine-induced neurotoxicity in dorsal root ganglion by interacting with inflammatory signaling pathway MMP3

Application of local anesthetic reagents, such as lidocaine (Lid), could cause significant neurotoxicity in spinal cord dorsal root ganglia neurons (DRGNs). In this study, we investigated the potential rescuing effect of cordycepin (CDC) in an in vitro explant model of lidocaine-induced apoptosis in DRGNs. Explant of rat neonatal DRGNs was prepared, and treated with Lid in vitro to induce neuronal apoptosis. Prior to Lid treatment, DRGN explant was pre-incubated with various concentrations of CDC to evaluate its rescuing effect on Lid-induced apoptosis. We found that, in cultured DRGNs, Lid caused significant neuronal apoptosis whereas pre-incubation of CDC rescued Lid-induced neurotoxicity. In addition, gene and protein expressions of Caspase-9 and Matrix metalloproteinase 3 (MMP3), a key component of inflammatory signal pathway, were both upregulated by Lid but downregulated by CDC pre-incubation. We further overexpressed MMP3 in cultured DRGNs. And discovered that forced MMP3 overexpression upregulated endogenous MMP3 and caspase-9, and reversed the rescuing effect of CDC on Lid-induced neurotoxicity in DRGNs. Therefore, we concluded that CDC has protective effect on local anesthesia induced spinal cord neurotoxicity, possibly through the inverse regulation on inflammatory signaling pathway.

Lin28a functionally modulates bupivacaine-induced dorsal root ganglion neuron apoptosis through TrkA activation

PurposeLin-28 Homolog A gene (Lin28a) is an important regulator in nerve system. In this study, we investigated the functional mechanism of Lin28a during the process of bupivacaine (BUP)-induced neuronal apoptosis of spinal cord dorsal root ganglion neurons (DRGNs). MethodsYoung mouse DRGNs were cultured in vitro and treated with series concentrations of BUP. Apoptosis was evaluated by TUNEL assay. Corresponding Lin28a mRNA and protein expressions were evaluated by qRT-PCR and western blot (WB) assays. Lin28a was downregulated by siRNA and its effect on BUP (5 mM)-induced DRGN apoptosis was measured by qRT-PCR, WB and TUNEL assays, respectively. Alternatively, Lin28a was upregulated in DRGNs. It’s effect on BUP (0.1 mM)-induced DRGN apoptosis was also measured. Finally, WB was used to examine Caspase-3/9 and TrkA protein expressions in Lin28a-downregualted and BUP-injured DRGN to explore Lin28a-associated signaling pathways. ResultsIn DRGN in vitro culture, 0.1 mM BUP induced moderate neuronal apoptosis while 5 mM BUP induced significant apoptosis. Lin28a mRNA and protein were both upregulated by BUP, in concentration-dependent manner. Functional assays showed that Lin28a downregulation rescued 5 mM BUP-induced neuronal apoptosis, whereas Lin28a upregulation aggravated 0.1 mM BUP-induced neuronal apoptosis in DRGNs. WB showed that Lin28a downregulation reduced Caspase-3/9 proteins and activated TrkA through phosphorylation in BUP-injured DRGNs. ConclusionLin28a is a potent regulator in BUP-induced neuronal apoptosis in DRGNs. The apoptotic protection by Lin28a inhibition is likely through the activation of TrkA signaling pathway.

Hydrogen Peroxide Induces Muscle Nociception via Transient Receptor Potential Ankyrin 1 Receptors.

H2O2 has a variety of actions in skin wounds but has been rarely studied in deep muscle tissue. Based on response to the transient receptor potential ankyrin 1 antagonists after plantar incision, we hypothesized that H2O2 exerts nociceptive effects via the transient receptor potential ankyrin 1 in muscle. Nociceptive behaviors in rats (n = 269) and mice (n = 16) were evaluated after various concentrations and volumes of H2O2 were injected into the gastrocnemius muscle or subcutaneous tissue. The effects of H2O2 on in vivo spinal dorsal horn neuronal activity and lumbar dorsal root ganglia neurons in vitro were evaluated from 26 rats and 6 mice. Intramuscular (mean ± SD: 1,436 ± 513 s) but not subcutaneous (40 ± 58 s) injection of H2O2 (100 mM, 0.6 ml) increased nociceptive time. Conditioned place aversion was evident after intramuscular (-143 ± 81 s) but not subcutaneous (-2 ± 111 s) injection of H2O2. These H2O2-induced behaviors were blocked by transient receptor potential ankyrin 1 antagonists. Intramuscular injection of H2O2 caused sustained in vivo activity of dorsal horn neurons, and H2O2 activated a subset of dorsal root ganglia neurons in vitro. Capsaicin nerve block decreased guarding after plantar incision and reduced nociceptive time after intramuscular H2O2. Nociceptive time after intramuscular H2O2 in transient receptor potential ankyrin 1 knockout mice was shorter (173 ± 156 s) compared with wild-type mice (931 ± 629 s). The greater response of muscle tissue to H2O2 may help explain why incision that includes deep muscle but not skin incision alone produces spontaneous activity in nociceptive pathways.

Нейропротекторный эффект комената кальция при окислительном стрессе и глутаматной цитотоксичности на модели культивируемых нейронов мозжечка и спинальных ганглиев

Изучено влияние нового фармакологического соединения кальциевой соли коменовой кислоты (CaK) на генерацию активных форм кислорода в модельной системе ЦФЛ (цитрат фосфатный буфер с добавлением люминола), на токсичность глутамата в диссоциированных культурах нейронов мозжечка крыс, а также его влияние на ростовые процессы культивируемых нейронов спинальных ганглиев эмбрионов кур в условиях окислительного стресса в сравнении с коменовой кислотой (КК). Установлено, что CaK повышает устойчивость культивируемых нейронов мозжечка к глутаматной цитотоксичности, проявляет выраженный нейротрофический эффект — защищает нейроны спинальных ганглиев эмбрионов кур от окислительного воздействия перекиси водорода, значительно снижает содержание свободных радикалов в модельной системе ЦФЛ (на 64,6 %, p &lt; 0,001). Антиокислительные свойства CaK практически не отличаются от КК, в тоже время максимальный нейропротекторный эффект CaK проявляется в более низких (1 и 10 мкМ), чем КК (1000 мкМ) концентрациях, а нейротрофическое действие, в отличие от КК, в более широком диапазоне концентраций: CaK — 0,01 и 0,001 мМ, КК — 0,01 мМ.

Abnormal neurofilament inclusions and segregations in dorsal root ganglia of a Charcot-Marie-Tooth type 2E mouse model.

Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy is the most prevalent inherited peripheral neuropathy and is associated with over 90 causative genes. Mutations in neurofilament light polypeptide gene, NEFL cause CMT2E, an axonal form of CMT that results in abnormal structures and/or functions of peripheral axons in spinal cord motor neurons and dorsal root ganglion neurons. We have previously generated and characterized a knock-in mouse model of CMT2E with the N98S mutation in Nefl that presented with multiple inclusions in spinal cord neurons. In this report, we conduct immunofluorescence studies of cultured dorsal root ganglia (DRG) from NeflN98S/+ mice, and show that inclusions found in DRG neurites can occur in embryonic stages. Ultrastructural analyses reveal that the inclusions are disordered neurofilaments packed in high density, segregated from other organelles. Immunochemical studies show decreased NFL protein levels in DRG, cerebellum and spinal cord in NeflN98S/+ mice, and total NFL protein pool is shifted toward the triton-insoluble fraction. Our findings reveal the nature of the inclusions in NeflN98S/+ mice, provide useful information to understand mechanisms of CMT2E disease, and identify DRG from NeflN98S/+ mice as a useful cell line model for therapeutic discoveries.