Transcriptional regulatory network during axonal regeneration of dorsal root ganglion neurons:laser-capture microdissection and deep sequencing

The key regulators and regeneration-associated genes involved in axonal regeneration of neurons after injury have not been clarified.In high-throughput sequencing,various factors influence the final sequencing results,including the number and size of cells,the depth of sequencing,and the method of cell separation.There is still a lack of research on the detailed molecular expression profile during the regeneration of dorsal root ganglion neuron axon.In this study,we performed lase r-capture microdissection coupled with RNA sequencing on dorsal root ganglion neurons at 0,3,6,and 12 hours and 1,3,and 7 days after sciatic nerve crush in rats.We identified three stages after dorsal root ganglion injury:early(3-12 hours),pre-regeneration(1 day),and regeneration(3-7 days).Gene expression patterns and related function enrichment res ults showed that one module of genes was highly related to axonal regeneration.We verified the up-regulation of activating transcription factor 3(Atf3),Kruppel like factor 6(Klf6),AT-rich inte raction domain 5A(Arid5α),CAMP responsive element modulator(Crem),and FOS like 1,AP-1 transcription factor Subunit(Fosl1) in dorsal root ganglion neurons after injury.Suppressing these transcription factors(Crem,Arid5o,Fosl1 and Klf6) reduced axonal regrowth in vitro.As the hub transcription factor,Atf3 showed higher expression and activity at the preregeneration and regeneration stages.G protein-coupled estrogen receptor 1(Gper1),inte rleukin 12a(Il12α),estrogen receptor 1(ESR1),and interleukin 6(IL6) may be upstream factors that trigger the activation of Atf3 during the repair of axon injury in the early stage.Our study presents the detailed molecular expression profile during axonal regeneration of dorsal root ganglion neurons after peripheral nerve injury.These findings may provide reference for the clinical screening of molecular targets for the treatment of peripheral nerve injury.

In vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate-induced neurotoxicity

Ciliary neurotrophic factor has neuroprotective effects mediated through signal transducer and Janus kinase（JAK） 2/activator of transcription 3（STAT3） and phosphatidylinositol 3-kinase（PI3 K）/Akt signaling pathways.Whether ciliary neurotrophic factor is neuroprotective for glutamate-induced excitotoxicity of dorsal root ganglion neurons is poorly understood.In the present study,the in vitro neuroprotective effects of ciliary neurotrophic factor against glutamate-induced excitotoxicity were determined in a primary culture of dorsal root ganglion neurons from Wistar rat embryos at embryonic day 15.Whether the JAK2/STAT3 and PI3 K/Akt signaling pathways were related to the protective effects of ciliary neurotrophic factor was also determined.Glutamate exposure inhibited neurite outgrowth,cell viability,and growth-associated protein 43 expression and promoted apoptotic neuronal cell death,all of which were reversed by the administration of exogenous ciliary neurotrophic factor.Additionally,preincubation with either JAK2 inhibitor AG490 or PI3 K inhibitor LY294002 blocked the neuroprotective effect of ciliary neurotrophic factor.These data indicate that the two pathways JAK2/STAT3 and PI3 K/Akt play major roles in mediating the in vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate-induced neurotoxicity.

Low-frequency electrical stimulation improves neurite outgrowth of dorsal root ganglion neurons in vitro via upregulating Ca^(2+)-mediated brain-derived neurotrophic factor expression

Short-term, low-frequency electrical stimulation of neural tissues significantly enhances axonal regeneration of peripheral nerves following injury. However, little is known about the mechanisms of electrical stimulation to induce neurite outgrowth. In the present study, short-term, low-frequency electrical stimulation, using identical stimulation parameters of in vivo experiments, was administered to in vitro dorsal root ganglion （DRG） neurons. Enhanced neurite outgrowth, as well as synthesis and release of brain-derived neurotrophic factor （BDNF）, were examined in electrical stimulation-treated DRG neuronal cultures. Because the effects of electrical stimulation on neuronal intracellular signaling molecules are less reported, classic calcium intracellular signals are directly or indirectly involved in electrical stimulation effects on neurons. Cultured DRG neurons were pretreated with the calcium channel blocker nifedipine, followed by electrical stimulation. Results suggested that electrical stimulation not only promoted in vitro neurite outgrowth, but also enhanced BDNF expression. However, nifedipine reduced electrical stimulation-enhanced neurite outgrowth and BDNF biosynthesis. These results suggest that the promoting effects of electrical stimulation on DRG neurite outgrowth could be associated with altered calcium influx, which is involved induction of neuronal BDNF expression and secretion.

The sensitivity of neurons with non-periodic activity to sympathetic stimulation in rat injured dorsal root ganglion

Objective The relationship between compressed dorsal root ganglion （DRG） neurons and firing pattern and sensitivity of neurons was studied in chronically the Hindmarsh-Rose （HR） neuronal model. Methods Spontane- ous activities from single fibers of chronically compressed DRG neurons in rats were recorded, and divided into periodic and non-periodic firing patterns. The sensitivity of the two kinds of firing pattern neuron to sympathetic stimulation （SS） was compared. Result It was found that 27.3% of periodic firing neurons and 93.2% of non-periodic firing neurons responded to SS respectively （ periodic vs non-periodic, P 〈 0.01 ）. The responses to SS with different stimulation time were greater non-periodic firing neurons than periodic firing neurons （P 〈 0.01 ）. The non-periodic firing neurons obviously responded to SS. After the firing pattern of these neurons transformed to periodic firing pattern, their responses to SS disappeared or decreased obviously. The HR neuronal model exhibited a significantly greater response to perturbation in non-periodic （chaotic） firing pattern than in periodic firing pattern. Conelusion The non-periodic firing neurons with deterministic chaos are more sensitive to external stimuli than the periodic firing neurons.

Silencing the enhancer of zeste homologue 2,Ezh2,represses axon regeneration of dorsal root ganglion neurons

Recovery from injury to the peripheral nervous system is different from that of the central nervous system in that it can lead to gene reprogramming that can induce the expression of a series of regeneration-associated genes.This eventually leads to axonal regeneration of injured neurons.Although some regeneration-related genes have been identified,the regulatory network underlying axon regeneration remains largely unknown.To explore the regulator of axon regeneration,we performed RNA sequencing of lumbar L4 and L5 dorsal root ganglion(DRG)neurons at different time points(0,3,6,12 hours,1,3 and 7 days)after rat sciatic nerve crush.The isolation of neurons was carried out by laser capture microscopy combined with NeuN immunofluorescence staining.We found 1228 differentially expressed genes in the injured sciatic nerve tissue.The hub genes within these differentially expressed genes include Atf3,Jun,Myc,Ngf,Fgf2,Ezh2,Gfap and Il6.We verified that the expression of the enhancer of zeste homologue 2 gene(Ezh2)was up-regulated in DRG neurons after injury,and this up-regulation differed between large-and small-sized dorsal root ganglion neurons.To investigate whether the up-regulation of Ezh2 impacts axonal regeneration,we silenced Ezh2 with siRNA in cultured DRG neurons and found that the growth of the newborn axons was repressed.In our investigation into the regulatory network of Ezh2 by interpretive phenomenal analysis,we found some regulators of Ezh2(including Erk,Il6 and Hif1a)and targets(including Atf3,Cdkn1a and Smad1).Our findings suggest that Ezh2,as a nerve regeneration-related gene,participates in the repair of the injured DRG neurons,and knocking down the Ezh2 in vitro inhibits the axonal growth of DRG neurons.All the experimental procedures approved by the Administration Committee of Experimental Animals of Jiangsu Province of China(approval No.S20191201-201)on March 21,2019.

Effects of TRPA1 activation and inhibition on TRPA1 and CGRP expression in dorsal root ganglion neurons

Transient receptor potential ankyrin 1(TRPA1) is a key player in pain and neurogenic inflammation, and is localized in nociceptive primary sensory dorsal root ganglion(DRG) neurons. TRPA1 plays a major role in the transmission of nociceptive sensory signals. The generation of neurogenic inflammation appears to involve TRPA1-evoked release of calcitonin gene-related peptide(CGRP). However, it remains unknown whether TRPA1 or CGRP expression is affected by TRPA1 activation. Thus, in this study, we examined TRPA1 and CGRP expression in DRG neurons in vitro after treatment with the TRPA1 activator formaldehyde or the TRPA1 blocker menthol. In addition, we examined the role of extracellular signal-regulated protein kinase 1/2(ERK1/2) in this process. DRG neurons in culture were exposed to formaldehyde, menthol, the ERK1/2 inhibitor PD98059 + formaldehyde, or PD98059 + menthol. After treatment, real-time polymerase chain reaction, western blot assay and double immunofluorescence labeling were performed to evaluate TRPA1 and CGRP expression in DRG neurons. Formaldehyde elevated mRNA and protein levels of TRPA1 and CGRP, as well as the proportion of TRPA1-and CGRP-positive neurons. In contrast, menthol reduced TRPA1 and CGRP expression. Furthermore, the effects of formaldehyde, but not menthol, on CGRP expression were blocked by pretreatment with PD98059. PD98059 pretreatment did not affect TRPA1 expression in the presence of formaldehyde or menthol.

Stimulatory Effects of NMDA on Intracellular Ca^(2+) Nonlinear Kinetic Model in Rat Dorsal Root Ganglion Neurons

The present study revealed the stimulatory effects of NMDA on intracellular ca 2+ concentration in rat dorsal root ganglion (DRG) neurons. Fura 3/AM, an intracellular calcium fluorescent indicator was used to monitor the fluctuation of 〔ca 2+ 〕 i. Here we present the evidence that (1) Confocal microscopy resolved the cells of three different sizes, based on which a cell diameter distribution histogram was drawn. The fluorescence signals originated from the cells of different sizes, small size (diameter<30μm), medium size (diameter between 30 to 50μm),and large size (diameter>50μm); presumably intracellular Ca 2+ concentration was different in the cells of different sizes. (2) The time related variation of fluorescence intensity was observed. In particular, the fluorescence intensity in 0 Ca 2+ bath solution was affected by the application of high ca 2+ solution. (3) In 0 ca 2+ bath solution the intracellular Ca 2+ concentration nonlinear properties of distinct diameter cells was described. (4) A kind of SETAR model was fitted with a medium sized cell.(5)The residuals from the fitted model were tested to see whether they were plausibly Gaussian. These findings indicated that in distinct types of cells intracellular Ca 2+ concentration had different characteristics in different DRG neurons, and contributed to different functions of these neurons. Besides, the established threshold autoregressive model can share intracellular ca 2+ with the main nonlinear kinetic

Effect of type-2 astrocytes on the viability of dorsal root ganglion neurons and length of neuronal processes

The role of type-2 astrocytes in the repair of central nervous system injury remains poorly un- derstood. In this study, using a relatively simple culture condition in vitro, type-2 astrocytes, differentiated from oligodendrocyte precursor cells by induction with bone morphogenetic pro- tein-4, were co-cultured with dorsal root ganglion neurons. We examined the effects of type-2 astrocytes differentiated from oligodendrocyte precursor cells on the survival and growth of dorsal root ganglion neurons. Results demonstrated that the number of dorsal root ganglion neurons was higher following co-culture of oligodendrocyte precursor cells and type-2 astrocytes than when cultured alone, but lower than that of neurons co-cultured with type-1 astrocytes. The length of the longest process and the length of all processes of a single neuron were shortest in neurons cultured alone, followed by neurons co-cultured with type-2 astroc^es, then neurons co-cultured with oligodendrocyte precursor cells, and longest in neurons co-cultured with type-1 astrocytes. These results indicate that co-culture with type-2 astrocytes can increase neuronal survival rate and process length. However, compared with type-1 astrocytes and oligodendrocyte precursor cells, the promotion effects of type-2 astrocytes on the growth of dorsal root ganglion neurons were weaker.

Pulsed electrical stimulation protects neurons in the dorsal root and anterior horn of the spinal cord after peripheral nerve injury

Most studies on peripheral nerve injury have focused on repair at the site of injury, but very few have examined the effects of repair strategies on the more proximal neuronal cell bodies. In this study, an approximately 10-mm-long nerve segment from the ischial tuberosity in the rat was transected and its proximal and distal ends were inverted and sutured. The spinal cord was subjected to pulsed electrical stimulation at T10 and L3, at a current of 6.5 m A and a stimulation frequency of 15 Hz, 15 minutes per session, twice a day for 56 days. After pulsed electrical stimulation, the number of neurons in the dorsal root ganglion and anterior horn was increased in rats with sciatic nerve injury. The number of myelinated nerve fibers was increased in the sciatic nerve. The ultrastructure of neurons in the dorsal root ganglion and spinal cord was noticeably improved. Conduction velocity of the sciatic nerve was also increased. These results show that pulsed electrical stimulation protects sensory neurons in the dorsal root ganglia as well as motor neurons in the anterior horn of the spinal cord after peripheral nerve injury, and that it promotes the regeneration of peripheral nerve fibers.

In vivo imaging of the neuronal response to spinal cord injury:a narrative review

Deciphering the neuronal response to injury in the spinal cord is essential for exploring treatment strategies for spinal cord injury(SCI).However,this subject has been neglected in part because appropriate tools are lacking.Emerging in vivo imaging and labeling methods offer great potential for observing dynamic neural processes in the central nervous system in conditions of health and disease.This review first discusses in vivo imaging of the mouse spinal cord with a focus on the latest imaging techniques,and then analyzes the dynamic biological response of spinal cord sensory and motor neurons to SCI.We then summarize and compare the techniques behind these studies and clarify the advantages of in vivo imaging compared with traditional neuroscience examinations.Finally,we identify the challenges and possible solutions for spinal cord neuron imaging.

Endogenous neurotrophin-3 promotes neuronal sprouting from dorsal root ganglia

In the present study, we investigated the role of endogenous neurotrophin-3 in nerve terminal sprouting 2 months after spinal cord dorsal root rhizotomy. The left L1-5 and L7-S2 dorsal root ganglia in adult cats were exposed and removed, preserving the L6 dorsal root ganglia. Neurotrophin-3 was mainly expressed in large neurons in the dorsal root ganglia and in some neurons in spinal lamina II. Two months after rhizotomy, the number of neurotrophin-3-positive neurons in the spared dorsal root ganglia and the density of neurite sprouts emerging from these ganglia were increased. Intraperitoneal injection of an antibody against neurotrophin-3 decreased the density of neurite sprouts. These findings suggest that endogenous neurotrophin-3 is involved in spinal cord plasticity and regeneration, and that it promotes axonal sprouting from the dorsal root ganglia after spinal cord dorsal root rhizotomy.

Effects of Intrathecally Administerd NaV1.8 Antisense Oligonucleotide on the Expression of Sodium Channel mRNA in Dorsal Root Ganglion

Neuropathic pain has been hypothesized to be the result of aberrant expression and function of sodium channels at the site of injury. To investigate the effects of NaV1.8 antisense oligonucleotide on the expression of sodium channel mRNA in dorsal root ganglion （DRG） neurons in chronic neuropathic pain. 24 Sprague-Dawley rats weighing 200--260 g were anesthetized with the intraperitoneal injection of 300 mg· kg^-1 choral hydrate. The CCI model was made by loose ligation of sciatic nerve trunk by 4--0 chromic gut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. A PE-10 catheter was implanted in subarachnoid space at lumbar region. On the 7th postoperative day the animals were randomly divided into 4 groups. The drugs were injected intrathecally twice a day for 5 consecutive days in group 2--4. The animals were decapitated 14 days after the surgery. The L4--L6 DRG of the operated side was removed and crushed, and total RNA was extracted with Trizol reagent. The contralateral side was used as control. The change of NaV1.8 sodium channel transcripts was determined by RT-PCR. Pain threshold was significantly lowered after CCI as compared with that in control group and was elevated 3 days after antisense oligonucleotide injection. Sensory neuron specific TTX-R sodium channel NaV1.8 transcript was down-regulated after antisense oligonucleotide injection at the dosage of 45 μg as compared with that in CCI group （P〈0.01）, and it was even greater at the dosage of 90 μg. The intrathecally injected NaV1.8 antisense oligonucleotide can reduce the mechanical allodynia and thermal hyperalgesia partially by downregulating the SNS transcript expression.

Physiological relevance of soma secretion in dorsal root ganglion neurons

Dorsal root ganglion (DRG) cells are primary sensory neurons and are important in pain. Recently, a distinct type of exocytosis, Ca2+ independent but voltage-dependent, is found

Combination of Quercetin, Cinnamaldehyde and Hirudin Protects Rat Dorsal Root Ganglion Neurons against High Glucose-Induced Injury through Nrf-2/HO-1 Activation and NF-κB Inhibition

Objective： TO examine the effects of the combination of quercetin （Q）, cinnamaldehyde （C） and hirudin （H）, a Chinese medicine formula on high glucose （HG）-induced apoptosis of cultured dorsal root ganglion （DRG） neurons. Methods： DRG neurons exposed to HG （45 mmol/L） for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species （ROS） level and apoptosis were determined. The expression of nuclear factor of Kappa B （NF- κB）, inhibitory kappa B α （IκBα ）, phosphorylated IκBα and Nf-E2 related factor 2 （Nrf2） were examined using reverse transcription-polymerase chain reaction （RT-PCR） and Western blot assay. The expression of hemeoxygenase-1 （HO-1）, interleukin-6 （IL-6）, tumor necrosis factor （TNF-α） and caspase-3 were also examined by RT-PCR and Western blot assay. Results： HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF- κB signaling pathway （P〈0.05）. Co-treatment with Q, C, H and their combination decreased HG-induced caspase-3 activation and apoptosis （P〈0.05 or P〈0.01）. The expressions of NF- κB, IL-6 and TNF-α were down- regulated, and Nrf2/HO-1 expression was up-regulated （P〈0.05 or P〈0.01）. QCH has better effect in scavenging ROS, activating Nrf-2/HO-1, and down-regulating the NF- κB pathway than other treatment group. Conclusions： DRG neurons＇ apoptosis was increased in diabetic conditions, which was reduced by QCH formula treatment. The possible reason could be activating Nrf-2/HO-1 pathway, scavenging ROS, and inhibition of NF- κB activation. The effect of QCH combination was better than each monomer or the combination of the two monomers.

Effects of Hirudin on High Glucose-Induced Oxidative Stress and Inflammatory Pathway in Rat Dorsal Root Ganglion Neurons

Objective:To investigate protective effects of hirudin on oxidative stress and apoptosis of spinal dorsal root ganglion cells in high-glucose rats at the cellular and molecular level.Methods:Dorsal root ganglion neurons(DRGn)were harvested from embryonic day in 15 SD rats,purified and identificated after primary culture.They were divided into the normal control group,high-glucose(HG)group,positive control(alpha-lipoic acid,ALA)group,low-dose hirudin group(H1),medium-dose hirudin group(H2)and high-dose hirudin group(H3).The control group was cultured by neuron specific culture medium,while the HG group was cultured by neuron specific culture medium and 20 mmol/L glucose(HG medium).The hirudin groups were cultured by HG medium+0.25 IU/mL hirudin(H1),HG medium+0.5 IU/mL hirudin(H2)and HG medium+1 IU/mL hirudin(H3).The ALA group was cultured by HG medium +100μmol/L ALA.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylt etrazolium bromide(MTT)assay was used to explore the optimum concentration and intervention time.Flow cytometry assay was used to detect the level of reactive oxygen series(ROS).Western blot and quantificational realtime polymerase chain reaction(qRT-PCR)were used to detect the expression of protein and mRNA of nuclear factor erythroid 2-related factor 2(Nrf-2),hemeoxygence-1(HO-1),nuclear factor-κB(NF-κB)and Caspase-3.TUNEL assay was used to test the apoptosis rate of different groups.Results:After 24 h of culture,the cell activity of hirudin and ALA groups were higher than that of HG group,and there was a statistical difference between the H1 group and HG group(P<0.05).In hirudin groups,the apoptosis rate of cells,the expression of activated Caspase-3 protein and Caspase-3 mRNA were lower than those of HG group(P<0.01),higher than those of ALA group(P<0.01 or P<0.05).The ROS level of hirudin groups was higher than that of ALA group(P<0.01),lower than that of HG group(P<0.01 or P<0.05).The expression of NF-κB(P65)protein in H3 group were lower than those of HG group(P<0.05).The expression of Nrf-2 protein in hirudin groups was higher than that of HG group(P<0.01),lower than that of ALA group(P<0.01 or P<0.05).The expression of HO-1 protein in hirudin groups was lower than that of ALA group(P<0.01 or P<0.05),higher than that of HG group(P<0.01 or P<0.05).Conclusions:The activity of DRGn cells can be promoted by hirudin under HG conditions.The effects of hirudin on the inhibition of HG on DRGn cells damage mainly include scavenging ROS,up-regulating Nrf-2/HO-1 pathway,inhibiting activation of NF-κB pathway,down-regulating the expression of and Caspase-3 and reducing DRGn cell apoptosis.

Modulation of dragon's blood on tetrodotoxin-resistant sodium currents in dorsal root ganglion neurons and identification of its material basis for efficacy

To clarify the modulation of dragon's blood on the tetrodotoxin-resistant (TTX-R) sodium currents in dorsal root ganglion (DRG) neurons and explore its corresponding material basis for the efficacy, using whole-cell patch clamp technique, the effects of dragon's blood and the combined effects of three components (cochinchinenin A, cochinchinenin B, and loureirin B) extracted from dragon's blood on the TTX-R sodium currents in acute-isolated DRG neurons of rats were observed. According to the operational definition of material basis for the efficacy of TCM established, the material basis of the modulation on the TTX-R sodium currents in DRG neurons of dragon's blood was judged from the experimental results. The drug interaction equation of Greco et al. was used to assess the interaction of the three components extracted from dragon's blood. This investigation demonstrated that dragon's blood suppressed the peak TTX-R sodium currents in a dose-dependent way and affected the activations of TTX-R sodium currents. The effects of the combination of cochinchinenin A, cochinchinenin B, and loureirin B were in good agreement with those of dragon's blood. Although the three components used alone could modulate TTX-R sodium currents, the concentrations of the three components used alone were respectively higher than those used in combination when the inhibition rates on the TTX-R sodium currents of them used alone and in combination were the same. The combined effects of the three components were synergistic. These results suggested that the interference with pain messages caused by the modulation of dragon's blood on TTX-R sodium currents in DRG neurons may explain some of the analgesic effect of dragon's blood and the corresponding material basis for the efficacy is the combination of cochinchinenin A, cochinchinenin B, and loureirin B.

Effects of dragon’s blood resin and its component loureirin B on tetrodotoxin-sensitive voltage-gated sodium currents in rat dorsal root ganglion neurons

Using whole-cell patch clamp technique on the membrane of freshly isolated dorsal root ganglion (DRG) neurons, the effects of dragons blood resin and its important component loureirin B on tetrodotoxin-sensitive (TTX-S) voltage-gated sodium currents were observed. The results show that both blood resin and loureirin B could suppress TTX-S voltage-gated sodium currents in a dose-dependent way. The peak current amplitudes and the steady-state activation and inactivation curves are also made to shift by 0.05% blood resin and 0.2 mmol/L loureirin B. These results demonstrate that the effects of blood resin on TTX-S sodium current may contrib-ute to loureirin B in blood resin. Perhaps the analgesic effect of blood resin is caused partly by loureirin B directly interfering with the nociceptive transmission of primary sensory neurons.

The effects of rises in external K^+ on the hyperpolarization-activated cation current I_h in rat dorsal root ganglion neurons

The effects of rises in external K+(Kext) were examined on the hyperpolarization-activated cation current(Ih) in rat dorsal root ganglion neurons using the whole-cell patch clamp technique.The results showed that Kext increased Ih in a certain concentration and voltage-dependent manner.At the basal Kext level(4 mmol/L),Ih had a maximal amplitude of 1085 ± 340 pA which was enhanced by ～45% and ～92% at 8 and 16 mmol/L Kext,respectively.The midpoint activation voltage was significantly shifted from -98 mV in the hyperpolarizing direction by 8 and 12 mV at 8 and 16 mmol/L Kext,respectively with alteration of the activation course of Ih.The short time constants of activation became longer with the increasing amplitude of the command potential upon rises in Kext.The long time constants became shorter.The reversal potentials were shifted in the positive direction without significant alterations upon rises in Kext.According to the functional role of Ih,Kext increased Ih,resulting in an enhanced neuronal excitability,which might produce activation potential abnormality and perhaps neuropathic pain involved.

Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion

The cell body or soma in the dosal root ganglion(DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons.In a rat model of radicular pain,an intraforaminal implantation of a rod that chronically compressed the lumbar DRG("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accompanied by hyperalgesia in the ipsilateral hind paw.By the 5th day after onset of CCD,there was a novel upregulation in neuronal expression of the chemokine,monocyte chemoattractant protein-1(MCP-1 or CCL2) and also its receptor,CCR2.The neurons developed,in response to topically applied MCP-1,an excitatory response that they normally do not have.CCD also activated non-neuronal cells including,for example,the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days.A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue.The release of ligands such as CCL2,in addition to possibly activating nociceptive neurons(maintaining the pain),may also act to preserve injured cells in the face of ischemia and hypoxia,for example,by promoting angiogenesis.Thus,somal hyperexcitability,as often said of inflammation,may represent a double edged sword.

FOXO3a as a sensor of unilateral nerve injury in sensory neurons ipsilateral, contralateral and remote to injury

Emerging evidence supports that the stress response to peripheral nerve injury extends beyond the injured neuron,with alterations in associated transcription factors detected both locally and remote to the lesion.Stress-induced nuclear translocation of the transcription factor forkhead class box O3a(FOXO3a)was initially linked to activation of apoptotic genes in many neuronal subtypes.However,a more complex role of FOXO3a has been suggested in the injury response of sensory neurons,with the injured neuron expressing less FOXO3a.To elucidate this response and test whether non-injured sensory neurons also alter FOXO3a expression,the temporal impact of chronic unilateral L4–6 spinal nerve transection on FOXO3a expression and nuclear localization in adult rat dorsal root ganglion neurons ipsilateral,contralateral or remote to injury relative to na?ve controls was examined.In na?ve neurons,high cytoplasmic and nuclear levels of FOXO3a colocalized with calcitonin gene related peptide,a marker of the nociceptive subpopulation.One hour post-injury,an acute increase in nuclear FOXO3a in small size injured neurons occurred followed by a significant decrease after 1,2 and 4 days,with levels increasing toward pre-injury levels by 1 week post-injury.A more robust biphasic response to the injury was observed in uninjured neurons contralateral to and those remote to injury.Nuclear levels of FOXO3a peaked at 1 day,decreased by 4 days,then increased by 1 week post-injury,a response mirrored in C4 dorsal root ganglion neurons remote to injury.This altered expression contralateral and remote to injury supports that spinal nerve damage has broader systemic impacts,a response we recently reported for another stress transcription factor,Luman/CREB3.The early decreased expression and nuclear localization of FOXO3a in the injured neuron implicate these changes in the cell body response to injury that may be protective.Finally,the broader systemic changes support the existence of stress/injury-induced humeral factor(s)influencing transcriptional and potentially behavioral changes in uninjured dorsal root ganglion neurons.Approval to conduct this study was obtained from the University of Saskatchewan Animal Research Ethics Board(protocol#19920164).