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.

Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats

Estrogen affects the generation and transmission of neuropathic pain,but the specific regulatory mechanism is still unclear.Activation of the N-methyl-D-aspartate acid receptor 1（NMDAR1） plays an important role in the production and maintenance of hyperalgesia and allodynia.The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain.A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats.These rats were then subcutaneously injected with 17β-estradiol,the NMDAR1 antagonist D（-）-2-amino-5-phosphonopentanoic acid（AP-5）,or both once daily for 15 days.Compared with injured drug na？ve rats,rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency,indicating increased sensitivity to mechanical and thermal pain.Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity（as assessed by immunohistochemistry） and protein（as determined by western blot assay） in spinal dorsal root ganglia.This 17β-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5,whereas AP-5 alone did not affect NMDAR1 expression.These results suggest that 17β-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve,and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.

Optimization of micropatterned poly(lactic-coglycolic acid) films for enhancing dorsal root ganglion cell orientation and extension

Nerve conduits have been a viable alternative to the ‘gold standard’ autograft for treating small peripheral nerve gap injuries. However, they often produce inadequate functional recovery outcomes and are ineffective in large gap injuries. Ridge/groove surface micropatterning has been shown to promote neural cell orientation and guide growth. However, optimization of the ratio of ridge/groove parameters to promote orientation and extension for dorsal root ganglion （DRG） cells on poly（lactic-co-glycolic acid） （PLGA） films has not been previously conducted. Photolithography and micro-molding were used to define various combinations of ridge/groove dimensions on PLGA films. The DRG cells obtained from chicken embryos were cultured on micropatterned PLGA films for cell orientation and migration evaluation.Biodegradation of the films occurred during the test period, however, this did not cause deformation or distortion of the micropatterns. Results from the DRG cell orientation test suggest that when the ridge/groove ratio equals 1 （ridge/groove width parameters are equal, i.e., 10 μm/10 μm （even））, the degree of alignment depends on the size of the ridges and grooves, when the ratio is smaller than 1 （groove controlled） the alignment increases as the ridge size decreases, and when the ratio is larger than 1 （ridge controlled）, the alignment is reduced as the width of the grooves decreases. The migration rate and neurite extension of DRG neurons were greatest on 10 μm/10 μm and 30 μm/30 μm micropatterned PLGA films. Based on the data, the 10 μm/10 μm and 30 μm/30 μm micropatterned PLGA films are the optimized ridge/groove surface patterns for the construction of nerve repair devices.

PKCε Mediates Substance P Inhibition of GABA_A Receptors-Mediated Current in Rat Dorsal Root Ganglion

The mechanism underlying the modulatory effect of substance P（SP） on GABA-activated response in rat dorsal root ganglion（DRG） neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA（1–1000 μmol/L） induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons（89.8 %） examined with whole-cell patch-clamp recordings. Bath application of GABA（1–1000 μmol/L） evoked a depolarizing response in 236 out of 257（91.8%） DRG neurons examined with intracellular recordings. Application of SP（0.001–1 μmol/L） suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1（NK1） receptors antagonist spantide but not by L659187 and SR142801（1 μmol/L, n=7）, selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C（PLC） inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca2＋-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the ＂pre-synaptic inhibition＂ evoked by GABA, which may explain its role in pain and neurogenic inflammation.

Differential Expression of Alpha-Adrenoceptor Subtypes in Rat Dorsal Root Ganglion after Chronic Constriction Injury

Summary： mRNAs of alpha-adrenoceptor （α-AR） subtypes are found in neurons in dorsal root ganglion （DRG） and change after peripheral nerve injury. In this study, the distribution of α-AR subtype proteins was studied in L5 DRG of normal rats and rats with chronic constriction injury of sciatic nerve （CCI）. Using immunofluorescence technique, it was found that α1A-, α1B-, and α2A-AR proteins were expressed in large, medium, and small size neurons in normal DRG, and significantly increased in all size neurons 14 days after CCI. α1D- and α2C-AR was also expressed in all size neurons in normal DRG. However, α1D-AR was significantly increased and α2C-AR was decreased in small size neurons 14 days post CCI. α2B-AR neurons were not detectable in normal and CCI DRG. Co-expression of α1A- and α2A-AR in the same neuron was observed in normal DRG and increased post CCI. Collectively, these results indicated that there is distinct distribution of α-AR subtypes in DRG neurons, and the distribution and levels of expression of α-AR subtypes change differently after CCI. The up-regulation of α-AR subtypes in DRG neurons may play an important role in the process of generating and transmitting neuropathic pain.

Brain-derived neurotrophic factor expression in dorsal root ganglion neurons in response to reanastomosis of the distal stoma after nerve grafting

Studies have shown that retreatment of the distal stoma after nerve grafting can stimulate nerve regeneration. The present study attempted to verify the effects of reanastomosis of the distal stoma, after nerve grafting, on nerve regeneration by assessing brain-derived neurotrophic factor expression in 2-month-old rats. Results showed that brain-derived neurotrophic factor expression in L2-4 dorsal root ganglia began to increase 3 days after autologous nerve grafting post sciatic nerve injury, peaked at 14 days, decreased at 28 days, and reached similar levels to the sham-surgery group at 56 days. Brain-derived neurotrophic factor expression in L2-4 dorsal root ganglia began to increase 3 days after reanastomosis of the distal stoma, 59 days after autologous nerve grafting post sciatic nerve injury, significantly increased at 63 days, peaked at 70 days, and gradually decreased thereafter, but remained higher compared with the sham-surgery group up to 112 days. The results of this study indicate that reanastomosis of the distal stoma after orthotopic nerve grafting stimulated brain-derived neurotrophic factor expression in L2.4 dorsal root ganglia.

Characteristics of neural growth and cryopreservation of the dorsal root ganglion using three-dimensional collagen hydrogel culture versus conventional culture

In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion(DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates(T-DRG) versus three-dimensional collagen hydrogels(C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis-and myelination-related genes were highly expressed in C-DRG, while epithelial–mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2′-deoxyuridine(EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B(S100 B) and lower α-smooth muscle actin(α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China(approval No. 2020-IRB16), on March 15, 2020.

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.

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.

Expression of gamma-aminobutyric acid type A receptor α_2 subunit in the dorsal root ganglion of rats with sciatic nerve injury

The γ-aminobutyric acid neurotransmitter in the spinal cord dorsal horn plays an important role in pain modulation through primary afferent-mediated presynaptic inhibition. The weakening of γ-aminobutyric acid-mediated presynaptic inhibition may be an important cause of neuropathic pain. γ-aminobutyric acid-mediated presynaptic inhibition is related to the current strength of γ-aminobutyric acid A receptor activation. In view of this, the whole-cell patch-clamp technique was used here to record the change in muscimol activated current of dorsal root ganglion neurons in a chronic constriction injury model. Results found that damage in rat dorsal root ganglion neurons following application of muscimol caused concentration-dependent activation of current, and compared with the sham group, its current strength and γ-aminobutyric acid A receptor protein expression decreased. Immunofluorescence revealed that γ-aminobutyric acid type A receptor α2 subunit protein expression decreased and was most obvious at 12 and 15 days after modeling. Our experimental findings confirmed that the y-aminobutyric acid type A receptor α2 subunit in the chronic constriction injury model rat dorsal root ganglion was downregulated, which may be one of the reasons for the reduction of injury in dorsal root ganglion neurons following muscimol-activated currents.

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.

Preemptive analgesic effects of low-dose ketamine on growth-associated protein expression in dorsal root ganglion of chronic constriction injury model rats

BACKGROUND： Ketamine is a noncompetitive N-methyl-D-aspartate （NMDA） receptor antagonists and plays an important role in the treatment of pain. OBJECTIVE： To analyze the preemptive analgesic effects of different doses of ketamine on growth-associated protein-43 （GAP43） expression in dorsal root ganglion in a rat model of chronic sciatic nerve constricted injury, and to study the differences between high-dose and low-dose ketamine DESIGN： Randomized controlled animal study. SETTING： Medical College of Shantou University. MATERIALS： Thirty-five adult male Sprague Dawley rats were provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine. Ketamine hydrochloride injection was provided by Hengrui Pharmaceutical Co., Ltd., Jiangsu. METHODS： This study was performed at the Immunological Laboratory, Medical College of Shantou University from September to December 2006. Model of chronic sciatic nerve constricted injury： after anesthesia, the right sciatic nerve was exposed and ligated l-cm distal to the ischiadic tuberosity with a No. 3-0 cat gut suture. Grouping and intervention： 35 rats were randomly divided into 4 groups： normal control group （n = 5）, chronic constriction injury （CCI） group （n = 10）, low-dose ketamine group （n = 10）, and high-dose ketamine group （n = 10）. Rats in the normal control group did not undergo any surgery or drug intervention. Rats in the CCI group received intraperitoneal injection of saline （1 mL）, and their sciatic nerves were ligated after 10 minutes. Rats in the low-dose ketamine group underwent intraperitoneal injection of ketamine （25 mg/kg） 10 minutes prior to ligation of sciatic nerve; while, rats in the high-dose ketamine group were given intraperitoneal injection of ketamine （50 mg/kg） 10 minutes prior to ligation of sciatic nerve. On the third and the seventh days after surgery, dorsal root ganglion were resected from the sciatic nerve and cut into sections. MAIN OUTCOME MEASURES： GAP-43 expression in dorsal root ganglion was detected by immunohistochemistry and image analysis system, as well as semi-quantitative analysis. RESULTS： Thirty-five Sprague Dawley rats were included in the final analysis. Qualitative analysis： GAP-43 expression in the CCI group was higher than in the normal control group. Quantitative analysis： after three post-operative days, GAP-43 expression in the CCI group was significantly higher than in the normal control group （t = 22.919, 7.319, P 〈 0.05）. GAP-43 expression in the low-dose and high-dose ketamine group was significantly lower than in the CCI group （t = 11.166, 26.474, P 〈 0.05）. After seven postoperative days, GAP-43 expression in the low-dose and high-dose ketamine groups was significantly lower than in the CCI group （t = 2.382, 5.016, P 〈 0.05）. CONCLUSION： Preoperative administration of ketamine inhibited the increased GAP-43 expression in dorsal root ganglion during neuropathic pain.

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.

Dorsal root ganglion progenitors differentiate to gamma-aminobutyric acid-and choline acetyltransferase-positive neurons

This study examined the isolation and differentiation of dorsal root ganglion progenitor cells for therapeutic use in neurodegenerative diseases. Rat embryonic dorsal root ganglia progenitors were isolated and purified using the differential adhesion method combined with cytosine arabinoside treatment. After culture in serum-free medium supplemented with B27, basic fibroblast growth factor and epidermal growth factor, these cells remained viable and survived for more than 18 months in vitro. Most cells differentiated to neurons that were immunoreactive for gamma-aminobutyric acid and choline acetyltransferase as detected by immunohistochemical staining. In addition, nerve growth factor and neurotrophic tyrosine kinase receptor expression were also observed in dorsal root ganglion progenitors and differentiated cells. K252a, an inhibitor that blocks nerve growth factor-induced signaling, inhibited cell survival, suggesting the possible existence of a nerve growth factor autocrine loop in these proliferating cells.

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.

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.

Implanting iodine-125 seeds into rat dorsal root ganglion for neuropathic pain: neuronal microdamage without impacting hind limb motion

The use of iodine-125 （L251） in cancer treatment has been shown to relieve patients＇ pain. Consid- ering dorsal root ganglia are critical for neural transmission between the peripheral and central nervous systems, we assumed that 125I could be implanted into rat dorsal root ganglia to provide relief for neuropathic pain. 125I seeds with different radioactivity （0, 14.8, 29.6 MBq） were im- planted separately through L4-5 and L5-6 intervertebral foramen into the vicinity of the L5 dorsal root ganglion, von Frey hair results demonstrated the mechanical pain threshold was elevated after implanting 125I seeds from the high radioactivity group. Transmission electron microscopy revealed that nuclear membrane shrinkage, nucleolar margination, widespread mitochondrial swelling, partial vacuolization, lysosome increase, and partial endoplasmic reticulum dilation were visible at 1,440 hours in the low radioactivity group and at 336 hours in the high radio- activity group. Abundant nuclear membrane shrinkage, partial fuzzy nuclear membrane and endoplasmic reticulum necrosis were observed at 1,440 hours in the high radioactivity group. No significant difference in combined behavioral scores was detected between preoperation and postoperation in the low and high radioactivity groups. These results suggested that the mechan- ical pain threshold was elevated after implanting 125I seeds without influencing motor functions of the hind limb, although cell injury was present.

Electroacupuncture upregulated platelet derived growth factor expression in spared dorsal root ganglion of cats

A bilateral spared dorsal root ganglion model was established in healthy adult cats by bilateral resection of L1-5 and L7-S2 dorsal root ganglia. L6 dorsal root ganglia were spared. Zusanfi （ST36） and Xuanzhong （BL39） or Futu （ST32） and Sanyinjiao （SP6） were alternatively electro-stimulated on the right leg. Immunohistochemical staining of anti-serum platelet-derived growth factor demonstrated that the number of total neurons and medium-small sized platelet-derived growth factor positive neurons was significantly decreased on the 7~ day following injury. After 7 days of acupuncture, the total number of positive and large neurons staining for platelet-derived growth factor on the acupuncture side significantly increased compared to the non-acupuncture side. After acupuncture for 14 days, the total positive and medium-small sized neurons significantly increased compared with the non-acupuncture side. Results indicate that acupuncture promoted the synthesis of platelet-dedved growth factor in spared dorsal root ganglia.

Effects of cyclooxygenase 2 inhibitor on growth-associated protein 43 and nerve growth factor expression in dorsal root ganglion during neuropathic pain development

BACKGROUND： Inflammatory responses in injured nerves have been recognized as important factors for initially sensitizing nociceptive neurons. Cyclooxygenase （COX） is the rate-limiting enzyme in prostaglandin synthesis, and COX-2 inhibitor is involved in mechanisms of analgesia and anti-inflammation. OBJECTIVE： To investigate the effects of COX-2 inhibitor on thermal and mechanical hyperalgesia, as well as expression of growth associated protein 43 （GAP-43） and nerve growth factor （NGF） in dorsal root ganglion, in a rat model of neuropathic pain due to chronic constriction injury. DESIGN, TIME AND SETTING： A randomized, controlled, comparison study that was performed at the Surgical Department and Pathological Laboratory, Second Affiliated Hospital of Shantou University Medical College from September 2006 to September 2007. MATERIALS： COX-2 inhibitor, Iornoxicam, was purchased from Nycomed Pharmaceutical （Austria）; rabbit anti-GAP-43, and rabbit anti-NGF polyclonal antibodies were purchased from Boster, Wuhan, China. METHODS： A total of 50 adult, Wistar rats were randomly assigned to four groups： normal control （n = 5）, model （n = 15）, normal saline control （n = 15）, and Iornoxicam treatment （n =15）. With exception of the control group, the sciatic nerve of all rats was loosely ligated to establish a model of chronic constriction injury. The model rats were divided into three subgroups according to varying post-operative survival periods： 3, 7 and 14 days （n = 5）, respectively. Rats in the Iornoxicam treatment group were intraperitoneally injected with 1.3 mg/kg lornoxicam every 12 hours throughout the entire experimental procedure. Rats in the normal saline control group were intraperitoneally injected with 1.3 mL/kg saline. MAIN OUTCOME MEASURES： Immunohistochemistry revealed expression of GAP-43 and NGF in the L5 dorsal root ganglions. Mechanical withdrawal threshold and thermal withdrawal latency were used to observe neurological behavioral changes in rats. RESULTS： The relative gray values of GAP-43- and NGF-positive neurons in the model group were remarkably increased compared with the normal control rats （P 〈 0.01）, while the relative gray values in the Iomoxicam treatment group were significantly less than the model and normal saline control groups （P 〈 0.01）. Mechanical withdrawal threshold and thermal withdrawal latency gradually decreased with increasing injury time in the model, normal saline control, and Iornoxicam treatment groups, and were significantly less than the normal control group （P 〈 0.05）. In addition, mechanical withdrawal threshold and thermal withdrawal latency were significantly greater in the Iornoxicam treatment group compared with the model and normal saline control groups （P 〈 0.05）. CONCLUSION： Intraperitoneal injection of the COX-2 inhibitor Iornoxicam attenuated mechanical and thermal hyperalgesia induced by sciatic nerve chronic constriction injury and inhibited the increased expression of GAP-43 and NGF.

Increased gene and protein expressions of the transient receptor potential vanilloid receptor 4 following sustained pure mechanical pressure on rat dorsal root ganglion neurons

Dorsal root ganglion （DRG） neurons from newborn Wistar rats cultured in vitro were pressurized with 20, 40, 80 or 120 mm Hg compressive Ioadings （1 mm Hg = 0.133 kPa） for 12, 24, 48 or 72 hours, respectively. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide test showed that pressures less than 80 mm Hg had no obvious impact on the activity of DRG neurons. The protein expression levels of transient receptor potential vanilloid receptor 4 （TRPV4）, transient receptor potential vanilloid receptor 1, transient receptor potential channel of melastatin type 8, and transient receptor potential subtype ankyrin 1 were assessed by western blot analysis. The mRNA expression of TRPV4 was assessed by real-time PCR. The results showed that sustained mechanical compression up-regulated TRPV4 mRNA and protein expression in the rat DRG neurons, in a time-dependent fashion. Similar changes were not found in the protein expression of transient receptor potential vanilloid receptor 1, transient receptor potential channel of melastatin type 8, and transient receptor potential subtype ankyrin 1. Images of cells using a laser scanning confocal microscope showed that the sustained mechanical pressure increased the number of responsive DRG neurons and was synergistic on the enhanced Ca^2＋ responses to the TRPV4 phorbol ester agonist 4a-phorbo112, 13-didecanoate and hypotonic solutions. These findings demonstrate that sustained mechanical compressive loading in vitro increases the expression of TRPV4 mRNA and protein in DRG neurons and sensitizes TRPV4 Ca^2＋ signals. Mechanical compression does not impact other ion channels in the transient receptor potential family.