Pain modulation effect on motor cortex after optogenetic stimulation in shPKCγ knockdown dorsal root ganglion-compressed Sprague-Dawley rat model.

Neuropathic pain can be generated by chronic compression of dorsal root ganglion (CCD). Stimulation of primary motor cortex can disrupt the nociceptive sensory signal at dorsal root ganglion level and reduce pain behaviors. But the mechanism behind it is still implicit. Protein kinase C gamma is known as an essential enzyme for the development of neuropathic pain, and specific inhibitor of protein kinase C gamma can disrupt the sensory signal and reduce pain behaviors. Optogenetic stimulation has been emerged as a new and promising conducive method for refractory neuropathic pain. The aim of this study was to provide evidence whether optical stimulation of primary motor cortex can modulate chronic neuropathic pain in CCD rat model. Animals were randomly divided into CCD group, sham group, and control group. Dorsal root ganglion-compressed neuropathic pain model was established in animals, and knocking down of protein kinase C gamma was also accomplished. Pain behavioral scores were significantly improved in the short hairpin Protein Kinase C gamma knockdown CCD animals during optic stimulation. Ventral posterolateral thalamic firing inhibition was also observed during light stimulation on motor cortex in CCD animal. We assessed alteration of pain behaviors in pre-light off, stimulation-light on, and post-light off state. In vivo extracellular recording of the ventral posterolateral thalamus, viral expression in the primary motor cortex, and protein kinase C gamma expression in dorsal root ganglion were investigated. So, optical cortico-thalamic inhibition by motor cortex stimulation can improve neuropathic pain behaviors in CCD animal, and knocking down of protein kinase C gamma plays a conducive role in the process. This study provides feasibility for in vivo optogenetic stimulation on primary motor cortex of dorsal root ganglion-initiated neuropathic pain.

Naked mole-rats lack cold sensitivity before and after nerve injury.

Neuropathic pain is a chronic disease state resulting from injury to the nervous system. This type of pain often responds poorly to standard treatments and occasionally may get worse instead of better over time. Patients who experience neuropathic pain report sensitivity to cold and mechanical stimuli. Since the nociceptive system of African naked mole-rats contains unique adaptations that result in insensitivity to some pain types, we investigated whether naked mole-rats may be resilient to sensitivity following nerve injury. Using the spared nerve injury model of neuropathic pain, we showed that sensitivity to mechanical stimuli developed similarly in mice and naked mole-rats. However, naked mole-rats lacked sensitivity to mild cold stimulation after nerve injury, while mice developed robust cold sensitivity. We pursued this response deficit by testing behavior to activators of transient receptor potential (TRP) receptors involved in detecting cold in naïve animals. Following mustard oil, a TRPA1 activator, naked mole-rats responded similarly to mice. Conversely, icilin, a TRPM8 agonist, did not evoke pain behavior in naked mole-rats when compared with mice. Finally, we used RNAscope to probe for TRPA1 and TRPM8 messenger RNA expression in dorsal root ganglia of both species. We found increased TRPA1 messenger RNA, but decreased TRPM8 punctae in naked mole-rats when compared with mice. Our findings likely reflect species differences due to evolutionary environmental responses that are not easily explained by differences in receptor expression between the species.

Regenerative activity of Hericium erinaceus on axonal injury model using in vitro laser microdissection technique.

OBJECTIVE: Peripheral nerve injury (PNI) is an important global health problem. Nerve Growth Factor (NGF) plays crucial role in the survival, growth, and maintenance of various neurons in the mammalian nervous system, human included. Hericium erinaceus (HE), an edible and medicinal mushroom, has been extensively studied for its neuroprotective properties. In this study, the neuroprotective and neurotogenic effects of HE and NGF were compared on mouse PNI model by using a laser microdissection technique. METHODS: Neuronal cultures were prepared from dorsal root ganglia (DRG) of 6-8 week aged mice, pretreated them with phosphate-buffered saline (PBS), NGF, HE, or the combination of NGF and HE. To model axonal injury in vitro, axons were cut (axotomy) with a microscope-controlled laser beam. Axotomized neurons were imaged under the microscope. Axotomized neurons' survival ratios were calculated using the propidium iodide (PI), which is a red-fluorescent nuclear dye. Their axon lengths were measured using the AxioVision 4.8 software. RESULTS: Although both HE and NGF have neuroprotective and regenerative effects on axotomized peripheral sensory neurons, HE exhibits a higher neuroprotective activity compared to the NGF. The combination of HE and NGF maximizes axonal regeneration ability of axotomized neurons. CONCLUSION: HE has capabilities of preventing the death of neurons and regenerating their axons in the experimental axonal injury model. Our findings provide experimental evidence that HE may serve as a neuroprotective and regenerative candidate for treating peripheral nerve injuries. Present study warrants further investigation of HE as a potential natural compound to remedy PNI.

Tabebuia aurea decreases hyperalgesia and neuronal injury induced by snake venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Tabebuia aurea (Silva Manso) Benth. & Hook. f. ex S. Moore is used as anti-inflammatory, analgesic and antiophidic in traditional medicine, though its pharmacological proprieties are still underexplored. In the bothropic envenoming, pain is a key symptom drove by an intense local inflammatory and neurotoxic event. The antivenom serum therapy is still the main treatment despite its poor local effects against pain and tissue injury. Furthermore, it is limited to ambulatorial niches, giving space for the search of new and more inclusive pharmacological approaches. AIM OF THE STUDY: evaluation of Tabebuia aurea hydroethanolic extract (HEETa) in hyperalgesia and neuronal injury induced by Bothrops mattogrossensis venom (VBm). MATERIALS AND METHODS: Stem barks from Tabebuia aurea were extracted with ethanol and water (7:3, v/v) to yield the extract HEETa. Then, HEETa was analyzed by LC-DAD-MS and its constituents were identified. Snake venoms were extracted from adult specimens of Bothrops mattogrossensis, lyophilized and kept at -20 °C until use. Male Swiss mice, weighting 20-25 g, were used to hyperalgesia (electronic von Frey), motor impairment (Rotarod test) and tissue injury evaluation (histopatology and ATF-3 immunohistochemistry). Therefore, three experimental groups were formed: VBm (1 pg, 1 ng, 0.3 µg, 1 µg, 3 and 6 µg/paw), HEETa orally (180, 540, 720, 810 or 1080 mg/kg; 10 mL/kg, 30 min prior VBm inoculation) and VBm neutralized (VBm: HEETa, 1:100 parts, respectively). In all set of experiments a control (saline group) was used. First, we made a dose-time-response course curve of VBm's induced hyperalgesia. Next, VBm maximum hyperalgesic dose was employed to perform HEETa orally dose-time-response course curve and analyses of VBm neutralized. Paw tissues for histopathology and DRGs were collected from animals inoculated with VBm maximum dose and treated with HEETa antihyperalgesic effective dose or neutralized VBm. Paws were extract two or 72 h after VBm inoculation and DRGs, in the maximum expected time expression of ATF-3 (72 h). RESULTS: From HEETa extract, glycosylated iridoids were identified, such as catalpol, minecoside, verminoside and specioside. VBm induced a time and dose dependent hyperalgesia with its highest effect seen with 3 µg/paw, 2 h after venom inoculation. HEETa effective dose (720 mg/kg) decreased significantly VBm induced hyperalgesia (3 µg/paw) with no motor impairment and signs of acute toxicity. HEETa antihyperalgesic action starts 1.5 h after VBm inoculation and lasted up until 2 h after VBm. Hyperalgesia wasn't reduced by VBm: HEETa neutralization. Histopathology revealed a large hemorragic field 2 h after VBm inoculation and an intense inflammatory infiltrate of polymorphonuclear cells at 72 h. Both HEETa orally and VBm: HEETa groups had a reduced inflammation at 72 h after VBm. Also, the venom significantly induced ATF-3 expression (35.37 ±â€¯3.25%) compared with saline group (4.18 ±â€¯0.68%) which was reduced in HEETa orally (25.87 ±â€¯2.57%) and VBm: HEETa (19.84 ±â€¯2.15%) groups. CONCLUSION: HEETa reduced the hyperalgesia and neuronal injury induced by VBm. These effects could be related to iridoid glycosides detected in HEETa and their intrinsic reported mechanism.

Bogijetong decoction and its active herbal components protect the peripheral nerve from damage caused by taxol or nerve crush.

BACKGROUND: Bogijetong decoction (BGJTD) is a herbal drug formulation used in the traditional Asian medicine to treat neuropathic insults associated with diabetes and anticancer therapy. To understand the biological basis of BGJTD on protective effects against neuropathy, we investigated physiological and biochemical responses of the sciatic nerves deranged by taxol injection or crush injury in the rats. METHODS: Dissociated Schwann cells and neurons were prepared from the sciatic nerve and dorsal root ganglia (DRG) respectively and were treated with taxol and BGJTD. The sciatic nerve in the rat was injected with taxol or given crush injury. Animals were then administered orally with BGJTD. Effects of BGJTD treatment on cultured cells and in vivo sciatic nerves and DRG tissues were examined by immunofluorescence staining and western blot analysis. Sciatic nerve regeneration was assessed by histological observation using retrograde tracing technique and by behavioral hot plate test. Eighteen different herbal components of BGJTD were divided into 4 subgroups and were used to select herbal drugs that enhanced neurite outgrowth in cultured neurons. RESULTS: Morphological abnormalities in the sciatic nerve axons and DRG tissue caused by taxol injection were largely improved by BGJTD treatment. BGJTD treatment enhanced neurite outgrowth in cultured DRG neurons and improved Schwann cell survival. Phospho-Erk1/2 levels were elevated by BGJTD administration in the injured- or taxol-injected sciatic nerves. Vimentin phosphorylation catalyzed by cell division cycle 2 (Cdc2) kinase was induced from Schwann cells in the sciatic nerves after taxol injection and crush injury, and phospho-vimentin levels were further upregulated by BGJTD treatment. Retrograde tracing of DiI-labeled DRG sensory neurons revealed growth-promoting activity of BGJTD on axonal regeneration. A drug group (Be) composed of 4 active herbal components which were selected by neurite growth-enhancing activity was as effective as BGJDT for the recovery of thermal sensitivity of the hind paws which had been suppressed by taxol administration. CONCLUSIONS: These data suggest that BGJTD and its active herbal components may protects the peripheral nerve from damage caused by taxol injection and nerve crush.

[Effect of 1-tetrahydropalmatine on spontaneous electric discharge induced by chronic dorsal root ganglion neurons compression in rats].

OBJECTIVE: To study the effect of 1-tetrahydropalmatine (1-THP) on the spontaneous electric discharge (SED) induced by chronic dorsal root ganglion neurons compression. METHODS: Using single fiber recording method, the SED of 84 neurons class A induced by compression were recorded. The effect of 1-THP on the SEDs and its relation with concentration were observed. RESULTS: In the 84 SED of neurons, 25 showed periodical rhythmicity (PR) and 59 showed non-periodic rhythmicity (non-PR). 1-THP (100 micromol/L) inhibited SED in 16.0% (4/25) of neurons with PR and 67.8% (40/59) of neurons with non-PR (P < 0.01) in an effect-dose dependent manner, the higher the concentration of 1-THP, the more the inhibition, with quicker inhibiting in initiation and longer time needed for recovery. SED in 57.1% neurons were recovered 20 min after elution, but unrecovered even after 3 h in the others. CONCLUSION: 1-THP shows inhibitory effect on the A-fiber SED induced by chronic dorsal root ganglion neurons compression.

Expression and regulation of Sef, a novel signaling inhibitor of receptor tyrosine kinases-mediated signaling in the nervous system.

Fibroblast growth factors (FGFs) signal via four distinct high affinity cell surface tyrosine kinase receptors, termed FGFR1-FGFR4 (FGFR-FGF-receptor). Recently, a new modulator of the FGF signaling pathway, the transmembrane protein 'similar expression to FGF genes' (Sef), has been identified in zebrafish and subsequently in mammals. Sef from mouse and human inhibits FGF mitogenic activity. In the present study, we analyzed the expression of Sef in distinct rat brain areas, in the spinal cord and in peripheral nerves and spinal ganglia using semi-quantitative RT-PCR. Furthermore, we studied the cellular expression pattern of Sef in intact spinal ganglia and sciatic nerves and, in addition, after crush lesion, using in situ hybridization and immunohistochemistry. Sef transcripts were expressed in all brain areas evaluated and in the spinal cord. A neuronal expression was found in both intact and injured spinal ganglia. Intact sciatic nerves, however, showed little or no Sef expression. Seven days after injury, high Sef expression was concentrated to the crush site, and Schwann cells seemed to be the source of Sef. The labeling pattern of up-regulated Sef was complementary to the patterns of FGF-2 and FGFR1-3, which were localized proximal and distal to the crush site. These results suggest an involvement of Sef during the nerve regeneration process, possibly by fine-tuning the effects of FGF signaling.

Electro-acupuncture induced NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion in cats subjected to removal of adjacent ganglia.

This study evaluated the effect of electro-acupuncture (EA) on the NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion (DRG) in cats subjected to bilateral removal of L1-L5 and L7-S2 DRG, using immunostaining, in situ hybridization and RT-PCR. The positive products of NGF, NT-3 protein and mRNA in the small and large neurons of spared L6 DRG in EA side increased greatly more than that of control side, while the increased BDNF was only noted in small and medium-sized neurons. RT-PCR demonstrated that the mRNA level for three factors was not influenced by EA in intact DRG, when a significant increase was seen in the spared L6 DRG of EA side. As it has been well known that DRG neurons project to the spinal cord wherein morphological plasticity has been present after DRG removal, the present results might have some bearing to the observed phenomenon.

Effects of electro-acupuncture on the expression of c-jun and c-fos in spared dorsal root ganglion and associated spinal laminae following removal of adjacent dorsal root ganglia in cats.

This study evaluated the plastic changes of c-jun and c-fos in the right sixth lumbar dorsal root ganglion (L6 DRG), Rexed's lamina II in representative spinal segments L3, L5, and L6 and in the nucleus dorsalis (ND) at L3 segments after electro-acupuncture (EA) in cats subjected to removal of L1-L5 and L7-S2 DRG. Following dorsal root ganglionectomy, there was a significant increase in the density of c-jun immunoreactivity in the neurons and glia in spinal lamina II and in the ND; there was also marked elevation in the expression of c-fos in ND. In both cases there was no change in the c-jun and c-fos immunoreactivity in the DRG. After EA in the operated animals, there was an up-regulation in the expression of c-jun in the L6 DRG and the associated spinal lamina II; however, increased c-fos expression was detected only in the L6 DRG. Western blot and RT-PCR were also performed to quantitatively explore the mRNA and protein expression changes in the spinal dorsal horn and associated DRG. Following partial deafferentation, there was a significant increase in the protein level of both c-jun and c-fos in the dorsal horn, while, in both cases there was no change in c-jun and c-fos protein and mRNA in the DRG. After EA in the operated animals, both c-jun protein and its mRNA in the L6 DRG as well as the associated dorsal horn of L6 spinal segment were upregulated, but increased c-fos protein and its mRNA was observed only in the L6 DRG. These findings suggested that c-jun and c-fos might be related to the acupuncture promoted spinal cord plasticity as reported previously.

Cell culture modeling to test therapies against hyperglycemia-mediated oxidative stress and injury.

The concept that oxidative stress is a key mediator of nerve injury in diabetes has led us to design therapies that target oxidative stress mechanisms. Using an in vitro model of glucose-treated dorsal root ganglion (DRG) neurons in culture, we can examine both free radical generation, using fluorimetric probes for reactive oxygen species, and cell death via the TUNEL assay. The cell culture system is scaled down to a 96-well plate format, and so is well suited to high-throughput screening. In the present study, we test the ability of three drugs, nicotinamide, allopurinol, and alpha-lipoic acid, alone and in combination to prevent DRG neuron oxidative stress and cell death. This combination of drugs is currently in clinical trial in type 1 diabetic patients. We demonstrate independent effects on oxidative stress and neuronal survival for the three drugs, and neuronal protection using the three drugs in combination. The data strengthen the rationale for the current clinical trial. In addition, we describe an effective tool for rapid preclinical testing of novel therapies against diabetic neuropathy.

Plasticity of gene expression in injured human dorsal root ganglia revealed by GeneChip oligonucleotide microarrays.

Root avulsion from the spinal cord occurs in brachial plexus lesions. It is the practice to repair such injuries by transferring an intact neighbouring nerve to the distal stump of the damaged nerve; avulsed dorsal root ganglia (DRG) are removed to enable nerve transfer. Such avulsed adult human cervical DRG ( [Formula: see text] ) obtained at surgery were compared to controls, for the first time, using GeneChip oligonucleotide arrays. We report 91 genes whose expression levels are clearly altered by the injury. This first study provides a global assessment of the molecular events or "gene switches" as a consequence of DRG injuries, as the tissues represent a wide range of surgical delay, from 1 to 100 days. A number of these genes are novel with respect to sensory ganglia, while others are known to be involved in neurotransmission, trophism, cytokine functions, signal transduction, myelination, transcription regulation, and apoptosis. Cluster analysis showed that genes involved in the same functional groups are largely positioned close to each other. This study represents an important step in identifying new genes and molecular mechanisms in human DRG, with potential therapeutic relevance for nerve repair and relief of chronic neuropathic pain.

Transcriptional upregulation of SCG10 and CAP-23 is correlated with regeneration of the axons of peripheral and central neurons in vivo.

We have compared SCG10 and CAP-23 expression with that of GAP-43 during axonal regeneration in the peripheral and central nervous systems (PNS, CNS) of adult rats. SCG10, CAP-23, and GAP-43 mRNAs were strongly upregulated by motor and dorsal root ganglion (DRG) neurons following sciatic nerve crush, but not after dorsal rhizotomy. When the sciatic nerve was cut and ligated to prevent reinnervation of targets, expression of all three mRNAs was prolonged. Neurons in the thalamic reticular nucleus and deep cerebellar nuclei transiently upregulated these mRNAs after axotomy, and showed prolonged upregulation of all three molecules when regenerating axons into peripheral nerve grafts inserted into the thalamus of cerebellum. Neurons in the dorsal thalamus and cerebellar cortex showed poor regenerative capacity and most did not upregulate any of these mRNAs. Thus, in both PNS and CNS neurons, the transcription of SCG10, CAP-23, and GAP-43 mRNAs is coregulated following axotomy and during regeneration. Signals from living peripheral nerve appear to maintain expression of all three mRNAs in regenerating neurons, and in PNS neurons downregulation correlates with target reinnervation. Thus, SCG10 and CAP-23, as well as GAP-43, are likely to be important neuronal determinants of regenerative ability.