Neuron-Microglia Interaction is Involved in Anti-inflammatory Response by Vagus Nerve Stimulation in the Prefrontal Cortex of Rats Injected with Polyinosinic:Polycytidylic Acid.

Injection of polyinosinic:polycytidylic acid (poly(I:C)) into experimental animals induces neuroimmunological responses and thus has been used for the study of neurological disorders such as anxiety, depression, and chronic fatigue. Here, we investigated the effects of vagus nerve stimulation (VNS) on poly(I:C)-induced neuroinflammation and associated behavioral consequences in rats. The microglia in the prefrontal cortex (PFC) displayed the activated form of morphology in poly(I:C)-injected rats and changed to a normal shape after acute VNS (aVNS). Production of phospho-NF-κB, phospho-IκB, IL-1ß, and cleaved caspase 3 was elevated by poly(I:C) and downregulated by aVNS. In contrast, phospho-Akt levels were decreased by poly(I:C) and increased by aVNS. Neuronal production of fractalkine (CX3CL1) in the PFC was markedly reduced by poly(I:C), but recovered by aVNS. Fractalkine interaction with its receptor CX3CR1 was highly elevated by VNS. We further demonstrated that the pharmacological blockade of CX3CR1 activity counteracted the production of IL-1ß, phospho-Akt, and cleaved form of caspase 3 that was modulated by VNS, suggesting the anti-inflammatory effects of fractalkine-CX3CR1 signaling as a mediator of neuron-microglia interaction. Behavioral assessments of pain and temperature sensations by von Frey and hot/cold plate tests showed significant improvement by chronic VNS (cVNS) and forced swimming and marble burying tests revealed that the depressive-like behaviors caused by poly(I:C) injection were rescued by cVNS. We also found that the recognition memory which was impaired by poly(I:C) administration was improved by cVNS. This study suggests that VNS may play a role in regulating neuroinflammation and somatosensory and cognitive functions in poly(I:C)-injected animals.

Vagus nerve stimulation modulates hippocampal inflammation caused by continuous stress in rats.

BACKGROUND: Previous studies have shown that vagus nerve stimulation (VNS) can attenuate inflammatory responses in peripheral tissues and also improve some neurological disorders and cognitive function in the brain. However, it is not clear how VNS is involved in neuropathological processes in brain tissues. Here, we investigated the regulatory effects of VNS on the production of proinflammatory cytokines in the hippocampus of an animal model of continuous stress (CS). METHODS: CS was induced by placing rats in cages immersed with water, and acute or chronic electrical stimulation was applied to the cervical vagus nerve of CS animals. Protein levels in the gastric and hippocampal tissues were measured by western blotting and protein signals analyzed by immunofluorescence staining. von Frey test and forced swimming test were performed to assess pain sensitivity and depressive-like behavior in rats, respectively. RESULTS: Levels of TNF-α, IL-1ß, and IL-6 in the gastric and hippocampal tissues were significantly increased in CS animals compared to the untreated control and downregulated by acute VNS (aVNS). Iba-1-labeled microglial cells in the hippocampus of CS animals revealed morphological features of activated inflammatory cells and then changed to a normal shape by VNS. VNS elevated hippocampal expression of α7 nicotinic acetylcholine receptors (α7 nAChR) in CS animals, and pharmacological blockade of α7 nAChR increased the production of TNF-α, IL-1ß, and IL-6, thus suppressing cholinergic anti-inflammatory activity that was mediated by VNS. Chronic VNS (cVNS) down-regulated the hippocampal production of active form of caspase 3 and 5-HT1A receptors and also decreased levels of TNF-α, IL-1ß, and IL-6 in the gastric and hippocampal tissues of CS animals. Pain sensitivity and depressive-like behavior, which were increased by CS, were improved by cVNS. CONCLUSIONS: Our data suggest that VNS may be involved in modulating pathophysiological processes caused by CS in the brain.

Phosphorylation of STAT3 by axonal Cdk5 promotes axonal regeneration by modulating mitochondrial activity.

Cyclin-dependent kinase 5 (Cdk5) is involved in neural organization and synaptic functions in developing and adult brains, yet its role in axonal regeneration is not known well. Here, we characterize Cdk5 function for axonal regeneration after peripheral nerve injury. Levels of Cdk5 and p25 were elevated in sciatic nerve axons after injury. Cdk5 activity was concomitantly induced from injured nerve and increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) on the serine 727 residue. Pharmacological and genetic blockades of Cdk5 activity phosphorylating STAT3 resulted in the inhibition of axonal regeneration as evidenced by reduction of retrograde labeling of dorsal root ganglion (DRG) sensory neurons and spinal motor neurons and also of neurite outgrowth of preconditioned DRG neurons in culture. Cdk5 and STAT3 were found in mitochondrial membranes of the injured sciatic nerve. Cdk5-GFP, which was translocated into the mitochondria by the mitochondrial target sequence (MTS), induced STAT3 phosphorylation in transfected DRG neurons and was sufficient to induce neurite outgrowth. In the mitochondria, Cdk5 activity was positively correlated with increased mitochondrial membrane potential as measured by fluorescence intensity of JC-1 aggregates. Our data suggest that Cdk5 may play a role in modulating mitochondrial activity through STAT3 phosphorylation, thereby promoting axonal regeneration.

Vagal afferent fibers contribute to the anti-inflammatory reactions by vagus nerve stimulation in concanavalin A model of hepatitis in rats.

BACKGROUND: Increasing number of studies provide evidence that the vagus nerve stimulation (VNS) dampens inflammation in peripheral visceral organs. However, the effects of afferent fibers of the vagus nerve (AFVN) on anti-inflammation have not been clearly defined. Here, we investigate whether AFVN are involved in VNS-mediated regulation of hepatic production of proinflammatory cytokines. METHODS: An animal model of hepatitis was generated by intraperitoneal (i.p.) injection of concanavalin A (ConA) into rats, and electrical stimulation was given to the hepatic branch of the vagus nerve. AFVN activity was regulated by administration of capsaicin (CAP) or AP-5/CNQX and the vagotomy at the hepatic branch of the vagus nerve (hVNX). mRNA and protein expression in target tissues was analyzed by RT-PCR, real-time PCR, western blotting and immunofluorescence staining. Hepatic immune cells were analyzed by flow cytometry. RESULTS: TNF-α, IL-1ß, and IL-6 mRNAs and proteins that were induced by ConA in the liver macrophages were significantly reduced by the electrical stimulation of the hepatic branch of the vagus nerve (hVNS). Alanine transaminase (ALT) and aspartate transaminase (AST) levels in serum and the number of hepatic CD4+ and CD8+ T cells were increased by ConA injection and downregulated by hVNS. CAP treatment deteriorated transient receptor potential vanilloid 1 (TRPV1)-positive neurons and increased caspase-3 signals in nodose ganglion (NG) neurons. Concomitantly, CAP suppressed choline acetyltransferase (ChAT) expression that was induced by hVNS in DMV neurons of ConA-injected animals. Furthermore, hVNS-mediated downregulation of TNF-α, IL-1ß, and IL-6 expression was hampered by CAP treatment and similarly regulated by hVNX and AP-5/CNQX inhibition of vagal feedback loop pathway in the brainstem. hVNS elevated the levels of α7 nicotinic acetylcholine receptors (α7 nAChR) and phospho-STAT3 (Tyr705; pY-STAT3) in the liver, and inhibition of AFVN activity by CAP, AP-5/CNQX and hVNX or the pharmacological blockade of hepatic α7 nAChR decreased STAT3 phosphorylation. CONCLUSIONS: Our data indicate that the activity of AFVN contributes to hepatic anti-inflammatory responses mediated by hVNS in ConA model of hepatitis in rats.

Cdk5 Phosphorylation of STAT3 in Dorsal Root Ganglion Neurons Is Involved in Promoting Axonal Regeneration After Peripheral Nerve Injury.

PURPOSE: The goal of this study is to investigate the role of cyclin-dependent kinase 5 (Cdk5) in axonal regeneration in dorsal root ganglion (DRG) neurons after peripheral nerve injury. METHODS: Crush injury was given on the sciatic nerve in rats. The DRG tissues were prepared 1, 3, and 7 days after injury and used for western blotting and immunofluorescence staining experiments. Primary DRG neurons were prepared and treated with Cdk5 inhibitor roscovitine or used for transfections with plasmid constructs. After immunofluorescence staining, neurite length of DRG neurons was analyzed and compared among experimental groups. In addition, roscovitine was injected into the DRG in vivo, and the sciatic nerve after injury was prepared and used for immunofluorescence staining to analyze axonal regeneration in nerve sections. RESULTS: Levels of Cdk5 and p25 were increased in DRG neurons after sciatic nerve injury (SNI). Levels of S727-p-STAT3, but not Y705-p-STAT3, were increased in the DRG. Immunofluorescence staining revealed that Cdk5 and STAT3 proteins were mostly colocalized in DRG neurons and Y705-p-STAT3 signals were localized within the nucleus area of DRG neurons. A blockade of Cdk5 activity by roscovitine or by transfection with dominant negative Cdk5 (dn-Cdk5) and nonphosphorylatable forms of STAT3 (S727A or Y705F) resulted in significant reductions of the neurite outgrowth of cultured DRG neurons. In vivo administration of roscovitine into the DRG markedly attenuated distal elongation of regenerating axons in the sciatic nerve after injury. CONCLUSION: Our study demonstrated that Cdk5 activity induced from DRG neurons after SNI increased phosphorylation of STAT3. The activation of Cdk5-STAT3 pathway may be involved in promoting axonal regeneration in the peripheral nerve after injury.

Augmented Buyang Huanwu Decoction facilitates axonal regeneration after peripheral nerve transection through the regulation of inflammatory cytokine production.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal formulation Buyang Huanwu Decoction (BYHWD) has been used to treat cardiovascular disorders including cerebral ischemia. Recent studies showed its effects on promoting axonal regeneration after nerve injury. However, compositional reformulation supplemented with herbal components that regulates inflammation may increase its efficacy for nerve repair. AIM OF THE STUDY: We prepared a new herbal decoction by adding selected herbal components to BYHWD (augmented BYHWD; ABHD) and investigated the effect of ABHD on the production of inflammatory cytokines and axonal regeneration using an animal model of nerve transection and coaptation (NTC). MATERIALS AND METHODS: A rat model of NTC was performed on the sciatic nerve. The sciatic nerve and dorsal root ganglion (DRG) were isolated and used for immunofluorescence staining and western blot analysis. DRG tissue was also used to prepare primary neuron culture and the length of neurites was analyzed. Sensorimotor nerve activities were assessed by rotarod and von Frey tests. RESULTS: Three herbal components that facilitated neurite outgrowth were chosen to formulate ABHD. ABHD administration into the sciatic nerve 1 week or 3 months after NTC facilitated axonal regeneration. Cell division cycle 2 (Cdc2) and brain-derived neurotrophic factor (BDNF) proteins were induced from the reconnected distal portion of the sciatic nerve and the levels were further elevated by in vivo administration of ABHD. Phospho-Erk1/2 level was increased by ABHD treatment as well, implying its role in mediating retrograde transport of BDNF signals into the neuronal cell body. Production of inflammatory cytokines IL-1ß and TNF-α was induced in the reconnected nerve but attenuated by ABHD treatment. Behavioral tests revealed that ABHD treatment improved functional recovery of sensorimotor activities. CONCLUSIONS: A newly formulated ABHD is effective at regulating the production of inflammatory cytokines and promoting axonal regeneration after nerve transection and may be considered to develop therapeutic strategies for peripheral nerve injury disorders.

Anti-Inflammatory Effects of Modified Buyang Huanwu Decoction.

METHODS: A cytotoxicity assay for BHD was performed using the MTT assay. Following treatment with BHD, mBHD-1, and mBHD-2 in the presence of lipopolysaccharide (LPS), nitric oxide (NO) secretion was detected in cell supernatants using a NO detection kit. The expression of proinflammatory mediators was detected using RT-PCR and western blotting. To verify the mechanism of mBHD, specific inhibitors of JNK (SP600125) or p38 (SB203580) were used for co-treatment with mBHD, and then the changes in NO and nitric oxide synthase (iNOS) were measured. RESULTS: Both mBHD-1 and mBHD-2 showed greater anti-inflammatory effects than BHD. Both mBHD-1 and mBHD-2 inhibited NO secretion and decreased the expression of IL-1ß, IL-6, TNF-α, and iNOS. Treatment with a p38 inhibitor and a JNK inhibitor in mBHD-1- and mBHD-2-treated cells resulted in inhibition of NO and iNOS. CONCLUSION: We provided the first experimental evidence that mBHD may be a more useful anti-inflammatory than BHD. High concentrations or long-term use of BHD may be harmful to inflammatory status. Therefore, the length of treatment and concentration should be considered depending on the targeted disease.

Acupuncture stimulation attenuates TNF-α production via vagal modulation in the concanavalin A model of hepatitis.

BACKGROUND: A growing body of evidence shows that neuronal activity is involved in modulating the efficacy of acupuncture therapy. However, it has been seldom investigated whether neuronal activity following acupuncture stimulation is effective at regulating hepatic inflammation. OBJECTIVE: Using the concanavalin A (ConA) model of hepatitis, we investigated the regulation of inflammatory cytokine tumor necrosis factor (TNF)-α in the liver tissue and the blood after acupuncture stimulation at ST36. METHODS: Mice were subjected to ConA injection, acupuncture stimulation at ST36 by manual acupuncture (MA) or electroacupuncture (EA) procedures, and vagotomy (VNX). Liver tissue and blood were collected for TNF-α analysis. TNF-α mRNA was analyzed by real-time polymerase chain reaction (PCR), and TNF-α, CD11b, CD68, and Erk1/2 proteins were analyzed by Western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay. RESULTS: TNF-α mRNA and protein were induced in CD11b-positive hepatic cells and the plasma at 6-24 h after ConA injection. The application of MA or EA was very effective at attenuating the production of TNF-α. Anti-inflammatory effects of acupuncture were greatly suppressed by VNX in ConA-injected animals, suggesting the requirement of vagus nerve activity in acupuncture-mediated anti-inflammatory responses. Electrical stimulation of the sciatic nerve (SNS) resulted in an anti-inflammatory effect similar to acupuncture stimulation. In parallel with TNF-α, production of phospho-Erk1/2, which was induced in the liver tissue, was downregulated by MA and EA in liver cells. CONCLUSION: The regulatory effects of acupuncture stimulation on inflammatory responses in the liver may be modulated through the activation of the vagus nerve pathway.

Effects of Acupuncture on Chronic Stress-Induced Depression-Like Behavior and Its Central Neural Mechanism.

Depression is a serious psychiatric disorder with an enormous socioeconomic burden, and it is commonly comorbid with pain, chronic fatigue, or other inflammatory diseases. Recent studies have shown that acupuncture is an effective therapeutic method for reducing depressive symptoms; however, the underlying mechanism remains unknown. In this study, we investigated the effects of acupuncture on chronic stress-induced depression-like behavior and its central neural mechanisms in the brain. We induced chronic restraint stress (CRS) in male C57BL/6 mice for 14 or 28 consecutive days. Acupuncture treatment was performed at KI10·LR8·LU8·LR4 or control points for 7 or 14 days. Depression-like behavior was assessed with the open field test. Then, brain neural activity involving c-Fos and serotonin-related mechanisms via the 5-HT1A and 5-HT1B receptors were investigated. Acupuncture treatment at KI10·LR8·LU8·LR4 points rescued the depressive-like behavior, while control points (LU8·LR4·HT8·LR2) and non-acupoints on the hips did not. Brain neural activity was changed in the hippocampus, cingulate cortex, motor cortex, insular cortex, thalamus, and the hypothalamus after acupuncture treatment. Acupuncture treatment increased expression of 5-HT1A receptor in the cortex, hippocampus, thalamus, and the hypothalamus, and of 5-HT1B in the cortex and thalamus. In conclusion, acupuncture treatment at KI10·LR8·LU8·LR4 was effective in alleviating the depressive-like behavior in mice, and this therapeutic effect was produced through central brain neural activity and serotonin receptor modulation.

Increased ER-mitochondria tethering promotes axon regeneration.

Translocation of the endoplasmic reticulum (ER) and mitochondria to the site of axon injury has been shown to facilitate axonal regeneration; however, the existence and physiological importance of ER-mitochondria tethering in the injured axons are unknown. Here, we show that a protein linking ER to mitochondria, the glucose regulated protein 75 (Grp75), is locally translated at axon injury site following axotomy, and that overexpression of Grp75 in primary neurons increases ER-mitochondria tethering to promote regrowth of injured axons. We find that increased ER-mitochondria tethering elevates mitochondrial Ca2+ and enhances ATP generation, thereby promoting regrowth of injured axons. Furthermore, intrathecal delivery of lentiviral vector encoding Grp75 to an animal with sciatic nerve crush injury enhances axonal regeneration and functional recovery. Together, our findings suggest that increased ER-mitochondria tethering at axonal injury sites may provide a therapeutic strategy for axon regeneration.

Hippocampal activation of 5-HT1B receptors and BDNF production by vagus nerve stimulation in rats under chronic restraint stress.

A growing body of evidence shows that the electrical stimulation of the vagus nerve can improve mental illness including depression. Here, we investigated whether the vagus nerve stimulation (VNS) is involved in regulating the responsiveness of hippocampal neurons in rats under chronic restraint stress (CRS). c-Fos protein signals were detected 2 hr after VNS in 5-HT1A receptor-positive neurons in the dorsal raphe nucleus (DRN) as well as in the nucleus tractus solitarius (NTS). Chronic VNS was performed on a daily basis for 2 weeks using an implanted microelectrode in rats that had undergone CRS for 2 weeks. We found that the levels of both 5-HT1B receptors and phospho-Erk1/2 were decreased in parallel in the hippocampal neurons of CRS animals and then increased to the baseline levels by chronic VNS. Hippocampal induction of 5-HT1B receptors and phospho-Erk1/2 by VNS was diminished after the injection of 5,7-dihydroxytryptamine (5,7-DHT), a neurotoxin of serotonergic neurons, into the DRN. Hippocampal production of brain-derived neurotrophic factor (BDNF) was also upregulated by VNS, but the treatment of 5,7-DHT abrogated the effects of VNS on BDNF induction. VNS in CRS animals improved the behavioral scores in forced swimming test (FST) compared to sham-stimulated control. Our results suggest that VNS-mediated serotonergic input via 5-HT1B receptors into the hippocampal neurons may activate BDNF pathway and improve depressive-like behaviors in CRS animals.

Adenine attenuates lipopolysaccharide-induced inflammatory reactions.

A nucleobase adenine is a fundamental component of nucleic acids and adenine nucleotides. Various biological roles of adenine have been discovered. It is not produced from degradation of adenine nucleotides in mammals but produced mainly during polyamine synthesis by dividing cells. Anti-inflammatory roles of adenine have been supported in IgE-mediated allergic reactions, immunological functions of lymphocytes and dextran sodium sulfate-induced colitis. However adenine effects on Toll-like receptor 4 (TLR4)-mediated inflammation by lipopolysaccharide (LPS), a cell wall component of Gram negative bacteria, is not examined. Here we investigated anti-inflammatory roles of adenine in LPS-stimulated immune cells, including a macrophage cell line RAW264.7 and bone marrow derived mast cells (BMMCs) and peritoneal cells in mice. In RAW264.7 cells stimulated with LPS, adenine inhibited production of pro-inflammatory cytokines TNF-α and IL-6 and inflammatory lipid mediators, prostaglandin E2 and leukotriene B4. Adenine impeded signaling pathways eliciting production of these inflammatory mediators. It suppressed IκB phosphorylation, nuclear translocation of nuclear factor κB (NF-κB), phosphorylation of Akt and mitogen activated protein kinases (MAPKs) JNK and ERK. Although adenine raised cellular AMP which could activate AMP-dependent protein kinase (AMPK), the enzyme activity was not enhanced. In BMMCs, adenine inhibited the LPS-induced production of TNF-α, IL-6 and IL-13 and also hindered phosphorylation of NF-κB and Akt. In peritoneal cavity, adenine suppressed the LPS-induced production of TNF-α and IL-6 by peritoneal cells in mice. These results show that adenine attenuates the LPS-induced inflammatory reactions.

Electroacupuncture therapy in inflammation regulation: current perspectives.

Although acupuncture therapy is increasingly used to treat diverse symptoms and disorders in humans, its underlying mechanism is not known well. Only recently have experimental studies begun to provide insights into how acupuncture stimulation generates and relates to pathophysiological responsiveness. Acupuncture intervention is frequently used to control pathologic symptoms in several visceral organs, and a growing number of studies using experimental animal models suggest that acupuncture stimulation may be involved in inducing anti-inflammatory responses. The vagus nerve, a principal parasympathetic nerve connecting neurons in the central nervous system to cardiovascular systems and a majority of visceral organs, is known to modulate neuroimmune communication and anti-inflammatory responses in target organs. Here, we review a broad range of experimental studies demonstrating anti-inflammatory effects of electroacupuncture in pathologic animal models of cardiovascular and visceral organs and also ischemic brains. Then, we provide recent progress on the role of autonomic nerve activity in anti-inflammation mediated by electroacupuncture. We also discuss a perspective on the role of sensory signals generated by acupuncture stimulation, which may induce a neural code unique to acupuncture in the central nervous system.

Enhanced peripheral nerve regeneration through asymmetrically porous nerve guide conduit with nerve growth factor gradient.

In this study, we fabricated a nerve guide conduit (NGC) with nerve growth factor (NGF) gradient along the longitudinal direction by rolling a porous polycaprolactone membrane with NGF concentration gradient. The NGF immobilized on the membrane was continuously released for up to 35 days, and the released amount of the NGF from the membrane gradually increased from the proximal to distal NGF ends, which may allow a neurotrophic factor gradient in the tubular NGC for a sufficient period. From the in vitro cell culture experiment, it was observed that the PC12 cells sense the NGF concentration gradient on the membrane for the cell proliferation and differentiation. From the in vivo animal experiment using a long gap (20 mm) sciatic nerve defect model of rats, the NGC with NGF concentration gradient allowed more rapid nerve regeneration through the NGC than the NGC itself and NGC immobilized with uniformly distributed NGF. The NGC with NGF concentration gradient seems to be a promising strategy for the peripheral nerve regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 52-64, 2018.

Bogijetong Decoction and Its Selected Formulation Are Involved in Alleviating Neuropathic Pain in a Rat Model of Chronic Constrictive Injury.

Bogijetong decoction (BGJTD) is a formulation that is used for the treatment of neuropathic pain caused by cancer therapy, diabetes, and peripheral nerve injury. In the previous study, we selected four herbal constituents from BGJTD, formulated new decoction (BeD), and demonstrated its efficacy on the neuroprotection of peripheral sciatic nerve in streptozotocin-induced diabetic animals. Here, we report attenuating effects of BGJTD and BeD on neuropathic pain. Neuropathic pain was induced by ligation of the sciatic nerve to generate chronic constrictive injury (CCI). BeD was more effective than BGJTD in alleviating neuropathic pain lasting 3 - 4 weeks after CCI. In vivo administration of BeD did not alter the levels of brain-derived neurotrophic factor (BDNF) which were strongly induced by CCI in the sciatic nerve but downregulated TrkB production in the sciatic nerve. Downregulation of TrkB signals by BeD was confirmed in cultured DRG neurons. BGJTD was more effective in attenuating TNF-α production in the sciatic nerve than BeD, whereas BeD increased IL-6 more efficiently than BGJTD. Furthermore, phopsho-Erk1/2 was increased in the sciatic nerve and dorsal root ganglia (DRG) after BeD treatment. Neurite outgrowth of primary DRG neurons prepared from rats which had undergone CCI for 7 days was significantly increased in BeD-treated group of animals compared to the control and BGJTD-treated groups. Compositional comparison of BeD revealed that the neurite outgrowth was facilitated by the treatments of Panax ginseng and Paeonia lactiflora. Together, these data suggest that BeD induces unique molecular response at the injury site and may trigger retrograde signaling into the neuronal cell body to modulate pain responses.

α6 and ß1 Integrin Heterodimer Mediates Schwann Cell Interactions with Axons and Facilitates Axonal Regeneration after Peripheral Nerve Injury.

Several isoforms of integrin subunits are expressed in Schwann cells and mediate Schwann cell interactions with axons. Here, we identify α6 and ß1 integrins as heterodimeric proteins expressed in Schwann cells and define their functions in axonal regeneration. α6 and ß1 integrins are induced in Schwann cells in the sciatic nerve after a crush injury, and the blocking of integrin activity by siRNA expression and by treatment with anti-integrin antibodies attenuates Schwann cell contact with cultured neurons and decreases neurite outgrowth. After nerve transection, the levels of α6 and ß1 integrins in the distal nerve stump are lower than those in the corresponding nerve area after a crush injury. Schwann cells prepared from the distal nerves 7 days after transection are less supportive of neurite outgrowth in co-cultured neurons than those prepared from the nerves 7 days after a crush injury. When the transected nerves are reconnected after a delay of 1 to 2 weeks, the induced levels of α6 and ß1 integrins in the reconnected distal nerves are significantly reduced compared to those in the nerves after a crush injury. These changes correlate with retarded axonal regeneration in animals that have experienced nerve transections and delayed coaptation, which implies an attenuated Schwann cell capacity to support axonal regeneration due to delayed Schwann cell contact with axons. The present data suggest that α6 and ß1 integrins induced in Schwann cells after nerve injury may play a role in mediating Schwann cell interactions with axons and promote axonal regeneration.

Facilitating effects of Buyang Huanwu decoction on axonal regeneration after peripheral nerve transection.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Asian medicine, Buyang Huanwu decoction (BYHWD) has been used for the treatment of cardiovascular and neurological disorders. Recent experimental studies have begun to provide evidence on the protective effects of BYHWD on injured peripheral nerves. AIM OF THE STUDY: To examine whether BYHWD was effective in inducing axonal regeneration after peripheral nerve transection, and if so, how it acted on the nerve. MATERIALS AND METHODS: The sciatic nerve in rats was transected and resutured 0, 1, or 4 weeks later. BYHWD was orally administered daily into the animals with nerve transection and coaptation (NTC). Axonal regeneration was measured by immunofluorescence staining of NF-200 and superior cervical ganglion 10 (SCG10) and by retrograde tracing method. Changes of protein levels in the sciatic nerve were analyzed by western blot analysis. Effects of BYHWD and its constituents on neurite outgrowth were analyzed in cultured dorsal root ganglion (DRG) neurons. Hot plate and treadmill training tests were performed to assess the levels of functional recovery after nerve injury. RESULTS: The rate of axonal regeneration was attenuated by delayed coaptation after transection, but improved by BYHWD treatment. Levels of phospho-Erk1/2 and Cdc2 phosphorylation of vimentin, measured as indicators of the activation of regenerating axons and supportive Schwann cells, were increased in the sciatic nerve of NTC animals, and their distribution in the proximal and distal nerves were affected by BYHWD treatment. Treatment of BYHWD during the period of chronic denervation significantly increased axonal regeneration when analyzed by immunofluorescence staining and retrograde tracing methods. Neurite outgrowth of DRG neurons cocultured with Schwann cells from the chronically transected sciatic nerves was enhanced by BYHWD treatment. Radix Paeoniae Rubra induced neurite outgrowth most efficiently among all herbal constituents of BYHWD. Finally, hot plate and treadmill training tests demonstrated that BYHWD administration significantly improved the sensorimotor nerve function in NTC animals. CONCLUSIONS: Our data suggest that BYHWD treatment may contribute to the timely interaction between regenerating axons and distal Schwann cells in the transected nerve and facilitate axonal regeneration.

Protective Effects of Bogijetong Decoction and Its Selected Formula on Neuropathic Insults in Streptozotocin-Induced Diabetic Animals.

Bogijetong decoction (BGJTD) is a mixture of herbal formulation which is used in the traditional Korean medicine for the treatment of neuropathic pain caused by diabetes. Here, we investigated the regulatory effects of BGJTD and its reconstituted decoction subgroups on the neuropathic responses in streptozotocin- (STZ-) induced diabetic animals. Be decoction (BeD) was formulated by selecting individual herbal components that induced neurite outgrowth most efficiently in each subgroup. BeD induced the neurite outgrowth in DRG neurons most efficiently among decoction subgroups and downregulated the production of TNF-α from the sciatic nerves in STZ-diabetic animals. While the levels of phospho-Erk1/2 were elevated in the sciatic nerves of STZ-diabetic animals by BGJTD and BeD treatments, p38 level was downregulated by BGJTD and BeD. A single herbal component of BeD induced neurite outgrowth comparable to BeD and was involved in the regulation of Erk1/2 activation and TNF-α production in DRG neurons. Oral administration of BGJTD and BeD in STZ-diabetic animals reduced the latency time responding to thermal stimulation. Our results suggest that the reconstituted formulation is as effective as conventional BGJTD in inducing biochemical and behavioral recoveries from the neuropathy in peripheral nerves and thus the experimental reductionism may be applied to develop the methodology for compositional analysis of herbal decoctions.

Modulation of hippocampal neuronal activity by So-ochim-tang-gamibang in mice subjected to chronic restraint stress.

BACKGROUND: So-ochim-tang-gamibang (SOCG) is a decoction formula which has been used to improve mental activity in traditional Korean medicine. The present study was performed to evaluate whether the treatment of SOCG was involved in activating hippocampal neurons in mice which were subjected to chronic restraint stress (CRS). METHODS: Mice were subjected to CRS for 2 weeks to induce depressive-like behaviors. SOCG was orally administered for the same period. mRNA expression in the hippocampus was analyzed by RT-PCR. Levels of serotonin receptor 5-HT1AR in the hippocampus were determined by western blotting and by immunofluorescence staining in coronal brain sections. Cultured neurons were prepared from the dorsal root ganglia (DRG) in mice to examine the effects of CRS and SOCG treatment on neurite outgrowth. Depressive-like behaviors of experimental animals were measured by open field test (OFT) and forced swimming test (FST). RESULTS: mRNA levels of serotonin 1A and 1B receptors (5-HT1AR and 5-HT1BR) were decreased in the hippocampus of CRS animals and increased by SOCG treatment. Signals of 5-HT1AR protein in CA3 pyramidal cells were decreased by CRS but elevated back to levels in control animals after SOCG treatment. Phospho-Erk1/2 protein in CA3 cells showed similar pattern of changes as in 5-HT1AR, suggesting coordinated regulation after SOCG treatment in CRS animals. Axonal growth-associated protein GAP-43 levels were also decreased by CRS and then increased by SOCG treatment. In vivo administration of SOCG improved neurite outgrowth of primary DRG neurons from CRS animals and also increased 5-HT1AR protein signals. Behavioral tests of open field and forced swimming showed that immobility time periods were significantly decreased by SOCG treatment. CONCLUSIONS: Our data suggest that SOCG treatment may increase synaptic responsiveness to serotonergic neuronal inputs by upregulating 5-HT1AR in the hippocampal neurons.