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The repair of large bone defects remains a significant challenge in clinical practice and requires bone grafts or substitute materials. In this study, we developed a unique hybrid bone scaffold comprising a three dimensional (3D)-printed metal plate for weight bearing and a biodegradable polymer tube serving as bone conduit. We assessed the long-term effect of the hybrid bone scaffold in repairing radial bone defects in a beagle model.Bone defects were created surgically on the radial bone of three beagle dogs and individually-tailored scaffolds were used for reconstruction with or without injection of autologous bone and decellularized extracellular matrix (dECM). The repaired tissue was evaluated by X-ray, micro-computed tomography, and histological observation 6 months after surgery. The functional integrity of hybrid bone scaffold-mediated reconstructions was assessed by gait analysis.In vivo analysis showed that the hybrid bone scaffolds maintained the physical space and bone conductivity around the defect. New bone was formed adjacent to the scaffolds. Addition of autologous bone and dECM in the polymer tube improved healing by enhancing bone induction and osteoconduction. Furthermore, the beagles' gait appeared normal by 4 months.The future of bone healing and regeneration is closely related to advances in tissue engineering. Bone production using autologous bone and dECM loaded on 3D-printed hybrid bone scaffolds can successfully induce osteogenesis and provide mechanical force for functional bone regeneration, even in large bone defects.

While mast cells (MCs) are previously well-known as a pathological indicator of pain, their role in alleviating pain is recently emerged in acupuncture research. Thus, this study systematically reviews the role of MC in acupuncture analgesia. Animal studies on MC changes associated with the acupuncture analgesia were searched in PubMed and EMBASE. The MC number, degranulation ratio and pain threshold changes were collected as outcome measures for meta-analyses. Twenty studies were included with 13 suitable for meta-analysis, most with a moderate risk of bias. A significant MC degranulation after acupuncture was indicated in the normal and was significantly higher in the pain model. In the subgroup analysis by acupuncture type, manual (MA) and electrical (EA, each P.00001) but not sham acupuncture had significant MC degranulation. Meta-regression revealed the linear proportionality between MC degranulation and acupuncture-induced analgesia (P.001), which was found essential in MA (P.00001), but not in EA (P = .45). MC mediators, such as adenosine and histamine, are involved in its mechanism. Taken together, skin MC is an essential factor for acupuncture-induced analgesia, which reveals a new aspect of MC as a pain alleviator. However, its molecular mechanism requires further study. PERSPECTIVE: This systematic review synthesizes data from studies that examined the contribution of skin MC in acupuncture analgesia. Current reports suggest a new role for skin MC and its mediators in pain alleviation and explain a peripheral mechanism of acupuncture analgesia, with suggesting the need of further studies to confirm these findings.

The Shenmen point (acupuncture point heart 7: HT7), located in the heart meridian, is frequently used to treat mental disorders, including drug addiction, anxiety, and depression. This study aimed to determine how HT7 regulates anxiety and negative emotions caused by repeated alcohol administration, focusing on the amygdala and paraventricular nucleus (PVN). Repeated administration of alcohol (ETOH, 2 g/kg, i.p. injection, 16% v/v) for 14 days increased the corticosterone (CORT) levels, and HT7 stimulation reduced the plasma CORT levels. HT7 stimulation mitigated anxiety-like behaviors and reduced 22-kHz ultrasonic vocalizations in rats receiving repeated ETOH injections. HT7 stimulation increased the amygdala expression of mature brain-derived neurotropic factor (mBDNF) and phosphorylated tropomyosin receptor kinase B (pTrkB) and decreased the PVN corticotropin-releasing hormone (CRH) expression. Amygdala microinjections of the TrkB antagonist ANA-12 (0.1 pmol/1 μL) reversed the increase in PVN CRH levels. The reduced PVN CRH levels were regulated by CRH-expressing neurons in the amygdala, and the increased amygdala CRH levels were affected by the HT7-stimulation induced increases in mBDNF. HT7 stimulation alleviates increased stress hormone levels and mitigates anxiety and negative emotions caused by repeated ETOH administration. These results provide scientific support for the clinical use of acupuncture to treat various alcoholism-induced diseases.

BACKGROUND: Angiogenesis and vasculogenesis are essential processes for successful tissue regeneration in tissue engineering and regenerative medicine. The adipose-derived stromal vascular fraction (SVF) is not only a source of adipose stem cells (ASC) but also a suitable source of microvascular endothelial cells because it is a rich capillary network. So, we propose a new hypothesis for isolating adipose-derived human microvascular endothelial cells (HMVEC-A) from the SVF and developed a dual isolation system that isolates two cell types from one tissue. METHOD: To isolate HMVEC-A, we analyzed the supernatant discarded when ASC is isolated from the adipose-derived SVF. Based on this analysis, we assumed that the SVF adherent to the bottom of the culture plate was divided into two fractions: the stromal fraction as the ASC-rich fraction, and the vascular fraction (VF) as the endothelial cells-rich fraction floating in the culture supernatant. VF isolation was optimized and the efficiency was compared, and the endothelial cells characteristics of HMVEC-A were confirmed by flow cytometric analysis, immunocytochemistry (ICC), a DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) uptake, and in vitro tube formation assay. RESULTS: Consistent with the hypothesis, we found a large population of HMVEC-A in the VF and isolated these HMVEC-A by our isolation method. Additionally, this method had higher yields and shorter doubling times than other endothelial cells isolation methods and showed typical morphological and phenotypic characteristics of endothelial cells. CONCLUSION: Cells obtained by the method according to our hypothesis can be applied as a useful source for studies such as tissue-to-tissue networks, angiogenesis and tissue regeneration, patient-specific cell therapy, and organoid chips.

A new concept, assembling cell-laden tissue modules, is for the first time proposed for soft tissue engineering. Adipose-vascular tissue modules composed of a synthetic polymer-based substructure and customized bioinks using planar 3D cell printing are engineered. Such tissue modules are systematically assembled into a synthetic polymer-based module holder fabricated with rotational 3D printing, resulting in the development of a flexible and volumetric tissue assembly. Whereas most of the previous studies about the construction of adipose tissue are limited to hypoxia, poor vascularization, rapid resorption, and mismatch in mechanical properties, it is aimed to realize the construction of nonhypoxic, flexible, and volume-stable tissue assembly in this study. The significance of engineered tissue assembly is proven through various in vitro and in vivo evaluations. In particular, stable volume and remarkable neovascularization/adipogenesis are observed in the implanted assembly over four weeks. Interestingly, the size of newly formed lipid droplets and the remodeled morphology in the assembly are comparable to those in native adipose tissue. As far as it is known, this work is a first report suggesting a cell printing-based tissue assembly for functional reconstruction of soft tissue.

Because human emotion varies greatly among individuals and is a qualitative factor, measuring it with any degree of accuracy is very difficult. Heart rate variability (HRV), which is used in evaluations of the autonomic nervous system (ANS), is used to evaluate human emotions. This study examines the validity of HRV as a tool to evaluate emotions using the International Affective Picture System (IAPS). For experimentation, five photos were selected for each of the categories of “happy,” “unhappy”, and “neutral” from among the images provided by the IAPS. The subjects were required to complete the Self-Assessment Manikin (SAM) after being shown each picture. We extracted the R-R interval (RRI) value of each photo from the PPG, as well as the valence, arousal, and dominance value of each photo from the SAM to analyze their correlation. As results, there was significant positive correlation with valence and significant negative correlation with dominance in the photo simulation associated with the “unhappy” emotion, only when the arousal value exceeded a critical value. Therefore, the findings of this study suggest that it is possible to use an HRV-based evaluation only when a high level of emotion is induced by visual stimulation.

BACKGROUND: The stromal vascular fraction (SVF) isolated from adipose tissue, which contains stem cells as well as other cell types, has been applied in various research fields. Although different enzymatic concentrations and treatment durations have been applied to isolate the SVF, optimal conditions have not been established. Thus, we aimed to establish the optimal conditions for isolation of the SVF from adipose tissue by automated systems. METHODS: The SVF was collected from removed adipose tissues of five donors during surgery. The SVF was treated with 0.1% or 0.2% collagenase type I for 20, 40, or 60 min. Then, colony forming unit (CFU) assays and flow cytometry were performed to characterize the adipose stem cells (ASCs). A cytokine array was used to investigate the correlation between colony-formation ability and the secretion of isolated ASCs. RESULTS: Treatment with 0.1% collagenase type I for 60 min resulted in a higher SVF yield, whereas treatment with 0.1% collagenase for 40 min resulted in higher CFU values. In addition, expression of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 in the SVF was higher in the high-CFU group than in the low-CFU group. CONCLUSION: The optimal conditions for isolation of the SVF from adipose tissue were treatment with 0.1% collagenase type I for 40 min. We identified the conditions required for efficient SVF isolation based on high CFU values, and our results will facilitate the development of automated systems.

This study was to investigate the effect of subcutaneous injection of the adipose stem cells (ASCs) with conditioned media (CM) in the treatment of acne vulgaris scar. We used Adult male New Zealand white rabbit ears as an animal model and induced acne formation by Kignman method. Adipose tissue was isolated and harvested from the scapula of rabbits, and ASCs were cultured and expanded until passage 1. There have four groups in our experiment, include phosphate buffered saline (PBS), ASCs with PBS (ASC + PBS), CM, and ASCs with CM (ASC + CM) group. This solution of 0.6 ml injected to subcutaneous in each group. ASC + PBS and ASC + CM groups were containing ASCs of 5.0 × 106 cells/ml. We analyzed the treatment of 4 groups to scar tissue after 2 and 4 weeks by hematoxylin and eosin stain, immunohistochemistry, and RNA expression level of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), and matrix metalloproteinase-2 (MMP-2). Also, the expression of keratin 16 (K16) was detected by western blot analysis. H&E stain showed that infiltration of inflammation cells was significantly reduced at 2 and 4 weeks, as well as re-epithelialization was improved in the ASC + CM group. The ASC + CM gourp was reduced both expression levels of TNF-α, IL-1α, and MMP-2 and K16 protein level. In conclusion, the ASCs with CM has a significant curative effect on acne vulgaris scar, more to the point, the CM has a key role on treatment. It could be applied to a therapeutic approach to regenerate to treat acne vulgaris scar.

Background The P300 is a major index used to evaluate improvements in brain function. Although a few studies have reported evaluating the effectiveness of manual acupuncture or electro-acupuncture by monitoring the P300, research in this field is not yet very active. The aim of this study was to investigate the effects of periodic low-frequency electrical stimulation applied to BL62 and KI6 on brain activity by analyzing the P300. Method The study was conducted as a randomized double-blind test of 55 subjects in their 50s, including 26 males and 29 females. Each subject received 12 sessions of stimulation over a one-month period. In each session, low-frequency electrical stimulation at an average of 24 μA and 2 Hz was applied to the acupuncture points BL62 and KI6, and event-related potentials (ERPs) were measured before the first session and after the last session of the electrical stimulation. Results The results of a chi-square test indicated that the double-blind test was conducted correctly. Compared to the Sham group, all the subjects in the Real stimulation group showed a tendency toward a decreasing P300 latency and increasing P300 amplitude after all 12 sessions of stimulation. In the women, the amplitude significantly increased at Fz, Fcz, Cz, Cpz, and Pz. Conclusions With this experiment, the low-frequency electrical stimulation of two acupuncture points (BL62 and K16) was confirmed to have a positive influence on the prevention of natural cerebral aging. Trial registration This study was registered at the Clinical Research Information Service (CRIS) of the National Research Institute of Health ( https://cris.nih.go.kr/cris/search/search_result_st01_en.jsp? , Registration Number: KCT0001940). The date of registration was June 9, 2016.

In keloids, the mechanism underlying the excessive accumulation of extracellular matrix after injury of the skin is unclear, and there is no effective treatment because of the incomplete understanding of their pathogenesis; thus, a high recurrence rate is observed. We studied a new marker of keloids to determine a new treatment strategy. First, the keloid gene expression profile (GSE44270) was analyzed (downloaded from the Gene Expression Omnibus database) and the new keloid marker candidate, epidermal growth factor (EGF)-like repeats and discoidin I-like domains 3 (EDIL3) which were upregulated in keloid samples was identified. Knockdown of EDIL3 is known to suppresses angiogenesis by downregulating relevant inhibitory factors that can limit the supply of survival factors to tumor cells from the circulation via the vascular endothelial cells. In keloids, the mechanism of action of EDIL3 may be similar to that in tumors; the inhibition of apoptosis in tumor cells via a reduction in the apoptosis of blood vessels by upregulating an angiogenic factor. To determine whether EDIL3 is involved in keloid formation, we performed knockdown of EDIL3 in keloid fibroblasts in vitro by transfection with anti-EDIL3 small interfering RNA (via microporation). EDIL3 was upregulated in keloid fibroblasts compared with normal fibroblasts in collagen type I, II and III. Our results indicate the control of EDIL3 expression may be a new promising treatment of keloid disease also the molecular targeting of EDIL3 may improve the quality of treatment and reduce the formation of keloids.

Results : In our pilot experiment, half of the participants showed significantly improved manipulation skills in complex lifting/thrusting techniques after training with AMES which is developed in this study, while the other half of the participants did not show significant improvements. Conclusions : The AMES could be useful in acupuncture-manipulation training for students. Our findings suggest that novice can improve sophisticated hand movement for acupuncture manipulation with sensorimotor learning using visual feedback.

Reactive oxygen species (ROS) have been implicated in the development of behavioral sensitization following repeated cocaine exposure. We hypothesized that increased ROS following cocaine exposure would act as signaling molecules in the mesolimbic dopamine (DA) system, which might play an important role in mediating the reinforcing effects of cocaine. The aim of this study was to evaluate cocaine enhancement of brain metabolic activity and the effects of ROS scavengers on cocaine self-administration behavior, cocaine-induced ROS production in the nucleus accumbens (NAc) and cocaine enhancement of DA release in the NAc. Metabolic neural activity monitored by temperature and oxidative stress were increased in NAc following cocaine exposure. Systemic administration of the ROS scavenger N-tert-butyl-α-phenylnitrone (PBN) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), either pre- or post-treatment, significantly decreased cocaine self-administration without affecting food intake. Infusion of TEMPOL into the NAc inhibited cocaine self-administration. Increased oxidative stress was found mainly on neurons, but not astrocytes, microglia or oligodendrocytes, in NAc of rats self-administering cocaine. TEMPOL significantly attenuated cocaine-induced enhancement of DA release in the NAc, compared to saline controls. TEMPOL had no effect on the enhancement of DA release produced by the DA transporter inhibitor GBR12909. Taken together, these findings suggest that enhancement of ROS production in NAc neurons contributes to the reinforcing effect of cocaine.

We attempted to investigate whether acupuncture stimulation at HT7 can have an effect on brain activation patterns and alcohol abstinence self-efficacy. Thirty-four right-handed healthy subjects were recruited for this study. They were randomly assigned into two groups: the HT7 (Shenmen) group and the LI5 (Yangxi) group. Acupuncture stimulation was performed using a block paradigm during fMRI scanning. Additionally, the Korean version of Alcohol Abstinence Self-Efficacy Scale (AASES) was used to determine the effect of acupuncture stimulation on self-efficacy to abstain from alcohol use. According to the result of fMRI group analysis, the activation induced by HT7 stimulation was found on the bilateral postcentral gyrus, inferior parietal lobule, inferior frontal gyrus, claustrum, insula, and anterior lobe of the cerebellum, as well as on the left posterior lobe of the cerebellum (p<0.001, uncorrected). According to the AASES analysis, the interaction effect for gender and treatment was marginally significant (F(1,30)=4.152, p=0.050). For female group, the simple main effect of treatment was significant (F(1,11)=8.040, p=0.016), indicating that the mean change score was higher in the HT7 stimulation than in the LI5 stimulation. Therefore, our study has provided evidence to support that HT7 stimulation has a positive therapeutic effect on the alcohol-related diseases.

Recent evidences demonstrated that endothelial-to-mesenchymal transition (EndMT) has a crucial role in cancer and is recognized as a unique source of cancer-associated fibroblasts (CAFs). Primo vascular system (PVS) is a new circulatory system which may play an important role in cancer metastasis and regeneration. In the current study, we applied previously established time-saving method to identify primo vessels and further investigated the immunocytochemical properties of primo vessels. Both primo vessels and primary primo vessel cells in the mesentery expressed endothelial markers and fibroblast markers. Double-labeling experiments demonstrated that endothelial and fibroblast markers are coexpressed in primo vessels. In addition, under the stimulation of TGF-β1in vitro,primary primo vessel cells differentiated into fibroblasts. Therefore, we found that primo vessels in the mesentery had a transitional structure between endothelium and mesenchymal. This is a new finding of EndMT in normal postnatal animals.

The primo vascular system (PVS) was first introduced by Bong-Han Kim via his five research reports. Among these the third report was most extensive and conclusive in terms of the PVS anatomy and physiology relating to the acupuncture meridians. His study results, unfortunately, were not reproduced by other scientists because he did not describe the materials and methods in detail. In 2002, a research team in Seoul National University reinitiated the PVS research, confirmed the existence of PVS in various organs, and discovered new characteristics of PVS. Two important examples are as follows: PVS was found in the adipose tissue and around cancer tissues. In parallel to these new findings, new methods for observing and identifying PVS were developed. Studies on the cell and material content inside the PVS, including the immune function cells and stem cells, are being progressed. In this review, Bong-Han Kim’s study results in his third report are summarized, and the new results after him are briefly reviewed. In the last section, the obstacles in finding the PVS in the skin as an anatomical structure of acupuncture meridian are discussed.

There is growing public interest in alternative approaches to addiction treatment and scientific interest in elucidating the neurobiological underpinnings of acupuncture. Our previous studies showed that acupuncture at a specific Shenmen (HT7) points reduced dopamine (DA) release in the nucleus accumbens (NAc) induced by drugs of abuse. The present study was carried out to evaluate the effects of HT7 acupuncture on γ-aminobutyric acid (GABA) neuronal activity in the ventral tegmental area (VTA) and the reinstatement of cocaine-seeking behavior. Using microdialysis and in vivo single-unit electrophysiology, we evaluated the effects of HT7 acupuncture on VTA GABA and NAc DA release and VTA GABA neuronal activity in rats. Using a within-session reinstatement paradigm in rats self-administering cocaine, we evaluated the effects of HT7 stimulation on cocaine-primed reinstatement. Acupuncture at HT7 significantly reduced cocaine suppression of GABA release and GABA neuron firing rates in the VTA. HT7 acupuncture attenuated cocaine-primed reinstatement, which was blocked by VTA infusions of the selective GABA

We investigated commonalities and differences in brain responses to enhanced bodily attention around acupuncture points with and without stimulation. Fourteen participants received acupuncture needles at both PC6 and HT7 acupoints in the left hand. To enhance bodily attention to acupoints, participants responded to the locations of stimulations in a two-alternative forced choice task. Two fMRI scans were taken in a block design: session 1 labeled with manual stimulation (genuine stimulation) and session 2 labeled with electro-acupuncture (pseudo-stimulation). To compare cortical activation patterns, data were analyzed using the Freesurfer software package. Both genuine-and pseudo-stimulation resulted in brain activations in the insula, anterior cingulate cortex, secondary somatosensory cortex, superior parietal cortex and brain deactivation in the medial prefrontal cortex, posterior cingulate cortex, inferior parietal cortex and the parahippocampus. Genuine acupuncture stimulation exhibited greater brain activation in the posterior insula, posterior operculum and the caudal part of the anterior cingulate cortex, compared with pseudo-stimulation. We demonstrated that enhanced bodily attention triggered by genuine acupuncture stimulation can activate the salience network and deactivate the default mode network regardless of the type of stimulation. The component of enhanced attention to a certain part of the body is significant in the brain response to acupuncture stimulation.

Purpose: The meridian system is originated from concept of invisible line which connects between internal organ and specific point in the body surface. And specific points are discovered by observing reaction of patients after pushing patient’s skin with doctor’s hand. And it is so called Ashipoints which means points make patient scream “Ah!”. In this study, we suppose that the ashi-points have relation with referred pain, and thus observed an aspect of revelation of referred pain area visualized by evans blue dye. Methods: SD-rats (250 g) were injected evans blue dye through caudal veinwhile anesthetizedwithmixture of Zoletil and Rom pun. 10minutes later, silicone guide and Q-tip absorbing mustard oil was inserted to the large intestine in depth of 3, 5. 7 cm from the anus. Evans blue signwas observed after 30minutes after insert of theQ-tip. The location, size and shape of evans blue sign was recorded in the chart developed to record the sign and charts weremerged by u sing the Adobe Photoshop program. Results: More than 90% of evans blue sign were located in the hind paw. Specially, many signs were located in the lateral side of the foot (border between the red and white flesh). Most of signs have shape of long ellipse, and headed samedirection. The sign mainly located in rows around navicular tubercle. The signshad tendencyof distinguishedby thedepthbut there were no significant border for distinguish the signs. Conclusion: In this study, we have visualized referred pain area by using evans blue dye, and confirmed that the signs has tendency of forming lines, and many acupuncture points are located in the same anatomical area. In spite of the results of this study, we have confirmed only one-way communication and to confirm two-way communication, we plan to ob serve treatment effects of the sign area. Contact: Osang KWON, logos319@kiom.re.kr

Peripheral bee venom (BV) administration produces 2 contrasting effects, nociception and antinociception. This study was designed to evaluate whether the initial nociceptive effect induced by BV injection into the Zusanli acupoint is involved in producing the more prolonged antinociceptive effect observed in the mouse formalin test, and whether capsaicin-sensitive primary afferents are involved in these effects. BV injection into the Zusanli point increased spinal Fos expression but not spontaneous nociceptive behavior. BV pretreatment 10 minutes before intraplantar formalin injection dose-dependently attenuated nociceptive behavior associated with the second phase of the formalin test. The destruction of capsaicin-sensitive primary afferents by resiniferatoxin (RTX) pretreatment selectively decreased BV-induced spinal Fos expression but did not affect BV-induced antinociception. Furthermore, BV injection increased Fos expression in tyrosine hydroxylase immunoreactive neurons in the locus caeruleus, and this expression was unaltered by RTX pretreatment. Finally, BV’s antinociception was blocked by intrathecal injection of 10 μg idazoxan, and this effect was not modified by RTX pretreatment. These findings suggest that subcutaneous BV stimulation of the Zusanli point activates central catecholaminergic neurons via capsaicin-insensitive afferent fibers without induction of nociceptive behavior. This in turn leads to the activation of spinal α2-adrenoceptors, which ultimately reduces formalin-evoked nociceptive behaviors. Perspective This study demonstrates that BV acupuncture produces a significant antinociception without nociceptive behavior in rodents, which is mediated by capsaicin-insensitive afferents and involves activation of central adrenergic circuits. These results further suggest that BV stimulation into this acupuncture point might be a valuable alternative to traditional electrical or mechanical acupoint stimulation.

The anti-inflammatory effect (AI) induced by peripheral injection of diluted bee venom (dBV) involves activation of spinal cord circuits and is mediated by catecholamine release from adrenal medulla, but the precise neuronal mechanisms involved are not fully understood. In a recent study, we demonstrated that an increase in spinal acetylcholine is involved in mediating the anti-inflammatory effect of dBV and that this mediation also involves adrenomedullary activation. The present study utilized the mouse air pouch inflammation model to evaluate the involvement of spinal acetylcholine receptors and sympathetic preganglionic neurons (SPNs) in dBV's anti-inflammatory effect (dBVAI). Intrathecal (IT) pretreatment with atropine (muscarinic cholinergic antagonist) but not hexamethonium (nicotinic cholinergic antagonist) significantly suppressed dBVAI on zymosan-evoked leukocyte migration. Subsequent experiments showed that IT pretreatment with methoctramine (a muscarinic receptor type 2; M(2) antagonist), but not pirenzepine (an M(1) antagonist) or 4-DAMP (an M(3) antagonist), suppressed the dBVAI. In addition, dBV stimulation specifically increased Fos expression in SPNs of the T7-T11, but not the T1-T6 or T12-L2 spinal cord segments, in animals with zymosan-induced inflammation. Moreover, IT methoctramine pretreatment suppressed this dBV-induced Fos expression specifically in SPNs of T7-T11 level. Peripheral sympathetic denervation using 6-hydroxydopamine (6-OHDA) treatment (which spares sympathetic adrenal medullary innervation) did not alter dBVAI. Collectively these results indicate that dBV stimulation leads to spinal cord acetylcholine release that in turn acts on spinal M(2) receptors, which via a hypothesized disinhibition mechanism activates SPNs that project to the adrenal medulla. This activation ultimately leads to the release of adrenal catecholamines that contribute to dBVAI.

Ki Joo Kim,1,2 Young Ae Joe,3 Min Kyoung Kim,1,2 Su Jin Lee,1 Yeon Hee Ryu,1,2 Dong-Woo Cho,4,5 Jong Won Rhie1,2 1Department of Plastic Surgery, College of Medicine, 2Department of Molecular Biomedicine, 3Cancer Research Institute and Department of Medical Lifescience, The Catholic University of Korea, Seoul, Republic of Korea; 4Department of Mechanical Engineering, Pohang University of Science and Technology, Gyeongbuk, Republic of Korea; 5Department of Integrative Bioscience and Bioengineering, Pohang University of Science and Technology, Gyeongbuk, Republic of Korea Background: Silicon dioxide composites have been found to enhance the mechanical properties of scaffolds and to support growth of human adipose tissue-derived stem cells (hADSCs) both in vitro and in vivo. Silica (silicon dioxide alone) exists as differently sized particles when suspended in culture medium, but it is not clear whether particle size influences the beneficial effect of silicon dioxide on hADSCs. In this study, we examined the effect of different sized particles on growth and mitogen-activated protein kinase signaling in hADSCs.Methods: Silica gel was prepared by a chemical reaction using hydrochloric acid and sodium silicate, washed, sterilized, and suspended in serum-free culture medium for 48 hours, and then sequentially filtered through a 0.22 µm filter (filtrate containing nanoparticles smaller than 220nm; silica NPs). hADSCs were incubated with silica NPs or 3 µm silica microparticles (MPs), examined by transmission electron microscopy, and assayed for cell proliferation, apoptosis, and mitogen-activated protein kinase signaling.Results: Eighty-nine percent of the silica NPs were around 50–120 nm in size. When hADSCs were treated with the study particles, silica NPs were observed in endocytosed vacuoles in the cytosol of hADSCs, but silica MPs showed no cell entry. Silica NPs increased the proliferation of hADSCs, but silica MPs had no significant effect in this regard. Instead, silica MPs induced slight apoptosis. Silica NPs increased phosphorylation of extracellular signal-related kinase (ERK)1/2, while silica MPs increased phosphorylation of p38. Silica NPs had no effect on phosphorylation of Janus kinase or p38. Pretreatment with PD98059, a MEK inhibitor, prevented the ERK1/2 phosphorylation and proliferation induced by silica NPs.Conclusion: Scaffolds containing silicon dioxide for tissue engineering may enhance cell growth through ERK1/2 activation only when NPs around 50–120 nm in size are included, and single component silica-derived NPs could be useful for bioscaffolds in stem cell therapy. Keywords: particle, ceramic, biomaterial, mesenchymal stem cells, ERK

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) from various sites are applied in tissue engineering and cell therapy. The condition of AT-MSCs depends on the donor's age, body mass index (BMI), and gender. AT-MSCs from 66 human donors were analyzed, and the cells were sorted according to donor age (10-19 years: n = 1; 20-29 years: n = 5; 30-39 years: n = 12; 40-49 years: n = 22; 50-59 years: n = 12; 60-69 years: n = 9, and 70 years or older: n = 5), BMI (under 25, 25-30, and over 30), and gender (19 males and 48 females). Additionally, AT-MSCs were compared to bone marrow MSCs and chorionic tissue-derived MSCs. We measured the MSC yield, growth rate, colony-forming units, multipotency, and surface antigens. AT-MSC proliferation was greater in cells isolated from individuals aged less than 30 years compared to the proliferation of AT-MSCs from those over 50 years old. BMI was correlated with osteogenic differentiation potency; increased BMI enhanced osteogenesis. Adipogenic differentiation was more strongly induced in cells isolated from donors aged less than 30 years compared to those isolated from other age groups. Also, a BMI above 30 was associated with enhanced adipogenic differentiation compared to cells isolated from individuals with a BMI below 25. Bone marrow MSCs were strongly induced to differentiate along both osteogenic and adipogenic lineages, whereas AT-MSCs predominantly differentiated into the chondrogenic lineage. Therefore, the type of regeneration required and variations among potential donors must be carefully considered when selecting MSCs for use in applied tissue engineering or cell therapy.

Reactive oxygen species (ROS) have been implicated in the development of behavioral sensitization following repeated cocaine exposure. We hypothesized that increased ROS following cocaine exposure would act as signaling molecules in the mesolimbic dopamine (DA) system, which might play an important role in mediating the reinforcing effects of cocaine. The aim of this study was to evaluate cocaine enhancement of brain metabolic activity and the effects of ROS scavengers on cocaine self-administration behavior, cocaine-induced ROS production in the nucleus accumbens (NAc) and cocaine enhancement of DA release in the NAc. Metabolic neural activity monitored by temperature and oxidative stress were increased in NAc following cocaine exposure. Systemic administration of the ROS scavenger N-tert-butyl-α-phenylnitrone (PBN) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), either pre- or post-treatment, significantly decreased cocaine self-administration without affecting food intake. Infusion of TEMPOL into the NAc inhibited cocaine self-administration. Increased oxidative stress was found mainly on neurons, but not astrocytes, microglia or oligodendrocytes, in NAc of rats self-administering cocaine. TEMPOL significantly attenuated cocaine-induced enhancement of DA release in the NAc, compared to saline controls. TEMPOL had no effect on the enhancement of DA release produced by the DA transporter inhibitor GBR12909. Taken together, these findings suggest that enhancement of ROS production in NAc neurons contributes to the reinforcing effect of cocaine.

Background. Primo vascular system is a new anatomical structure whose research results have reported the possibility of a new circulatory system similar to the blood vascular system and cells. Electrophysiology, which measures and analyzes bioelectrical signals tissues and cells, is an important research area for investigating the function of tissues and cells. The bioelectrical study of the primo vascular system has been reported by using modern techniques since the early 1960s by Bonghan Kim. This paper reviews the research result of the electrophysiological study of the primo vascular system for the discussion of the circulatory function. We hope it would help to study the electrophysiology of the primo vascular system for researchers. This paper will use the following exchangeable expressions: Kyungrak system = Bonghan system = Bonghan circulatory system = primo vascular system = primo system; Bonghan corpuscle = primo node; Bonghan duct = primo vessel. We think that objective descriptions of reviewed papers are more important than unified expressions when citing the papers. That said, this paper will unify the expressions of the primo vascular system.

A primo vessel was observed in the abdominal cavity in the lung cancer mouse model, and its function as an extra metastatic path was observed. In this work, we found a primo vessel accompanying a blood vessel emanating from a tumor in the skin. We also presented simple and efficient criteria to distinguish a primo vessel from a blood or a lymph vessel and from a nerve. The criteria for using DAPI and Phalloidin will be useful in clinical situations to find and identify the primo vessels among the blood vessels, lymph vessels, or nerves in the tissue surrounding a tumor such as a melanoma or breast cancer.

We for the first time reported evidence for the existence of a novel network, a PVS, abovethe epicardium of the rat heart. (1) We were consecutively able to visualize the PVs and the PNs above the epicardial spaces of five rats’ hearts by using Cr-Hx spraying or injection. (2) Hematoxylin and eosin (H&E) and toluidine blue staining of the PVs and the PNs showed that they consisted of a basophilic matrix; specifically the PNs contained several mast cells, some of which were degranulating into pericardial space. Also, 4′, 6-diamidino-2 phenylindole (DAPI) images of the PVs and the PNs showed that they contained various kinds of cells. (3) Transmission electron microscopic (TEM) longitudinal image of the PVs showed that the sinuses contained many granules with high-electron-density cores in parallel with putative endothelial cells. (4) TEM images of the PNs demonstrated that they consisted of lumen-containing cells surrounded by fibers and that they had mast cells that were degranulating toward the epicardium of the rat heart. The above data suggest that mast-cells-containing novel network exists above the epicardium of the rat heart.

Fluorescent nanoparticles (FNPs) which were injected into a tumor tissue flowed out through the blood and lymph vessels. The FNPs in blood vessels remained only in the order for few minutes while those in lymph vessels remained for a long time disappearing completely in 25 hours. We found a primo vessel inside a lymph vessel near a blood vessel, and FNPs remained in the primo vessel for longer than 25 hours. In addition, we examined in detail the residual time characteristics of lymph vessels because it could be useful in a future study of fluid dynamical comparison of the three conduits. These residual time characteristics of FNPs in the three kinds of vessels may have implications for the dynamics of nanoparticle drugs for cancer chemotherapy.

We studied the biopotential of acupuncture points in six meridians and its standard error to verify how reliable it is to measure electric potential on skin for investigating electrical properties of acupuncture points. Twenty healthy subjects were participated in this study for each meridian. Five-phase acupuncture points were used for recording then smoothing and normalizing were performed to analyze data. There was a positive correlation between standard error of biopotentials and acupuncture depth. It means acupuncture points close to the skin provide more reliable results than ones with deeper acupuncture depth when investigating electrophysiological characteristics of meridians.

Using spontaneously hypertensive rats (SHR), this study investigated whether electroacupuncture (EA) could reduce early stage hypertension by examining nitric oxide (NO) levels in plasma and nitric oxide synthase (NOS) levels in the mesenteric resistance artery. EA was applied to the acupuncture point Governor Vessel 20 (GV20) or to a non-acupuncture point in the tail twice weekly for 3 weeks under anesthesia. In conscious SHR and normotensive Wistar Kyoto (WKY) rats, blood pressure was determined the day after EA treatment by the tail-cuff method. We measured plasma NO concentration, and evaluated endothelial NO syntheses (eNOS) and neuronal NOS (nNOS) protein expression in the mesenteric artery. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were lower after 3 weeks of GV20 treatment than EA at non-acupuncture point and no treatment control in SHR. nNOS expression by EA was significantly different between both WKY and no treatment SHR control, and EA at GV20 in SHR. eNOS expression was significantly high in EA at GV 20 compared with no treatment control. In conclusion, EA could attenuate the blood pressure elevation of SHR, along with enhancing NO/NOS activity in the mesenteric artery in SHR.

We present the first investigation of electrical corrosion in acupuncture needles after electrical stimulation. Using scanning electron microscopy, we observed the occurrence of electrochemical corrosion on the surface of stainless steel 304 acupuncture needles after electrical stimulation in the tibial muscles of rats. Biphasic pulse electrical stimuli with 10-Hz frequency, 1-mA intensity and 1-ms pulse width were applied to the needles. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method labels fragmented DNA. Positive staining using this test indicates apoptotic cells in electrically stimulated tissues. The risk of electrical corrosion was found to be less in bipolar, short-duration, low-current or voltage and short-period stimulation than in monopolar, long-duration, high-current or voltage and long-period stimulation. Evaluation with a scanning electron microscope revealed that electrical stimulation can increase the electrical corrosion of stainless steel 304 acupuncture needles. In biocompatibility studies of stainless steel 304 acupuncture needles for electrical stimulation, TUNEL-positive cells were detected in the tibial muscle within 5 days after electrical stimulation. The results of this study demonstrate that the corrosion products of stainless steel 304 acupuncture needles might affect the post-electrical stimulation tissue response.

The free radical is involved in neuronal cell death in human neurodegenerative disease. Dammishimgyu (DMSG)-herbal acupuncture has been used to treat neurological disorders in Korea. The present study was aimed to investigate the neuroprotective effect of DMSG-herbal acupuncture against H(2)O(2)-induced apoptosis in human neuroblastoma cell line, SH-SY5Y.The neuroprotective effect of DMSG-herbal acupuncture on H(2)O(2) induced apoptosis was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, reverse transcription-polymerase chain reaction (RT-PCR), western blots and nitrite assay.In this study, 100 microM H(2)O(2)-treated cells decreased the cell viability with apoptotic features and increased the production of nitric oxide (NO). However, 0.1% DMSG treatment after exposure to 100 microM H(2)O(2) inhibited both H(2)O(2)-stimulated mRNA and protein expressions of BCL2-associated X protein (BAX) and caspase 3 apoptosis-related cysteine peptidase (CASP3). In addition, 0.1% DMSG treatment inhibited the increased NO production induced by H(2)O(2).These results suggest that DMSG-herbal acupuncture shows protective effect against H(2)O(2)-induced neuronal damage.

This study was designed to investigate the involvement of inducible nitric oxide synthase (iNOS) in electroacupuncture (EA)-induced analgesia.Two and 100 Hz EA stimulation were applied at acupoint ST 36 (Zusanli) in iNOS knockout mice (n = 28). Needles were inserted 5 mm in depth in ST36. After insertion, the needles were fixed in situ with adhesive tape. EA was applied after the basal threshold determination. The EA parameters were set as follows: constant square wave current output (pulse width: 0.6 ms at 2 Hz and 0.2 ms at 100 Hz) and 2 mA intensities. EA was performed for 30 minutes and tail-flick latencies (TFLs) were evaluated every 15 minutes for 1 hour.In 2 Hz EA stimulation, the tail-flick response (TFR) of wild-type mice for durations of 0, 15, 30, 45 and 60 minutes were 2.70 +/- 0.26, 4.19 +/- 0.37, 4.17 +/- 0.34, 3.57 +/- 0.27 and 3.39 +/- 0.32 seconds of TFLs. Meanwhile, iNOS -/- mice showed 4.10 +/- 0.33, 4.77 +/- 0.24, 5.26 +/- 0.30, 4.48 +/- 0.33 and 5.00 +/- 0.41 seconds of TFLs. In 100 Hz EA stimulation, the TFR of wild-type mice were 3.01 +/- 0.24, 4.67 +/- 0.31, 4.76 +/- 0.25, 4.04 +/- 0.45 and 4.26 +/- 0.30 seconds of TFLs. The iNOS -/- mice were 4.33 +/- 0.16, 5.29 +/- 0.28, 5.06 +/- 0.35, 4.52 +/- 0.17 and 4.80 +/- 0.28 seconds of TFLs. Wild-type mice exhibited 63.9% increase in TFL compared to the baseline after 2 Hz EA, whereas the iNOS knockout mice exhibited 32.9% increase in TFL. The TFL after 100 Hz EA showed similar trends: 66.5% increase in TFL in wildtype mice and 18.3% increase in the iNOS knockout mice.The present findings suggested that iNOS may play a crucial role in both low- and high-frequency EA-induced analgesic effects.

Although acupuncture has been widely used for complementary therapeutic approaches to treat inflammatory diseases and inflammation-induced pain, the potential anti-inflammatory effects of acupuncture treatment remain controversial in clinical trials, and the underlying mechanisms are still unclear.The objective was to determine whether electroacupuncture (EA) is able to suppress the peripheral inflammatory response (e.g., zymosan-induced leukocyte migration into air pouch). As part of a mechanistic approach, it was further evaluated whether endogenous opioid systems are involved in the "EA-induced anti-inflammatory effect" (EA-AI).EA (1 or 120 Hz) was performed bilaterally in the Zusanli acupoint (ST36) or in a nonacupoint (gluteal muscle) for 30 min in ICR mice under anesthetic condition. The number of leukocytes that migrated into the air pouch was counted 4 hours after zymosan injection. EA was performed at 0, 0.5, 1, or 2 hours prior to zymosan injection, respectively. To evaluate opioid involvement in EA-AI, intrathecal naloxone (36 microg/mouse) and intraperitoneal naloxone methiodide (30 mg/kg) were administered 10 min before EA stimulation.Both the 1 and 120 Hz frequencies of EA into Zusanli acupoint at the same time with zymosan injection significantly reduced leukocyte migration into the air pouch as compared with those of control groups (i.e., anesthetic control and needling control into Zusanli acupoint without electrical stimulation). The EA stimulation into nonacupoint did not produce any significant anti-inflammatory effect. EA treatment at 0.5 hours prior to zymosan injection also produced an anti-inflammatory effect but 1 and 2 hours prior to zymosan injection did not elicit any effect. Peripheral opioid blockage significantly reversed EA-AI, whereas spinal opioid blockage did not alter EA-AI.EA can suppress peripheral inflammation through a peripheral opioid mechanism. To achieve the full effectiveness of EA, repeated application is recommended for the treatment of a variety of inflammatory diseases.

Several lines of evidence indicate significant interactions between the immune and nervous systems. Our recent study reveals that 'bee venom (BV) induced anti-inflammatory effect' (BVAI) was produced by sympathetic preganglionic neuronal activation and subsequent adrenomedullary catecholamine release in a zymosan-induced inflammation model. However, the specific peripheral input and the supraspinal neuronal systems that are involved in this BVAI remain to be defined. Here we show that subcutaneous BV injection into left hind limb significantly reduces zymosan-induced leukocyte migration and that this effect is completely inhibited by denervation of the left sciatic nerve. This BVAI was not affected by the destruction of capsaicin-sensitive primary afferent fibers using either neonatal capsaicin or resiniferatoxin (RTX) pretreatment. BV injection into the left hind limb significantly increased Fos expression in the contralateral locus coeruleus (LC) in non-inflamed mice. In zymosan-inflamed mice, BV injection produced a further increase in LC Fos expression as compared with non-inflamed mice. This BV-induced Fos increase in the LC was not affected by RTX pretreatment. Pharmacological blockage of central noradrenergic activity by either central chemical sympathectomy (i.c.v. 6-hydroxydopamine) or alpha2 adrenoceptor antagonism (i.c.v. idazoxan) completely blocked BVAI. Taken together, these results suggest that BVAI is mediated by peripheral activation of capsaicin-insensitive primary afferent fibers and subsequent central noradrenergic activation including the LC.

With chromium-hematoxylin staining, we found evidence for the existence of novel age-dependent network structures in the dura mater of rat brains. Under stereomicroscopy, we noticed that chromium-hematoxylin-stained threadlike structures, which were barely observable in 1-week-old rats, were networked in specific areas of the brain, for example, the lateral lobes and the cerebella, in 4-week-old rats. In 7-week-old rats, those structures were found to have become larger and better networked. With phase contrast microscopy, we found that in 1-week-old rats, chromium-hematoxylin-stained granules were scattered in the same areas of the brain in which the network structures would later be observed in the 4- and 7-week-old rats. Such age-dependent network structures were examined by using optical and transmission electron microscopy, and the following results were obtained. The scattered granules fused into networks with increasing age. Cross-sections of the age-dependent network structures demonstrated heavily-stained basophilic substructures. Transmission electron microscopy revealed the basophilic substructures to be clusters with high electron densities consisting of nanosized particles. We report these data as evidence for the existence of age-dependent network structures in the dura mater, we discuss their putative functions of age-dependent network structures beyond the general concept of the dura mater as a supporting matrix.