A Guide Book for Children and Adolescents to Experience Anatomy and Clinics / 체질인류학회지

The purpose of this study is to enable children and adolescents to experience anatomy and clinics. For the purpose, the ways to use the anatomy educational resources (comics, 3-dimensional images, and 2-dimensional images) and diagnostic tools (stethoscope, sphygmomanometer, pen light, and reflex hammer) were described in a guide book. Following the guide book, students experienced anatomy and clinics in a course of the science museum. They learned anatomy with the comics, then did virtual dissection with the 3-dimensional and 2-dimensional images. Sequentially, with the diagnostic tools, they listened to heart sound, measured blood pressure, and performed light reflex and knee jerk. Through this study, we have found that anatomy and clinics should be experienced pleasantly. The complimentary guide book is expected to be further improved in future, so as to achieve better experience at home, science museum, and school.

Effects of DA-9701, a Novel Prokinetic Agent, on Phosphorylated Extracellular Signal-Regulated Kinase Expression in the Dorsal Root Ganglion and Spinal Cord Induced by Colorectal Distension in Rats

BACKGROUND/AIMS: DA-9701, a standardized extract of Pharbitis Semen and Corydalis Tuber, is a new prokinetic agent that exhibits an analgesic effect on the abdomen. We investigated whether DA-9701 affects visceral pain induced by colorectal distension (CRD) in rats. METHODS: A total of 21 rats were divided into three groups: group A (no CRD+no drug), group B (CRD+no drug), and group C (CRD+DA-9701). Expression of pain-related factors, substance P (SP), c-fos, and phosphorylated extracellular signal-regulated kinase (p-ERK) in the dorsal root ganglion (DRG) and spinal cord was determined by immunohistochemical staining and Western blotting. RESULTS: The proportions of neurons in the DRG and spinal cord expressing SP, c-fos, and p-ERK were higher in group B than in group A. In the group C, the proportion of neurons in the DRG and spinal cord expressing p-ERK was lower than that in group B. Western blot results for p-ERK in the spinal cord indicated a higher level of expression in group B than in group A and a lower level of expression in group C than in group B. CONCLUSIONS: DA-9701 may decrease visceral pain via the downregulation of p-ERK in the DRG and spinal cord.

Contralaterally transplanted human embryonic stem cell-derived neural precursor cells (ENStem-A) migrate and improve brain functions in stroke-damaged rats

The transplantation of neural precursor cells (NPCs) is known to be a promising approach to ameliorating behavioral deficits after stroke in a rodent model of middle cerebral artery occlusion (MCAo). Previous studies have shown that transplanted NPCs migrate toward the infarct region, survive and differentiate into mature neurons to some extent. However, the spatiotemporal dynamics of NPC migration following transplantation into stroke animals have yet to be elucidated. In this study, we investigated the fates of human embryonic stem cell (hESC)-derived NPCs (ENStem-A) for 8 weeks following transplantation into the side contralateral to the infarct region using 7.0T animal magnetic resonance imaging (MRI). T2- and T2*-weighted MRI analyses indicated that the migrating cells were clearly detectable at the infarct boundary zone by 1 week, and the intensity of the MRI signals robustly increased within 4 weeks after transplantation. Afterwards, the signals were slightly increased or unchanged. At 8 weeks, we performed Prussian blue staining and immunohistochemical staining using human-specific markers, and found that high percentages of transplanted cells migrated to the infarct boundary. Most of these cells were CXCR4-positive. We also observed that the migrating cells expressed markers for various stages of neural differentiation, including Nestin, Tuj1, NeuN, TH, DARPP-32 and SV38, indicating that the transplanted cells may partially contribute to the reconstruction of the damaged neural tissues after stroke. Interestingly, we found that the extent of gliosis (glial fibrillary acidic protein-positive cells) and apoptosis (TUNEL-positive cells) were significantly decreased in the cell-transplanted group, suggesting that hESC-NPCs have a positive role in reducing glia scar formation and cell death after stroke. No tumors formed in our study. We also performed various behavioral tests, including rotarod, stepping and modified neurological severity score tests, and found that the transplanted animals exhibited significant improvements in sensorimotor functions during the 8 weeks after transplantation. Taken together, these results strongly suggest that hESC-NPCs have the capacity to migrate to the infarct region, form neural tissues efficiently and contribute to behavioral recovery in a rodent model of ischemic stroke.

Multiple 5-Hydroxytryptamine (5-HT) Receptors Are Involved in the Melittin-induced Nociceptive Responses in Rat I. Role of Peripheral 5-HT Receptor

Melittin-induced tonic pain model is characterized by local inflammation, edema, spontaneous flinchings, and sustained mechanical hypersensitivity. These nociceptive responses are mediated through selective activation of capsaicin-sensitive primary afferent fibers by melittin. The present study was undertaken to elucidate the role of peripheral 5-hydroxytryptamine (5-HT) receptors in the melittin-induced nociceptive responses. Changes in mechanical threshold, flinching behaviors and paw thickness were measured in rat intraplantarly injected with melittin (40microgram/paw) alone or treated together with melittin and 5-HT receptor antagonists. WAY-100635 (100microgram & 200microgram/paw), isamoltane hemifumarate (100microgram & 200microgram/paw), methysergide maleate (60microgram, 120microgram & 200microgram/paw) and ICS-205,930 (100microgram & 200microgram/paw) were intraplantarly injected 20 min before melittin injection. All 5-HT receptor antagonists tested in this experiment significantly attenuated the ability of melittin to reduce mechanical threshold and to induce flinching behaviors. 5-HT receptor antagonists, except ICS-205,930, had mild inhibitory effect on melittin-induced edema. These experimental findings suggest that multiple peripheral 5-HT receptors are involved in the melittin-induced nociceptive responses.

Effects of 3-aminobenzamide (3-AB) on the alterations in primary afferent and spinal neurons induced by spinal nerve injury in rats / 대한정형외과연구학회지

PURPOSE: The authors studied the effect of the 3-AB, an inhibitor of poly(ADP-ribose)polymerase (PARP), on the changes of primary afferents and spinal cord after spinal nerve injury. METHOD: The L5 and L6 spinal nerve of the rats were cut, and 3-AB (10 mg/Kg) was injected intraperitoneally once per day. The animals were sacrificed 3 days, 7 days, 14 days and 28 days after nerve injury. Binding of isolectin B4 (IB4) and immunohistochemistry (IHC) of CGRP for the changes in primary afferents, IHC of NK1 for sensory neurons, and of cleaved caspase 3 and NeuN for the apoptotic changes in spinal neurons were performed. RESULT: Decreased binding of IB4 and immunoreactivity (IR) for CGRP, increase of IR for NK1, and cleaved caspase 3 in both neurons and glia in ipsilateral dorsal horn were observed after spinal nerve injury. These changes were attenuated, especially at between 3 days and 14 days, by administration of 3-AB. CONCLUSION: It is suggested that inhibition of PARP by 3-AB may attenuate alterations of primary afferents and spinal neurons, at least in early stage, after spinal nerve injury.

Effects of 3-aminobenzamide (3-AB) on the alterations in primary afferent and spinal neurons induced by spinal nerve injury in rats / 대한정형외과연구학회지

PURPOSE: The authors studied the effect of the 3-AB, an inhibitor of poly(ADP-ribose)polymerase (PARP), on the changes of primary afferents and spinal cord after spinal nerve injury. METHOD: The L5 and L6 spinal nerve of the rats were cut, and 3-AB (10 mg/Kg) was injected intraperitoneally once per day. The animals were sacrificed 3 days, 7 days, 14 days and 28 days after nerve injury. Binding of isolectin B4 (IB4) and immunohistochemistry (IHC) of CGRP for the changes in primary afferents, IHC of NK1 for sensory neurons, and of cleaved caspase 3 and NeuN for the apoptotic changes in spinal neurons were performed. RESULT: Decreased binding of IB4 and immunoreactivity (IR) for CGRP, increase of IR for NK1, and cleaved caspase 3 in both neurons and glia in ipsilateral dorsal horn were observed after spinal nerve injury. These changes were attenuated, especially at between 3 days and 14 days, by administration of 3-AB. CONCLUSION: It is suggested that inhibition of PARP by 3-AB may attenuate alterations of primary afferents and spinal neurons, at least in early stage, after spinal nerve injury.

Comparative Study on the Nociceptive Responses Induced by Whole Bee Venom and Melittin

The present study was undertaken to confirm whether melittin, a major constituent of whole bee venom (WBV), had the ability to produce the same nociceptive responses as those induced by WBV. In the behavioral experiment, changes in mechanical threshold, flinching behaviors and paw thickness (edema) were measured after intraplantar (i.pl.) injection of WBV (0.1 mg & 0.3 mg/paw) and melittin (0.05 mg & 0.15 mg/paw), and intrathecal (i.t.) injection of melittin (6microgram). Also studied were the effects of i.p. (2 mg & 4 mg/kg), i.t. (0.2microgram & 0.4microgram) or i.pl. (0.3 mg) administration of morphine on melittin- induced pain responses. I.pl. injection of melittin at half the dosage of WBV strongly reduced mechanical threshold, and increased flinchings and paw thickness to a similar extent as those induced by WBV. Melittin- and WBV-induced flinchings and changes in mechanical threshold were dose- dependent and had a rapid onset. Paw thickness increased maximally about 1 hr after melittin and WBV treatment. Time-courses of nociceptive responses induced by melittin and WBV were very similar. Melittin-induced decreases in mechanical threshold and flinchings were suppressed by i.p., i.t. or i.pl. injection of morphine. I.t. administration of melittin (6microgram) reduced mechanical threshold of peripheral receptive field and induced flinching behaviors, but did not cause any increase in paw thickness. In the electrophysiological study, i.pl. injection of melittin increased discharge rates of dorsal horn neurons only with C fiber inputs from the peripheral receptive field, which were almost completely blocked by topical application of lidocaine to the sciatic nerve. These findings suggest that pain behaviors induced by WBV are mediated by melittin-induced activation of C afferent fiber, that the melittin- induced pain model is a very useful model for the study of pain, and that melittin-induced nociceptive responses are sensitive to the widely used analgesics, morphine.

Carrageenan-Induced Hyperalgesia Is Partially Alleviated by Endomorphin-1 Locally Delivered into Inflamed Paws In Rat

This study was performed to test whether endomorphin-1 has analgesic effect, when locally administrated into inflamed peripheral tissue. Carrageenan suspension (0.5%) was injected intraplantarly into the right paw of Sprague-Dawley male rats, and the rats were subjected to a series of mechanical stimuli with von Frei filaments before and after the injection. Carrageenan-injected rats showed typical inflammatory hyperalgesic signs and decrease of withdrawal threshold, peaked at 3 to 6 hours after the injection and lasted more than 3 days. Endomorphin-1 was intraplantarly injected with carrageenan, simultaneously or 3~4 hours after carrageenan. Simultaneous injection of endomorphin-1 with carrageenan significantly reduced hyperalgesia and thd analgesic effect was prolonged up to 8 hours. The delivery of endomorphin-1 (50 microgram) into the inflamed area after 3 to 4 hours of carrageenan injection significantly increased the threshold of hyperalgesic mechanical withdrawal response, but only partially. Intrathecal treatment of endomorphin-1 completely reversed carrageenan-induced hyperalgesia. This report is the first to show that peripherally delivered endomorphin-1 relieved inflammatory hyperalgesia. But a control through peripheral mu-opioid receptors appears to be not sufficient for complete pain treatment.

Antagonists of NMDA Receptor, Calcium Channel and Protein Kinase C Potentiate Inhibitory Action of Morphine on Responses of Rat Dorsal Horn Neuron

he present study was designed to examine whether the co-application of morphine with Ca2+ channel antagonist (Mn2+, verapamil), N-methyl-D-aspartate (NMDA) receptor antagonist (2-amino-5-phosphonopentanoic acid[AP5], Mg2+) or protein kinase C inhibitor (H-7) causes the potentiation of morphine- induced antinociceptive action by using an in vivo electrophysiological technique. A single iontophoretic application of morphine or an antagonist alone induced weak inhibition of wide dynamic range (WDR) cell responses to iontophoretically applied NMDA and C-fiber stimulation. Although there was a little difference in the potentiating effects, the antinociceptive action of morphine was potentiated when morphine was iontophoretically applied together with Mn2+, verapamil, AP5, Mg2+ or H-7. However, the potentiating action between morphine and each antagonist was not apparent, when the antinociceptive action evoked by morphine or the antagonist alone was too strong. These results suggest that the potentiating effect can be caused by the interaction between morphine and each antagonist in the spinal dorsal horn.

Effects of Norepinephrine on the Mechanoreceptors of the Urinary Bladder in the Cat / 대한마취과학회지

BACKGROUND: Recent findings suggest that a coupling between the somatic and sympathetic nervous system is critical not only for the development but also for the maintenance of pain behavioral changes. However, studies on the effect of sympathetic efferent system on sensory receptors in the visceral organ that is more dependent on the autonomic nervous system are lacking. This study examined whether norepinephrine (NE) had an influence on the mechanoreceptors in the feline urinary bladder. METHODS: Ten adult male cats were used and anesthetized with alpha-chloralose and artificially ventilated. A cannula with the pressure transducer was inserted through the urethra to apply mechanical stimuli and monitor the pressure of bladder. A tiny cannula inserted into the bilateral side branches of vesical arteries were used as a route for a NE (10A.M 9:40 01-10-08 bilaterally) injection. Nerve fiber recordings were obtained from the distal stump of the pelvic nerve. RESULTS: After the NE injection, the response of mechanoreceptors (n = 13) to the isotonic pressure stimulus (50 - 60 mmHg) decreased significantly (p < 0.05) in terms of sensitivity (i.e., ratio of nerve activity change to urinary bladder pressure change). The responses to pressure stimuli after an injection of an alpha1 adrenoceptor blocker (terazosin) reversed the effect of NE. The responses of mechanoreceptors to isotonic pressure stimulus were not affected significantly by NE with preinjection of an alpha2 adrenoceptor blocker (yohimbine). CONCLUSIONS: These results suggest that NE may have influence on the sensitivity of mechanoreceptors in the normal feline urinary bladder via an alpha1 adrenoceptor.

Increased response of hypogastric nerve fibers to bradykinin by mustard oil-induced uterine inflammation in the rat

It is well known that the inflammation of somatic tissues, bladder and colon can alter the sensitivity of primary afferents innervating these tissues. To see if uterine afferents also show altered sensitivity, we examined their responses to the algesic agent bradykinin before and after induction of uterine inflammation. Inflammation was induced by injecting the mustard oil into the uterine lumen of adult female rats. After induction of inflammation, the response latency to bradykinin did not change, but the duration and peak of the response and integrated impulse discharges during the response period increased significantly. Furthermore, after inflammation, the level of resting discharges of the afferents was much higher. These results are consistent with the idea that the inflammation can sensitize the uterine afferents.

Alterations in substance P and CGRP immunoreactivities in the uterus following the induction of inflammation in the rats / 대한산부인과학회잡지

OBJECTIVE: Recently some reports suggested substance P and CGRP might be important factors for inflammation and hyperalgesia. This study was performed to see whether substance P or CGRP containing nerve fibers might be changed by mustard oil-induced inflammation. METHODS: After injection of mustard oil(5%) into uterine lumen, the uteri were removed and examined with immunohistochemical methods for substance P and CGRP. RESULTS: In the normal uterus, most of the substance P- or CGRP-immunoreactive nerve fibers were observed along the vascular structure and some in the myometrium, only few in the endometrium. Mustard oil did not changed this pattern of nerve fiber distribution but after 48 hrs, the amount of substance P or CGRP immunoreactive nerve fibers were greatly reduced compared with the normal uterus. It is not clear whether the decrease of substance P and CGRP immunoreactive fibers in the uterus was resulted from the depletion of the neuropeptides in the nerve fibers or the retraction of nerve fibers. CONCLUSIONS: These results suggest that the inflammation should cause the change of nerve fibers included in the nociception. This change may attribute the generation of inflammation and inflammatory hyperalgesia.

Electrophysiological study on the modulation of uterine mechanoreceptor by substance P / 대한산부인과학회잡지

OBJECTIVE: In inflammation, hyperalgesia is a common phenomenon but its mechanism has not been clarified. Recently some reports suggested substance P might be important factors for inflammatory hyperalgesia in somatic tissue. This study was performed to see whether substance P modulate the activities of uterine afferent fibers in the hypogastric nerve of the cat. METHODS: While recording the electrical activities of nerve fibers, mechanical stimuli were applied as balloon distention using balloon inserted into uterine lumen before and during substance P infusion through uterine artery. RESULTS: Substance P increased the responses to balloon distension of uterus in 14 uterine mechanoreceptive afferent fibers of 24 over 10% compared to before substance P infusion, and decreased the responses of 3. And L-703,606, the neurokinin 1 receptor antagonist failed the modulation of mechano sensitive response by substance P and reduced the spontaneous activities. CONCLUSIONS: These results suggest that substance P modulated the activities of uterine nerve fibers and their responses to mechanical stimulus. It is hypothesized that this kind of modulation of afferent nerve fibers by substance P may be important for the development of inflammatory hyperalgesia.

Identification of mutagenic site of c-H-ras oncogene damaged by N-acetoxyacetylaminofluorene(AAAF)

A molecularly cloned human cellular H-ras (c-H-ras) oncogene(pbc N1 plasmid) was treated with N-acetoxyacetylaminofluorene (AAAF) in vitro and subcloned into E.coli. This was done to identify the mutational changes at specific codons of the gene. Guanine nucleotides were identified as the major AAAF binding site of the DNA adduct formed. Base changes in codons 12 and 61 were determined by the analysis of restriction fragment length polymorphism (RFLP) and site specific oligonucleotide hybridization. RFLP was observed due to the loss of the Hpall recognition site at codon 11 and 12 of AAAF-treated c-H-ras gene. Hybridization of AAAF treated c-H-ras with 32P-labeled oligonucleotide probes for the mutant alleles of codon 61 showed no substitutions at codon 61. From these results, it is assumed that AAAF treatment in vitro caused mutation at codon 12 but not at codon 61 of the c-H-ras oncogene and that codon 12 is the primary target of mutation by AAAF

Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. I. Purification and some properties of AP DNA endonucleases in rat liver chromatin

Three kinds of apurinic/apyrimidinic (AP) DNA endonuclease, APcI, APcII, APcIII, were purified from rat liver chromatin through 1M KCl extraction, DEAE-trisacryl ion exchange chromatography. Sephadex G-150 gel filtration and AP DNA cellulose affinity chromatography. Activities of the purified APcI, APcII and APcIII were 62.5, 83.3 and 52.0 EU/mg of protein, respectively. Molecular weights of APcI, APcII and APcIII, each consisting of a single polypeptide, were 30,000, 42,000 and 13,000, and isoelectric points of them were 7.2, 6.3 and 6.2, respectively. Three enzymes showed different substrate specificities; APcI acted only on AP DNA, and APcII acted on both AP DNA and UV DNA, while APcIII acted on 3'-methyl-4-monomethylaminoazobenzene (3'-Me MAB) DNA adduct as well as AP DNA and UV DNA. These results indicate that three kinds of AP DNA endonuclease present in rat liver chromatin have structural and functional diversities.

Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. II. Kinetic properties of AP DNA endonucleases in rat liver chromatin

An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.