Homologous Assay Development for Detection of Dexamethasone using HRP as Label Enzyme

Background: Dexamethasone is a synthetic corticosteroid similar to cortisol produced naturally by the adrenal glands. As an anti- inflammatory and immunosuppressive agent, it is used in many diseases such as rheumatoid arthritis and allergic anaphylactic shock, and its suppression test to diagnose Cushing's syndrome. Its further use includes its administration before antibiotics in bacterial meningitis, antitumor treatment, for treatment of glucocorticoid resistance, Addison抯 disease, and congenital adrenal hyperplasia. The drug is abused by using it in animal husbandry as a growth promoter and in horse sports to enhance their performance. Methods: In this study, the development of homologous ELISA using Dexamethasone-21-hemisuccinate (DEX-21-HS)-Bovine serum albumin antiserum and Dexamethasone-21-hemisuccinate (DEX-21-HS)-Horseradish peroxidase enzyme conjugate has been done. The n-hydroxysuccinimide ester method was used to prepare the immunogen and enzyme conjugate. Results: The sensitivity 0.25 ng/mL, affinity 2.8x10-8 L/mol and ED50 4.98 ng/mL of the assay were found. The cross-reactivity of the assay was checked and found with three steroids (Corticosterone- 1.13%, Progesterone- 2.25% and Prednisolone- 6.3%) out of 48 structurally related steroids. Then, analytical variables of the developed assay were studied, such as recovery (98.55% to 105.08%), precision (Inter and Intra- assay coefficient of variation <9.28%), correlation (R2= 0.98) by utilizing a commercially available Dexamethasone kit for comparison. Conclusion: This study concluded that low-cost indigenous ELISA for Dexamethasone had been developed, which can give results within 75-80 minutes.

Heme peroxidases are responsible for the dehydrogenation and oxidation metabolism of harmaline into harmine / 中国天然药物

Harmaline and harmine are β-carboline alkaloids with effective pharmacological effects. Harmaline can be transformed into harmine after oral administration. However, enzymes involved in the metabolic pathway remain unclear. In this study, harmaline was incubated with rat liver microsomes (RLM), rat brain microsomes (RBM), blood, plasma, broken blood cells, and heme peroxidases including horseradish peroxidase (HRP), lactoperoxidase (LPO), and myeloperoxidase (MPO). The production of harmine was determined by a validated UPLC-ESI-MS/MS method. Results showed that heme peroxidases catalyzed the oxidative dehydrogenation of harmaline. All the reactions were in accordance with the Hill equation. The reaction was inhibited by ascorbic acid and excess H2O2. The transformation of harmaline to harmine was confirmed after incubation with blood, plasma, and broken blood cells, rather than RLM and RBM. Harmaline was incubated with blood, plasma, and broken cells liquid for 3 h, and the formation of harmine became stable. Results indicated an integrated metabolic pathway of harmaline, which will lay foundation for the oxidation reaction of dihydro-β-carboline. Moreover, the metabolic stability of harmaline in blood should not be ignored when the pharmacokinetics study of harmaline is carried out.

Rapid and sensitive high-throughput determination method for alanine-dependent transaminase / 中国药科大学学报

@#A new method for determining transaminase activity based on the color change of the reaction solution was established, by using alanine-dependent transaminase VfTA from Vibrio fluvialis JS17 as the research object coupled with pyruvate oxidase and horseradish peroxidase. After the optimization of the conditions, the linear relationship between VfTA activity units and the absorbance at 400 nm was investigated. This method was also applied to determine the activity of commercial transaminase ATA117. The results showed that the detection limit of transaminase VfTA activity was up to 0. 45 U/mL and the detection limit of ATA117 activity was up to 0. 5 U/mL. The transaminase activity could be quickly judged according to the color depth of the reaction solution.

Electrochemical Immunosensor for Microcystin-LR Detection Based on Nano-composite Material Immobilization and Enzymatic Amplification / 分析化学

A competitive electrochemical immunosensor based on the nano-composite material immobilization and enzymatic amplification was designed for detection of microcystin-LR. Gold nanoparticles/carboxylated multi-walled carbon nanotubes (AuNPs/c-MWCNTs) composite film, which formed by electrodepositing of AuNPs on the C-MWCNTs modified glassy carbon electrode,was used for the immobilization of the antibody of microcystin-LR (anti-MCLR). Horseradish peroxidase (HRP) was introduced onto the nanocomposite interface by HRP blocked sensing interface and specific capture of antibody with target. It could be employed to catalyze the reduction of H2O2, and to block the possible remaining active sites as well. A competitive immunoreaction between antigen and MCLR-HRP was used for target analysis. In the presence of H2O2and hydroquinone (HQ),MCLR could be indirectly detected with differential pulse voltammetry (DPV) method by determining the reduction current of HQ. Under the optimal conditions, the proposed immunosensor exhibited wide linear ranges in the concentration ranges of 0.1-100.0 μg/L, with a detection limit of 0.038 μg/L (S/N = 3,n=8). The immunosensor showed good specificity, stability and sensitivity. It was used to determine MCLR in real water samples with the recoveries of 72.9%-117.3%.

Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles

Background: Iron magnetic nanoparticles have attracted much attention. They have been used in enzyme immobilization because of their properties such as product is easily separated from the medium by magnetic separation. The present work was designed to immobilize horseradish peroxidase on Fe3O4 magnetic nanopraticles without modification. Results: In the present study, horseradish peroxidase (HRP) was immobilized on non-modified Fe3O4 magnetic nanoparticles. The immobilized HRP was characterized by FT-IR spectroscopy, scanning electron microscopy, and energy dispersive X-ray. In addition, it retained 55% of its initial activity after 10 reuses. The optimal pH shifted from 7.0 for soluble HRP to 7.5 for the immobilized HRP, and the optimal temperature shifted from 40°C to 50°C. The immobilized HRP is more thermostable than soluble HRP. Various substrates were oxidized by the immobilized HRP with higher efficiencies than by soluble HRP. Km values of the soluble and immobilized HRP were 31 and 45 mM for guaiacol and 5.0 and 7.0 mM for H2O2, respectively. The effect of metals on soluble and immobilized HRP was studied. Moreover, the immobilized HRP was more stable against high concentrations of urea, Triton X-100, and isopropanol. Conclusions: Physical immobilization of HRP on iron magnetic nanoparticles improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent, and water-miscible organic solvent.

Chemiluminescence Immunoassay for Quantitative Analysis of Prostate Specific Antigen Complexed toα1-Antichymotrypsin in Human Serum / 分析化学

Eight mouse hybridoma cell lines which stably secreted monoclonal antibodies ( McAbs ) against human prostate-specific antigen-α1-antichymotrypsin complex ( PSA-ACT ) were obtained through hybridoma technique. After purification, the immunological characters of 8 McAbs were identified and classified by epitopes analysis through indirect enzyme-linked immunosorbent assay ( ELISA) . A pair of McAbs was chosen from above 8 McAbs, based on which a highly sensitive, simple and rapid chemiluminescence enzyme immunoassay ( CLEIA) was developed for determination of PSA-ACT in human serums using the lumino-H2 O2 reaction catalyzed by horseradish peroxidase ( HRP) as the chemiluminescence system. Several experiment factors such as coating buffer, coating concentration, dilution ratio of PSA-ACT-HRP complex, incubation time, immunoreaction protocol and chemiluminescence reaction time were optimized. The results showed that the linear range of the proposed method for PSA-ACT determination was 0-40 ng/mL (R2=0. 9943), with the detection limit of 0. 53 ng/mL. The inter-assay relative standard deviations (RSDs) were 4. 6%-6. 6%, and intra-assay RSDs were 5 . 7%-8 . 0%. The recoveries of PSA-ACT at three spiked levels in serum samples were 95. 4%-104. 2%. The proposed method exhibited a cross-reactivity of 0. 6% with free-PSA. The proposed method is stable, sensitive, rapid and simple, and provides a foundation for the development of PSA-ACT CLEIA kit and shows great value in clinical auxiliary diagnosis of prostate cancer.

Establishment of double antibody sandwich ELISA for pro-gastrin releasing peptide and its application / 医学研究生学报

Objective The value of pro-gastrin releasing peptide ( PGRP) which is the tumor marker of small cell lung canc-er has become a hot topic in recent years .The research was to build a new enzyme-linked immune sorbent assay ( ELISA) method ai-ming at detecting the concentration of PGRP in patients′serum. Methods We utilized synthetic PGRP epitopes for the screening of the monoclonal antibodies , labeled the screened monoclonal antibodies with horseradish peroxidase by modified sodium iodide method , and then established double antibody sandwich ELISA which could be used to detect the serum concentrations of PGRP in cancer pa -tients. Results We successfully screened E 12 mAb which could be served as the coating antibody and ED 1 mAb as the labeled anti-body.The standard antibody density range of new ELISA was 33 pg/mL~1.7 ×104 pg/mL.The comparison experiments between our method and the commercially available ELISA kit showed no significant difference ( P>0.05).The specificity of our method was 50%, and the sensitivity was 100%, while IBL kit was 92.2% and 100% respectively. Conclusion New ELISA can be used to detect the serum PGRP concentration in patients with small cell lung cancer .

The brainstem localization of gastric preganglionic parasympathetic neurons in the Agouti (Dasyprocta leporina) / Localización del tronco encefálico de las neuronas parasimpáticas preganglionares gástricas en el agutí (Dasyprocta leporina)

This study was designed to determine qualitatively, the source of gastric vagal nerve fibres in the Agouti. A total of 18 male and female adult agoutis were used for the present investigation. Following anaesthesia, laparotomy was performed and the stomach exteriorized. Multiple intramuscular injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were then made into different areas of the stomach in the experimental animals. The control animals were divided into four groups of two animals each. The first group had intraperitoneal injection of the tracer, the second had intramuscular injection of normal saline, the third group had injection of tracer into the hepatic portal vein and the last group had injection of the tracer into the gastric walls followed immediately by bilateral vagotomy. Following a survival period offive to seven days, the animals were sacrificed by transcardial perfusion, first with normal saline followed by fixative and finally with 20% buffered sucrose. Following perfusion, the brainstem was extracted from the brain, immersed in 20% buffered sucrose and kept refrigerated overnight for cryoprotection. The brainstems were subsequently sectioned serially, processed for WGA-HRP neurohistochemistry and then analysed under light and dark-field illuminations. The analysis of the sections taken from the experimental animals revealed bilateral presence of WGA-HRP labelled neurons in the dorsal motor nucleus of the vagus nerve (DMNV) and the nucleus ambiguus (nA) of the medulla oblongata. No labelled neurons were seen in any of the sections taken from the control animals. The implications of the findings are discussed.

Este estudio fue diseñado para determinar cualitativamente el origen de las fibras gástricas del nervio vago en el agutí. Un total de 18 agutíes adultos masculinos y femeninos fueron utilizados para la presente investigación. Después de la anestesia, se realizó una laparotomía y se sacó el estómago al exterior. Luego se hicieron múltiples inyecciones intramusculares de aglutinina de germen de trigo con peroxidasa de rábano (WGA-HRP) en diferentes áreas del estómago de los animales experimentales. Los animales del control fueron divididos en cuatro grupos de dos animales cada uno. Al primer grupo se le puso una inyección intraperitoneal del marcador; al segundo se le administró una inyección intramuscular de solución salina normal; al tercer grupo se le inyectó el marcador en la vena porta hepática; y al último grupo se le puso la inyección del marcador en las paredes gástricas, seguida inmediatamente por una vagotomía bilateral. Tras un periodo de supervivencia de cinco a siete días, los animales fueron sacrificados por perfusión transcardíaca, primero con solución salina normal, seguida de fijador, y finalmente con sacarosa tamponada al 20%. Después de la perfusión, el tronco encefálico fue extraído del cerebro, inmerso en sacarosa tamponada al 20%, y mantenido en refrigeración durante la noche para su crioprotección. Los tronos encefálicos fueron luego seccionados en serie, procesados para para el análisis neuro-histoquímico mediante aglutinina de germen de trigo con peroxidasa de rábano, y analizados entonces bajo iluminaciones de campo de luz y campo oscuro. El análisis de las secciones tomadas de animales experimentales reveló la presencia bilateral de neuronas etiquetadas WGA-HRP en el núcleo motor dorsal del nervio vago (DMNV) y en el núcleo ambiguo (nA) de la médula oblonga. No se observaron neuronas etiquetadas en ninguna de las secciones tomadas de los animales de control. Se discuten las implicaciones de los hallazgos.

Kinetics of enzymatic modification of quercetin with cysteine by horseradish peroxidase.

The kinetic mechanism of enzymatic modification of flavonol quercetin with L-cysteine by horseradish peroxidase (HRP) was studied. Reaction of modification of quercetin was followed by recording spectral changes over time at 380 nm. All reactions were performed in 100 mM phosphate buffer pH, 6.0 at 20ºC. Kinetic parameters were determined from graphics of linear Michaelis-Menten equation. The values obtained at specified intervals were: Vmax = 0.17 ÷ 0.91 ΔA380/min, Km = 0.023 ÷ 0.5 mM, kcat = 0.21 ÷ 1.14 ΔA380/min∙nM-1 and Vmax/Km = 0.83 ÷ 26.55 ΔA380/min∙mM-1. It was found that all investigated reactions of the modification of quercetin with L-cysteine by HRP followed an ordered mechanism. We propose that HRP initially reacts with H2O2 than with quercetin and finally with L-cysteine, leading to the introduction of L-cysteine in the structure of quercetin.

Efecto de la peroxidasa sobre la resistencia de unión de una resina compuesta al esmalte dental posblanqueamiento / Effect of peroxidase on composite resin adhesion to dental enamel after whitening

Introducción: se ha reportado que el oxígeno residual liberado por los agentes blanqueadores interfieren en la adhesión de las resinas compuestas a la estructura dental, razón por la cual se tiene como objetivo comparar la resistencia de unión al corte (RUC) de una resina compuesta al esmalte dental posblanqueamiento con peróxido de hidrógeno al 38%, antes y después de tratar la superficie con la enzima peroxidasa previo a la adhesión. Métodos: se seleccionaron 45 premolares humanos sanos, divididos entres grupos de 15 dientes cada uno. Grupo 1: control (solo adhesión); grupo 2: blanqueamiento y adhesión; grupo 3: blanqueamiento, aplicación de peroxidasa y adhesión. Posterior al tratamiento se midió la RUC en la máquina de ensayos Shimadzu, para determinar diferencia estadísticamente significativa entre los tres grupos con nivel de confianza de 95% y con valor de p < 0,05. Resultados: el grupo control obtuvo la RUC de 12,8 Mpa (± 3,2), el grupo con blanqueamiento tuvo el promedio de 3,5 Mpa (± 1,43) y el grupo con blanqueamiento y aplicación de peroxidasa presentó promedio de 12,2 Mpa (± 3,12). Conclusiones: los valores de RUC disminuyeronsignificativamente con la aplicación de peróxido de hidrógeno al 38%, sin embargo, se logró el aumento significativo al aplicar la peroxidasa previa a la adhesión.

Introduction: some reports suggest that residual oxygen released by whitening agents interfere with composite resinadhesion to dental structure. The objective of this study is therefore to compare composite resins’ shear bond strength to dental enamel after whitening using 38% hydrogen peroxide pre- and post- treatment with peroxidase enzyme before adhesion. Methods: a total of 45 healthy human premolars were selected and divided into three groups of 15 teeth each. Group 1: control group (only adhesion); group 2: whitening and adhesion; group 3: whitening, application of peroxidase, and adhesion. After treatment, shear bond strength was measured with a Shimadzu testing machine in order to determine significant statistical differences among the three groups, with a confidence level of 95% and p < 0.05. Results: the control group obtained a shear bond strength of 12.8Mpa (± 3.2), the whitening group showed an average 3.5 Mpa (± 1.43), and the group treated with whitening and peroxidase presented an average 12.2 Mpa (± 3.12). Conclusions: shear bond strength values significantly decreased with application of 38% hydrogen peroxide; however, a significant increase was also obtained byapplying peroxidase before adhesion.

New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA / 药物分析学报

Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA).The procedure used for HRP detection in EIA is critical for sensitivity and precision.This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate.The principle of the assay is as follow:sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol.The dimer is fluorescent and can be detected sensitively at ex.347 nm,em.427 nm.The measurable range of HRP was 1.0 × 10-18 to 1.0 × 10-15 mol/assay,with a detection limit of 1.0 × 10-18 tmol/assay.The coefficient of variation (CV,n=8) was examined at each point on the standard curve,with a mean CV percentage of 3.8％.This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae)

Astilbin (5,7,3’,4’-tetrahydroxy-2,3-dihydroflavonol-3-ß-o-rhamnoside), a flavonoid with a large range of biological activities, was isolated from Dimorphandra mollis, a shrub common to the Brazilian Cerrado. The purpose of this study is to verify the effects of astilbin on myeloperoxidase (MPO) and horseradish peroxidase (HRP), and its antioxidant activity against hypochlorous acid (HOCl) and total antioxidant activity (TAC) by the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+). Astilbin inhibited MPO and HRP activities in a concentration-dependent relationship and effectively scavenged HOCl. The TAC by ABTS•+ of astilbin (IC50 ~ 20 mM) was higher than that of uric acid, which was used as a positive control. These data demonstrate that astilbin is a potent antioxidant and that it inhibits MPO and HRP activities efficiently.

2-Amino-3-hydroxylpyridinE-H2O2-Horseradish Peroxidase Voltammetric EnzymE-Linked Immunoassay for Detection of Carcionembryonic Antigen in Human Serum / 分析化学

2-Amino-3-hydroxylpyridinE-H2O2-horseradish peroxidase (HRP) voltammetric enzymE-linked immunoassay based on N-heterocyclic substrate has been successfully applied for the detection of carcionembryonic antigen(CEA) in human serum. 2-Amino-3-hydroxylpyridine is oxidized with H2O2 catalyzed by HRP, and the resulting electroactive product produces a sensitive voltammetric peak at the potential of 0.36 V(vs. SCE) in Britton-Robinson (B-R) buffer solution. By this voltammetric peak, free HRP can be measured and immunoassay of HRP label can be developed. The enzymE-catalyzed reaction conditions and voltammetric detection conditions have been optimized, and the electrode procedure of the enzymatic product was investigated. The selected optimum reaction conditions were that the reaction medium was pH 6.0 B-R buffer solution for 10 mL reaction solution containing 1.0 mL of 0.2 mol/L B-R buffer solution, 3.0 mL of 8.0 mmol/L 2-amino-3-hydroxylpyridine solution and 1.5 mL of 0.5 mmol/L H2O2 solution, and the reaction time was 30 min at 37 ℃. The optimum detection conditions were that the supporting electrolyte was pH 7.0 B-R buffer solution for 10 mL of the overall detection solution containing 5 mL of reaction solution and 1.0 mL of 0.2 mol/L B-R buffer solution. The optimum instrumental conditions for the detection were chosen as follows: the initial potential, 0.00 V; the final potential, -0.80 V; the potential scanning rate, 400 mV/s; the mercury drop standing time, 7 s. Under the optimum conditions, the linear range for detection of free HRP was 4.0×10-4-1.0 μg/L with a detection limit of 0.12 μg/L. Based on the new immunoassay system, the linear range of the detection to CEA was 0.50-80 μg/L and the detection limit was 0.50 μg/L, which is 10 times lower than that of traditional spectrophotometric enzymE-linked immunosorbent assay method. The 2-Amino-3-hydroxylpyridinE-H2O2-HRP voltammetric enzymE-linked immunoassay new system exhibits excellent performance having wider linear range and lower detection limit. The new method is inexpensive and simple. It has a great potential in clinical diagnosis.

Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase / 西安交通大学学报·英文版

Objective: To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods: Human leukemia cell line K562 were exposed to indole-3-acetic acid (IAA) at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase(HRP) at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate (DCFH-DA) uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. Results: IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9%, which was 5 times that of control(P<0.01). Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion: The combination of IAA and HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.

Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase / 药物分析学报

Objective To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods Human leukemia cell line K562 were exposed to indole-3-acetic acid (IAA) at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase(HRP) at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate (DCFH-DA) uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. Results IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9%, which was 5 times that of control(P＜0.01). Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion The combination of IAA and HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.

Projections from respiratory neurons of medulla oblongata to the motor neuron pool of phrenic nerve in rat:An HRP retrograde tracing study / 中国康复理论与实践

@#ObjectiveTo observe the projections from respiratory neurons of medulla oblongata to the motor neuron pool of phrenic nerve in rat. MethodsBy using horseradish peroxidase (HRP) retrograde tracing technique after injecting HRP into phrenic nerve, retrograde labelled neurons were found in C3-C5 segment. Then, HRP was injected into the area where the phrenic motor neurons occupied, retrograde labelled neurons were found in medulla oblongata. ResultsPhrenic motor neurons locate in the C3-C5 segment ipsilaterally, occuping the intermediate portion of anterior horn and appearing typical motor neurons. Retrograde labelled neurons were found bilaterally in medulla oblongata, the neurons were located in nucleus retroambigualis (RNA), ventramedial area to nucleus retrofacialis (NRF). ConclusionThe phrenic motor neurons may receive direct projection of respiratory neuron in medulla oblongata, the projecting neurons are distributed in RNA and NRF area.

Effects of Cervical Sympathetic Nerve on the Axoplasmic Transport of Trigeminal Nerve in Rats / 复旦学报（医学版）

Purpose　To explore the effects of cervical sympathetic nerve on the axoplasmic transport of the trigeminal nerve.　Methods　48 Wistar rats were used with the right side as experimental side and the left side as control side.5 μl of 30% horseradish peroxidare(HRP) solution was injected into the symmetrical areas on both sides of the infraorbital regions.Then 0.4 ml of suspension made up of 0.2 ml 0.5% bupivacaine and 0.2 ml hydroprednisone-A was injected into C5 transverse process on the right side,and 0.4 ml of 0.9% normal saline on the control side.The animals survived for 4,6,8,10,12,14h,and were killed after perfusing through the ascending aorta.The superior cervical sympathetic ganglia and the trigeminal ganglia on both sides frozen sectioned,and treated with TMB method.The HRP labeled cells in the sections were observed under light microscope.The positive labeled cells were classified and counted.The sum of mean area and the mean optical density of HRP labeled cells in superior cervical sympathetic ganglia and the trigeminal ganglia on both sides were analysed by image pattern analysis system.　Results　The labeled cells were found in the trigeminal ganglia of the experimental sides after 6 h,the control side,8 h.The velocity of HRP axoplasmic transport of the experimental side was (5.50±0.95)mm/h,the control side (3.99±0.81)mm/h(P<0.01).The sum of mean area and the product of the sum of mean area and the mean optical density of HRP labeled cells in the trigeminal ganglia of the experimental side were larger than those of the control side (P<0.01).The labeled cells were found in the superior cervical sympathetic ganglia on both sides after 8 h.The sum of mean area and the mean optical density of HRP labeled cells in the superior cervical sympathetic ganglia on the control sides were larger than those of the experimental sides (P<0.01).　Conclusions　Cervical sympathetic nerve can affect the velocity of the axoplasmic transport of the trigeminal nerve.The cervical local block slows accelerates the axoplasmic transport of the cervical sympathetic nerve and the axoplasmic transport of the trigeminal nerve.

Relationship of the Branches of Cervical Dorsal Root Ganglia and Sympathetic Ganglia in Dominating the Zygapophyseal Joints of Rats / 复旦学报（医学版）

Purpose　To study the relationship between sympathetic nerve and the branches of primary sensory neurons in dominating cervical zygapophyseal joints with the HRP retrograde tracing methods.To explore the mechanism of the external intervertebral foramen's cryical nerve compression syndrome with neck-shoulder pain and the symptom of head and face regions.　Methods　8 Wistar rats were used with the right side as experimental side and the left side as control side.5 μl of 30% HRP solution was injected into the C5/C6 cervical zygapophyseal joint capsule of the right side by microinjection syringe and 5 μl of 0.9% normal saline was injected into the left side as control.The animals survived for 48 hours and were killed by perfusing through ascending aorta.The C1-T1 DRG,cervical sympathetic ganglia and trigeminal ganglia on both sides were sectioned by frozen section method,and treated with TMB method.The HRP labeled cells in sections were observed under optical microscope.The classification and count of HRP labeled cells in DRG and cervical sympathetic ganglia on experimental sides were analysed by image pattern analysis system.　Results　There were HRP labeled cells in middle cervical ganglia,inferior cervical ganglia (stellate ganglia) and C5-C7 DRG on experimental sides after HRP injection.Most of the labeled cells were small or middle size.The sum of mean area and the mean optical density of HRP labeled cells were larger in the middle cervical ganglia and C6 DRG than that in inferior cervical ganglia(stellate ganglia) and C5 or C7 DRG separately (P<0.01).There wasn't any HRP labeled cell in C1-T1 DRG of control side and in trigeminal ganglia.　Conclusions　The cervical zygapophyseal joint mainly dominated by the sensory branches of the three cervical nerves next to it and by the branches of the sympathetic nerves.It may be related to the causing of neck-shoulder pain and the symptoms of head and face regions of patients with the external intervertebral foramen's cervical nerve compression

Localization of sympathetic and sensory nerves innervating heart in the cat using HRP and WGA-HRP as neuronal tracers / 대한해부학회지

The origin of sympathetic and sensory nerves innervating heart in the cat was investigated using HRP (Horseradish peroxidase) and WGA-HRP (Wheat germ agglutinin-horseradish peroxidase) as neuronal tracers. The neural tracers were injected into subepicardial layer and myocardium of the right atrium, left atrium, right ventricle and left ventricle, respectively. Labeled sympathetic neuronal cell bodies were found in superior cervical ganglia, middle cervical ganglia, stellate ganglia and 4th and 5th thoracic ganglia, mainly in middle cervical ganglia and stellate ganglia. Heavier labeled neuronal cell bodies were found in the middle cervical ganglia and stellate ganglia when the neural tracers were injected into left atrium, right ventricle and left ventricle. Labeled sensory neuronal cell bodies were found in nodose ganglia and T1-T6 spinal ganglia, mainly in T1-T5 spinal ganglia. Heavier labeled neuronal cell bodies were found in the nodose ganglia when the neural tracers were injected into left atrium and right ventricle. These results may provide a neuroanatomical data on origin of sensory nerves innervating the heart of the cat.

Origings of Dorsal Root Ganaglion Cells Innervating Anterior and Posterior Cruciate Ligaments of the Rat Knee Joint

PURPOSE: The present study was designed to examine the distribution of dorsal root ganglion(DRG) cells innervating the anterior and posterior cruciate ligaments of the Sprague-Dawley rat knee joint. MATERIALS AND METHODS: Fluoro-gold(FG) was used to identify the distribution of DRG cells innervating the ligaments, and horseradish peroxidase(HRP) was used to measure the DRG cell size innervating the ligaments. RESULTS: Neural tracers-labelled DRG cells were found ipsilaterally only in the lumbosacra1 DRGs. FG-labelled DRG cells innervating the anterior and posterior cruciate ligaments were found from the 1st lumbar DRG to the 1st sacral DOR(L1-Sl). The majority of FG-labelled DRG cells innervating the poste-rior cruciate ligaments were located in the L4, and the majority innervating the anterior cruciate ligaments were found in the L3, The size of HRP-labelled DRG cells innervating the cruciate ligaments was below 800 micromiter (c), showing that these cells were small. CONCLUSION: This study indicates that the DRG origin of sensory nerves is different in each cruciate ligament of the knee joint. But the size and the type innervating each ligament is similar.