Enrichment of miR-17-5p enhances the protective effects of EPC-EXs on vascular and skeletal muscle injury in a diabetic hind limb ischemia model

BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo. METHODS: EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXsmiR-17-5p. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXsmiR-17-5p were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXsmiR-17-5p. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis. RESULTS: In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXsmiR-17-5p was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXsmiR-17-5p could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXsmiR-17-5p were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXsmiR-17-5p could be abolished by a PI3K inhibitor (LY294002). CONCLUSION: Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.

Effect of Thbs4 gene editing BMSCs transplantation on VEGF and Ang-1 in diabetic hind limb ischemia rats and related mechanism / 国际生物医学工程杂志

Objective To investigate the effects and the mechanism of thrombospondin 4 (Thbs4) gene-edited bone marrow mesenchymal stem cells (BMSCs) transplantation on vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in diabetic rats with hind limb ischemia. Methods Thirty Sprague-Dawley rats were randomly divided into the model group, BMSCs treatment group and Thbs4-BMSCs treatment group on average. After constructing the typeⅡdiabetic rat model with hind limb ischemia, 100μl normal saline, BMSCs suspension and Thbs4-BMSCs suspension (cell number: 2×106) were locally injected into the ischemic injury area of rats for the model group, BMSCs group and Thbs4-BMSCs group, respectively. The rats were sacrificed on the 14th day after stem cell transplantation, and the muscle tissues near the ischemic area were collected. The relative expression of VEGF and p-Smad2/3 protein was detected by Western Blot. The Ang-1 protein expression was detected by immunofluorescence staining. The levels of related genes were detected by qRT-PCR, and the von Willebrand Factor (vWF) protein expression was detected by immunohistochemistry staining. Results The relative expression levels of VEGF, Ang-1 and vWF protein in the Thbs4-BMSCs group were significantly higher than those in the model group and BMSCs group (VEGF protein:P<0.01 and P<0.05). The mRNA expression of VEGF and Ang-1 were significantly up-regulated, the differences were statistically significant(VEGF mRNA:all P<0.01;Ang-1:P<0.01 and P<0.05). The expression of p-Smad2/3 protein in the Thbs4-BMSCs group was significantly higher than that in the model group and the BMSCs treatment group (all P<0.01). The expression of p-Smad2/3 protein was significantly decreased after the addition of p-Smad2/3 pathway inhibitor, the differences were statistically significant (P<0.05). Conclusions Thbs4-BMSCs transplantation can effectively promote angiogenesis in diabetic rats with hind limb ischemia, and the effect of angiogenesis may be related to the activation of Smad2/3 signaling pathway.

Study of anticonvulsant activity of acetazolamide on albino rats and its influence on anticonvulsant activity of sodium valproate

Background: Epilepsy is a common neurological disorder. 30-40% of patients will continue to have seizures despite the use of antiepileptic drugs either alone or in combination. The present study is undertaken to evaluate the anticonvulsant activity of Acetazolamide (ACZ) in albino rats and its influence on anticonvulsant activity of sodium valproate.Methods: Albino rats (150-200gms) of male sex were randomly selected, from central animal facility, MMCRI, Mysore. They were divided into 6groups (per model) of 6 rats each, control group-normal saline 0.5ml, standard group-sodium valproate (300mg/kg), dose 1-ACZ (8.75mg/kg), dose 2-ACZ (17.5mg/kg) and dose 3-ACZ (35mg/kg), dose 4-ACZ (8.75mg/kg) with sodium valproate (150mg/kg). The anti-convulsant activity was screened using MES model and PTZ model.Results: Results were analysed by ANOVA followed by post hoc Fisher’s LSD test. The ACZ has shown anticonvulsant activity at the dose of 17.5mg/kg and 35mg/kg body weight and combination of ACZ 8.75mg/kg with sodium valproate 150mg/kg both in MES model and PTZ model. The anticonvulsant activity of ACZ was less when compared to Sodium Valproate in both MES model and PTZ model. The anticonvulsant activity of combination, ACZ 8.75mg/kg with Sodium valproate 150mg/kg was comparable and more significant when compared to standard drug alone in MES model and PTZ model.Conclusions: The ACZ has shown anticonvulsant activity in MES model and PTZ induced seizure model of epilepsy. This study has shown that ACZ potentiated the effect of sodium valproate and can be used as add on drug with sodium valproate in epilepsy.

Acupoint injection of BMSCs combined with Chinese herbs for capillary density in ischemic hind limb of diabetes mellitus rats / 中国针灸

OBJECTIVE@#To observe the effect of acupoint injection of bone mesenchymal stem cells (BMSCs) combined with Chinese herbs of benefiting for activating blood circulation for capillary density and arterioles density in skeletal muscle in ischemic hind limb of diabetes mellitus (DM) rats.@*METHODS@#A total of 80 rats were randomized into a normal sham operation group (10 rats) and a model group (70 rats). Disposable intraperitoneal injection of streptozotocin (STZ, 50.0 mg/kg) was used to establish DM model, and the rats in the model group were randomized into 7 subgroups, 10 rats in each one. The subgroups were the DM sham operation group, DM ischemic group, Chinese herb group (intragastric herbs of benefiting for activating blood circulation), local injection group (BMSCs local injection), local injection + Chinese herb group (BMSCs local injection combined with intragastric herbs of benefiting for activating blood circulation), acupoint injection group (BMSCs acupoint injection), acupoint injection + Chinese herb group (BMSCs acupoint injection combined with intragastric herbs of benefiting for activating blood circulation). The local injection was phosphate buffer (PBS) injection at the equidistant 5 points along the line between the ischemic tissue and the normal tissue a time. The acupoints were "Sanyinjiao" (SP 6), "Zhaohai" (KI 6), "Huantiao" (GB 30), "Housanli" (ST 36) and "Yanglingquan" (GB 34). 100 μL BMSCs with 1×10/mL was totally injected at the above acupoints for one rat, 20 μL an acupoint. 1.5 kg/L Chinese herbs were applied by intragastric administration, including 120 g Radix Astragali, 120 g Codonopsis, 48 g Radix Glycyrrhiza, 120 g Angelica sinensis, 120 g Blood Rattan, 48 g Achyranthes bidentata. Intragastric distilled water was used in the other non-Chinese herb groups. The expressions of α-smooth muscle actin (α-actin), latelet endothelial cell adhesion molecule (CD31) and von willebrand factor (vWF) in the skeletal muscle were detected with immunohistochemical SP two-step method.@*RESULTS@#Twenty-one days after intervention, the expressions of α-actin and CD31 on the operation hind limb were higher than those on the healthy hind limb in all the groups, except the Chinese herb group (<0.05<0.01). The vWF expressions on the operation side were lower than those on the healthy side in the Chinese herb group, the local injection group, the local injection + Chinese herb group and the acupoint injection + Chinese herb group (<0.05, <0.01). The α-actin expression on the operation side in the acupoint injection + Chinese herb group was higher than those in the normal sham operation group, DM sham operation group, the DM ischemic group and the local injection group (<0.05, <0.01). The CD31 expressions in the acupoint injection group, the acupoint injection + Chinese herb group, local injection + Chinese herb group were higher than those in the normal sham operation group, DM sham operation group and DM ischemic group (<0.05, <0.01). The CD31 expression in the acupoint injection + Chinese herb group was higher than those in the Chinese herb group and the local injection group (both <0.05). The vWF expressions in the local injection + Chinese herb group, the acupoint injection group and the acupoint injection + Chinese herb group lower than those in the DM sham operation group and the DM ischemic group (<0.05, <0.01).@*CONCLUSION@#schemia increases the expressions of the vascular density related factors of α-actin and CD31. It is more obvious for the increasing expressions of α-actin and CD31, and decreasing expression of vWF with the interventions of simple BMSCs injection and simple Chinese herbs of benefiting for activating blood circulation, especially with the combination of the above tow methods. It is indicated that acupoint injection of BMSCs combined with Chinese herbs of benefiting for activating blood circulation can improve the angiogenesis of ischemic tissue.

An outbreak of Australian Stringhalt associated with skin lesions in Colombia / Un brote de Arpeo Australiano asociado con lesiones de piel en Colombia / Um surto de Arpejo Australiano acompanhado de lesões de pele na Colômbia

Summary Anamnesis: eight Silla Argentina breed horses imported from Argentina presented with an abnormal gait and skin lesions. Clinical and laboratory findings: all cases showed different degrees of hind limb hyperflexion, atrophy of the lateral digital muscles, skin lesions, and poor body condition. They underwent several clinical and laboratory examinations, including endoscopy of the upper respiratory tract, skin and liver biopsy, blood count, biochemical profile, and electromyography that showed evidence of a neuropathy and dermatitis. Treatment approach: the horses were treated with phenylbutazone and thiamine. The response was patient dependent with different degrees of recovery resulting. Conclusion: this is the first report of an outbreak of bilateral Australian Stringhalt in Colombia and the first associated to skin lesions according to the veterinary medicine scientific literature.

Resumen Anamnesis: ocho caballos de raza silla Argentina importados de Argentina presentaron un andar anormal y lesiones de piel. Hallazgos clínicos y de laboratorio: todos los animales presentaron hiperflexión de los miembros anteriores de forma bilateral, en diferentes grados de severidad; atrofia del músculo digital lateral, lesiones de piel y condición corporal deficiente. Se realizaron endoscopias, biopsias de piel, hematología, química sanguínea, electromiografías que indicaron una neuropatía y dermatitis. Abordaje terapéutico: los caballos fueron tratados con fenilbutazona y tiamina, con una respuesta variable entre los animales. Conclusión: este es el primer reporte de un brote de arpeo australiano en Colombia y el primero asociado a lesiones de piel de acuerdo a la literatura científica médico-veterinaria.

Resumo Anamnese: oito cavalos de raça Silla Argentina foram importados a partir da Argentina por una marcha anormal e lesões cutâneas. Achados clínicos e de laboratório: todos os animais apresentaram hiperflexão bilateral dos membros anteriores com diferentes graus de gravidade; atrofia do músculo digital lateral, lesões de pele e condição corporal baixa. Foram efectuadas endoscopias, biópsias de pele, hematologia química sanguínea e electromiografias que indicaram uma neuropatia e uma dermatitis. Abordagem terapêutica: os cavalos foram tratados com fenilbutazona e tiamina obtendo uma resposta variável entre os animais. Conclusão: este é o primeiro relatório de arpejo Australiano reportado na Colômbia e o primeiro associado com lesões de pele de acordo com a literatura científica médico-veterinária.

Esqueletogênese em punaré (Thrichomys laurentinus- Rodentia, Echimyidae) / Skeletogenesis in punare (Thrichomys laurentinus - Rodentia, Echimyidae)

O objetivo deste estudo foi descrever o desenvolvimento do esqueleto do punaré (Thrichomys laurentinus). Para tanto, foram utilizados 11 embriões e 12 fetos em diferentes estágios de desenvolvimento, sendo divididos em 4 grupos de acordo com o período gestacional. As amostras foram obtidas no Centro de Multiplicação de Animais Silvestres da Universidade Federal Rural do Semi-Árido, Mossoró-RN, Brasil. Após fixados em formol (10%) ou glutaraldeído (2,5%), foi realizada a analise morfológica com auxílio de lupa, sendo as características macroscópicas fotodocumentadas. Análises de raios-x e coloração por alizarina red foram realizadas para melhor compreensão do desenvolvimento ósseo. Nas análises de raio-x os embriões não apresentaram nenhuma radiopacidade, ao contrário dos fetos que apresentavam radiopacidade gradual ao longo dos grupos. No grupo II houve aumento de radiopacidade na região da coluna vertebral e das regiões mandibular e maxilar. No grupo III a radiopacidade estava aumentada nos membros pélvicos, nas costelas e na região frontal e no grupo IV nos membros torácicos e nas regiões occipital, temporal e frontal do crânio. Tais características foram confirmadas pelas analises histológicas e pela técnica de Alizarina Red. Com isso podemos concluir que o conhecimento acerca da embriologia do sistema ósseo normal é fundamental para o entendimento dos efeitos adversos causados pela nutrição e uso de drogas durante o desenvolvimento.(AU)

The aim of this study was to describe the skeletogenesis in punaré (Thrichomys laurentinus). We used 11 embryos and 12 fetuses in different stages of development, allocated into 4 groups. Samples were obtained from the Multiplication Center of Wild Animals, at Federal Rural University of the Semi-Arid, Mossoro/RN, Brazil. After fixed in formalin (10%) or glutaraldehyde (2.5%) the morphological analysis was performed with a magnifying glass, and the macroscopic characteristics were photographed. Analysis of X-rays and alizarin red staining was made to better understand the development of bone structures. In x-ray analysis, it was possible to verify that the embryos showed no radiopacity, unlike fetuses that had gradual radiopacity along of the groups. In group II, there was an increase in radiopacity in the spine, mandibular and maxillary regions. In group III, the radiopacity was increased in the hind limbs, ribs and in the frontal region, and group IV showed higher radiopacity in the thoracic limbs and occipital, temporal and frontal skull. These characteristics were confirmed by histological and alizarin red analysis. We concluded that the knowledge of normal skeletal embryology is critical for understanding of adverse effects caused by nutrition and use of drugs during the development.(AU)

Effect of Naoxintong capsule on endothelial progenitor cell mobilization and homing following bone marrow transplantation in a mouse hind limb ischemia model / 中国中药杂志

Endothelial progenitor cells (EPCs) are precursor cells of endothelial cells. Signal molecules produced by ischemia and hypoxia can promote mobilization of bone marrow EPCs to peripheral circulation and formation of novel blood vessels in tissues that are damaged during heart attack. Naoxintong capsule (NXT) has the functions of promoting blood circulation, removing blood stasis, promoting the circulation of qi and relieving pain. The various components in NXT have protective effects on blood vessels and can effectively improve the symptoms of ischemia. However, its effect on EPCs is not clear. To study the intervention effect of NXT on mobilization and homing of peripheral blood EPCs, green fluorescent protein (GFP) transgenic mice were used for bone marrow transplantation (BMT) and then unilateral hind limb ischemia model (UHLI) were constructed. For BMT, wild-type ICR mice were irradiated by CS137 and then injected with 4×106 bone marrow cells isolated from GFP mice. The bone marrow reconstitution of recipients was assessed by quantification of GFP bone marrow-derived cells (BMDC) from transplanted mice 4 weeks after BMT. The UHLI model was duplicated by ligating femoral artery and divided into three groups: the model group, the NXT group (model+NXT) and the positive control group (model+simvastatin). Flow cytometry was used to detect the proportion of GFP positive cells and the peripheral blood EPCs levels at 1, 3, 7, 14 days before and after surgery. Ischemic tissue of gastrocnemius muscle was excised at 3 and 7 days after operation for immunofluorescence staining to detect the number of GFP+ cells. The bone marrow chimerism was achieved at day 28 after BMT. There was no significant difference in the percentage of GFP positive cells between BMT mice and GFP transgenic mice. NXT and simvastatin could significantly increase the number of peripheral blood EPCs 1,3 days after surgery. Three and seven days after operation, the number of homing EPCs was significantly higher in NXT group and positive control group than that in model group (P<0.001). In conclusion, NXT can obviously promote the mobilization and homing of EPCs.

S2-1 Effects of CO2-enriched water bath treatment on blood flow and angiogenesis in ischemic hind limb / 日本温泉気候物理医学会雑誌

Objectives: Peripheral artery disease (PAD) is a major health problem; however, no satisfactory intervention is available for its treatment. This study was undertaken to investigate the effects as well as mechanisms of the CO2-enriched water bath (CEWB) treatment on blood flow in the ischemic hind limb. Experimental Model: For inducing PAD, the femoral artery was occluded for 5 weeks in rats. The animals were treated with or without CEWB at 37°C for 4 weeks (20 min daily; 5 days per week) starting one week after the artery occlusion. CEWB was prepared by using Carbothera (Mitsubishi Rayon Engineering Tokyo). The blood flow was measured by Pulse Wave Doppler Ultrasound technique before and after the ligation as well as at the end of 4 weeks treatment. The angiogenesis (formation of new blood vessels) in the skeletal muscle was studied by histological examination. Results: The peak, mean or minimal blood flow was not detected in the untreated ischemic hind limb animals due to arterial ligation. However, the values for blood flow were about 50% of the control values upon treatment with CEWB; 67% of the ligated animals showed positive blood flow by CO2 treatment. Morphological examination of the treated ischemic skeletal muscle revealed a 3-fold increase in small artery count. Although plasma triglycerides were decreased and plasma NO concentration was increased in the ischemic animals, CEWB treatment produced no effects on these parameters. No mortality or changes in body wt, heart rate and plasma glucose, cholesterol or high density lipoproteins were seen in the control and experimental animals. Conclusion: This study demonstrates the beneficial effect of CEWB treatment on blood flow in hind limb PAD. Furthermore, it is suggested that this beneficial action of CO2 therapy may be due to the formation of new blood vessels in the ischemic skeletal muscle.

The relationship between hind limb ischemia of diabetic rats and endothelial nitric oxide synthase uncoupling / 中华内分泌外科杂志

Objective To investigate the relationship between vascular injury of diabetic rats with hind limb ischemia and endothelial nitric oxide synthase(eNOS) uncoupling.Methods 15 SD rats were randomly divided into 3 groups:control group (n =5),diabetes group (n =5) and diabetes ischemia group (n =5).Two weeks after the diabetic rat model and hind limb ischemia of diabetic rat model were set up,Enzyme linked immunosorbent assay(ELISA) was used to detect nitric oxide(NO) expression in the rat femoral artery; Dihydroethidium(DHE) fluorescent probe was used to detect superoxide(O2-) production in the frozen section of femoral artery; and Western blot was used to examine GTP cyclohydrolase 1 (GTPCH-1)expression in femoral artery.Results ①Compared with the control group,NO expression of femoral artery in diabetes group was significantly reduced,diabetes ischemia group decreased more significantly,and the difference between the 3 groups had statistical significance(P =0.000,P =0.000,P =0.001).②Frozen section showed:compared with the control group,O2-expression of diabetes group significantly increased,but not as much as diabetes hind limb ischemia group.③Western blot results showed that compared with the control group,the expression of GTPCH-1 in diabetes group reduced,diabetes hind limb ischemia group decreased more significantly,and the difference between the 3 groups had statistical significance (P =0.001,P =0.000,P =0.012).Conclusion eNOS uncoupling is present in diabetic rats with hind limb ischemia and vascular injury.

The experimental model of severe crush injury in rats by two hindlimbs compression / 中华急诊医学杂志

Objective To investigate a reproducible model of severe crush injury (CI) in rats.Methods A total of 50 clean grade male SD rats were randomly (random number) divided into 5 groups.Both hindlimbs of anesthetized rats were compressed by blocks weighing 3.5 kg,for 6 hours and followed by 3 hours of reperfusion on a specially notched device (group SP,n =10),ordinary compression (group NM,n =10) and simple control (group SHAM,n =10).Arterial tension,serum lactate,and potassium (K+),serum myoglobin (MB),aspartate transferase (AST) and alanine transferase (ALT),BUN and Cr were measured at 10 minutes after cannulaton,and 3 hours after release from compression.Muscles and kidneys were evaluated morphologically.Group D and E were treated in the same way and were observed for 72 h to get the survival rate of the NM group and the specially notched compression group.The SPSS 17.0 statistical software was used for statistical analysis,repeated-measures ANOVA analysis for the differences between groups,Kaplan Meier-estimator for survival analysis.Results The Specially notched compression produced a greater increase in serum lactate (F =39.626,P ＜ 0.05),AST (F =24.965,P ＜ 0.05),ALT (F =19.096,P＜0.05),BUN (F=7.938,P＜0.05),CR (F=14.787,P＜0.05) and MB (F=16.840,P ＜0.05) by the end of experiment than NM group and simple control group.The direct cellular damage and ischemia-reperfusion injury were found under microscope.In crush injury caused by specially notched compression there was acute tubular necrosis found at 24 hours after injury.Mortality rate in the NM group was 20％,whereas mortality rate reached 90％ in rats with specially notched compression (P ＜0.05).Conclusions It successfully developed a severe crush injury model in experimental rats,suggesting it is worthwhile to popularization.

S2-1Effects of CO₂-enriched water bath treatment on blood flow and angiogenesis in ischemic hind limb / 日本温泉気候物理医学会雑誌

<b>Objectives:</b> Peripheral artery disease (PAD) is a major health problem; however, no satisfactory intervention is available for its treatment. This study was undertaken to investigate the effects as well as mechanisms of the CO₂-enriched water bath (CEWB) treatment on blood flow in the ischemic hind limb.<BR><b>Experimental Model: </b>For inducing PAD, the femoral artery was occluded for 5 weeks in rats. The animals were treated with or without CEWB at 37°C for 4 weeks (20 min daily; 5 days per week) starting one week after the artery occlusion. CEWB was prepared by using Carbothera (Mitsubishi Rayon Engineering Tokyo). The blood flow was measured by Pulse Wave Doppler Ultrasound technique before and after the ligation as well as at the end of 4 weeks treatment. The angiogenesis (formation of new blood vessels) in the skeletal muscle was studied by histological examination.<BR><b>Results:</b> The peak, mean or minimal blood flow was not detected in the untreated ischemic hind limb animals due to arterial ligation. However, the values for blood flow were about 50% of the control values upon treatment with CEWB; 67% of the ligated animals showed positive blood flow by CO₂ treatment. Morphological examination of the treated ischemic skeletal muscle revealed a 3-fold increase in small artery count. Although plasma triglycerides were decreased and plasma NO concentration was increased in the ischemic animals, CEWB treatment produced no effects on these parameters. No mortality or changes in body wt, heart rate and plasma glucose, cholesterol or high density lipoproteins were seen in the control and experimental animals.<BR><b>Conclusion:</b> This study demonstrates the beneficial effect of CEWB treatment on blood flow in hind limb PAD. Furthermore, it is suggested that this beneficial action of CO₂ therapy may be due to the formation of new blood vessels in the ischemic skeletal muscle.

Entrenamiento microquirúrgico básico para realizar un modelo animal de alotrasplante compuesto vascularizado / Basic microsurgical training for a vascularized composite allotransplantation animal model

Introduction: rat hind limb transplantation is a complex animal model of vascularized composite allo-transplantation (VCA). A basic microsurgical training is required prior to the implementation of this model. Aim: to propose a training program for the acquisition of basic skills to perform a microsurgical VCA model. Animals and Methods: the training program was conducted in two stages. First, at the dry lab, basic suturing skills with 9-0 to 11-0 nylon sutures were practiced, reproduced from surgical videos performed by experts. In a second stage, at the wet lab using 13 Lewis rats, 6 hind limb microsurgical dissections were performed and the important steps for transplantation were identified: 10 end to end femoral artery anastomoses with 10-0 nylon interrupted suture; 10 end to end femoral vein anastomoses with 10-0 nylon suture; 3 femoral vein interposition in the femoral artery; 6 end to end sciatic nerve neurorrhaphy; 4 femur osteosynthesis with 21g needle and wire cerclage. Anastomotic patency rate and anastomotic surgical time were recorded. Results: arterial and venous patency rate was 100 and 90 percent respectively. Surgical time decreased from 49 to 24 minutes on arterial anastomoses and from 55 to 25 minutes on venous anastomoses after completion of the training program. When a vein interposition was performed, an immediate patency rate of 100 percent was obtained. Conclusion: a successful staged training model of basic microsurgical skills was performed, in order to perform a VCA model.

Introducción: el trasplante de extremidad posterior de la rata es un modelo microquirúrgico de alotras-plante compuesto vascularizado (ACV), que requiere para su implementación de un entrenamiento microquirúrgico básico sistematizado. Objetivo: comunicar un modelo de entrenamiento microquirúrgico básico para adquirir las habilidades y destrezas que permitan realizar un modelo microquirúrgico de ACV Animales y Métodos: el entrenamiento se realizó en 2 etapas: la primera, en laboratorio en seco. A partir de vídeos de microcirugía, se practicó las destrezas para maniobrar suturas de nylon de 9-0 a 11-0. En la segunda etapa in vivo con animales, (13 ratas Lewis), se realizaron: 6 disecciones microquirúrgicas de extremidad posterior para reconocimiento de estructuras anatómicas; 10 anastomosis término-terminal (T-T) de arteria femoral con sutura interrumpida 10-0; 10 anastomosis T-T de vena femoral; 3 interposiciones de vena femoral en arteria femoral; 6 neurorrafias T-T de nervio ciático; 4 fijaciones óseas de fémur con aguja 21 g y cerclaje con alambre. Se evaluó la permeabilidad de las anastomosis y los tiempos quirúrgicos. Resultados: anastomosis arteriales: la permeabilidad inmediata fue de un 100 por ciento. Los tiempos de anastomosis disminuyeron de 49 a 24 min con el entrenamiento. Anastomosis venosas: la permeabilidad inmediata fue de un 90 por ciento. Los tiempos de anastomosis disminuyeron paulatinamente de 55 a 25 min. Interposiciones venosas: se logró una permeabilidad de 100 por ciento. Conclusión: una estrategia de entrenamiento por etapas, es una forma factible de entrenamiento en microcirugía. Este entrenamiento permite obtener una aceptable permeabilidad anastomótica, paso crítico para realizar modelos de ACV.

Experimental Hind Limb & Inguinal-Femur Osteocutaneous Flap Model in Rats for Composite Tissue Transplantation

PURPOSE: Composite tissue allotransplantation (CTA) is a newly raised field as a treatment of severe body disfigurements. But in the point that it is transplant of functional organ not vital organ, it has limitation of using immunosuppressant for lifetime. Therefore, recent studies on CTA are focused on the reduction of risk of immunosuppression by inducing immunotolerance, developing new regimen and so on. So, appropriate experimental models to solve the problems are needed. We have performed CTA experiment using hind limb of rats which is frequently used CTA animal model. There were many trials and errors when actually conducting experiment. Hence the authors are to state the experiences in our own experiments. METHODS: Total 13 Sprague Dawley rats (SD rats) were used. In 10 rats, hind limb allotransplantation were performed and in 3 rats, inguinal-femur osteocutanoeus flap operation were conducted. RESULTS: Out of total 13 rats, on the day of operation 4 rats died, and 1dayafter operation 2 rats died. The remainder 7 rats were euthanized on the 3rd day after operation. Autophagy occurred in 3 rats. The dead experimental models were experienced in the early stage of study. CONCLUSION: We could increase survival rate through appropriate anesthesia, maintaining body temperature, supplement of water and pain control.

Treatment of posterior malleolar fractures by means of limited open reduction, poking reduction and QWIX cannulated screw internal fixation / 中华创伤杂志

Objective To evaluate the clinical effect of limited open reduction, poking reduction and QWIX cannulated screw internal fixation in treatment of posterior malleolar fracture. Methods A total of 14 patients with posterior malleolar fracture were treated by limited open reduction, poking reduction and QWIX cannulated screw internal fixation. Injury causes included traffic accidents in five patients, fall from height injury in four and inversion sprain in five. According to fracture type classification methods of Weber and Danis, there were two patients with type A3, seven with type B3 and five with type C3. Func-tional exercise was carried out based on postoperative exercise plan. Regular follow-up was performed to e-valuate the functional recovery of the ankle joint. Results All patients were followed up for 6-18 months (average 9.5 months). One patient with sural nerve injury returned to normal after six months. Twelve pa-tients showed excellent range of motion of the ankle joint and normal gait three months after operation. In 12 patients, dorsiflexion of the ankle joint was > 20° and plantar flexion > 40° six months after operation, which was approximately similar to contralateral normal ankle joint. The ankle joint function of all patients returned to normal six months after operation. According to Baird-Jackson ankle joint function evaluation, the result was excellent in 12 patients, good in one and fair in one, with excellence rate of 93%. Con-clusion The limited open reduction, poking reduction and internal fixation of QWIX caunulated screw are effective method for treatment of posterior malleolar fractures.

Cardiovascular Effects of Hind-limb Unweighing in Mice / 대한마취과학회지

BACKGROUND: Orthostatic intolerance is a debilitating problem that can occur after prolonged bed-rest, exposure to microgravity, and in the elderly. This study examined the integrated cardiovascular response to baroreceptor activation in a hind-limb unweighing (HLU) mouse model of microgravity to test the hypothesis that both the pressor and contractility response are attenuated in HLU mice. METHODS: C57BL/6 mice (25-30 g body wt, 8-10 wk old) were exposed to HLU for 2 weeks. A bilateral carotid artery occlusion and open-loop baroreceptor stimulus was performed to measure the myocardial contractile responses using a left ventricular micromanometer-conductance catheter in the mice. In isolated myocytes simultaneous sarcomere shortening and calcium transient were measured in response to increasing concentrations of the beta-agonist isoproterenol. RESULTS: In the controls, bilateral carotid artery occlusion increased the heart rate and mean arterial pressure. These responses were markedly attenuated in the HLU mice. A bilateral carotid artery occlusion also increased the slope of the end-systolic pressure volume relationship (Ees) by 70 +/- 11% and the slope was markedly attenuated to 10 +/- 8% in the HLU mice. Isoproterenol increased the sarcomere shortening in both control and HLU mice in a dose-dependent manner. However the contractile response to isoproterenol was significantly attenuated in the HLU mice than the controls. CONCLUSIONS: Both the pressor and myocardial contractile responses appear to be impaired in a mouse model of microgravity.

Change in blood flow of rats with diabetes mellitus after the acute hind limb ischemia / 基础医学与临床

Objective To establish the acute hind limb ischemia on Wistar rat with diabetes mellitus.MethodsThirty diabetes rats were induced by intraperitoneal STZ (50 mg/kg) injection, as well as the blood glucose level tested over 16.8 mmol/L. The rats were ligated on the left femoral artery, then the blood perfusion on the hind limbs ischemia was measured by LDPI after the operation. Results The fasting plasma glucose level on 22 Wistar rats(81.5%) was kept above 16.8 mmol/L, and the hind limb blood perfusion would recover slowly to the level of the right side from 1 to 14 day (P

Recovery law of hind limb function following complete spinal cord transection injury in rats / 中华创伤杂志

Objective To observe the recovery process of hind limb function following complete spinal cord transection injury and discuss corresponding mechanism. Methods The spinal cords of 15 SD rats were transected at T9 plane and a 3 mm spinal cord removed for a complete transection. The functional recovery of the hind limb was evaluated using CBS (combined behavioral score) and Basso Beattle Bresnahan (BBB) 1, 2, 4 and 6 weeks after injury. At the 6th week after injury, also experiment was done on histology, immunohistochemistry, motor evoked potential (MEP) of spinal cord and repeated transection. Results The hind limb function of the injured animals recovered at different degrees, most obvious at the 4th week and BBB was up to 12 at the 6th week. MEP result was related to the site of the stimulating electrode. No MEP was recorded when the stimulating electrode was placed above the injured site. But when the stimulating electrode was placed below the injury level, MEP was normal. The again transection of the spinal cord above the injury level took no effect on the recovered hind limb. However, again transection below the injury level resulted in complete paralysis. Histologic results showed that the injured site was filled with glial scars without axons. There existed a little scattered positive fibers of neurofilament protein 200 (NF200). Conclusions After complete spinal cord injury, there is a significant spontaneous recovery of the hind limb function that is a kind of autonomous functions of the injured distal spinal cord unrelated to repair of descending conducting fasciculus of the spinal cord. The distal spinal cord is intact in function.

Effects of Local Corticosteroid Injection and Weight Bearing on Injured Achilles Tendon in a Rat Model

OBJECTIVE: To evaluate the effects of local triamcinolone (TAC) injection and weight bearing on healing process of the injured Achilles tendon. METHOD: The right Achilles tendons in 54 rats, Sprague-Dawley (about 200 g), were each sutured after transection and allocated into three groups according to the amount of the local injection of TAC (none, 0.25 mg, and 1.25 mg respectively), and subsequently each group divided into three subgroups by the type of weight bearing {normal weight bearing (NWB), treadmill exercise (7-8 m/min, 10 min/day) from day 7 for 7 days, and hind limb immobilization respectively}. On 15th day rats were sacrificed, and then diameters of both injured and uninjured tendon, numbers of fibroblasts on injured tissues and he percentage of matured fibroblasts by microscopy were evaluated. RESULTS: The diameter of the hypertrophied neotendon in groups injected with TAC 1.25 mg was significantly decreased, but not in the others. There were no differences in numbers of fibroblast. As the amount of TAC increased in groups done with NWB and treadmill exercise, the percentages of matured fibroblasts significantly decreased. CONCLUSION: The local TAC injection on acute tendon injuries had deleterious effects on healing process of tendon, and follow-up study about the types of weight bearing is needed.

Effect of Endurance Exercise during Dexamethasone Treatment on the Attenuation of Atrophied Hind-limb Muscle Induced by dexamethasone in Rats / 간호학회지

The purpose of this study was to determine the effect of regular exercise during dexamethasone injection in the body weight, weight of hind-limb muscles, myofibrillar protein content and glutamine synthetase activity. 180-200g female Wistar were divided into four groups: control, exercise, dexamethasone injection(dexa), and exercise during dexamethasone injection(D+E) group. The dexa group received daily subcutaneous injection of dexamethasone at a dose of 4mg/kg body weight for 7days. The exercise group ran on a treadmill for 60min/day(20minutes every 4 hours) at 10m/min and a 10degrees grade. The control group received daily subcutaneous injection of normal saline at a dose of 4mg/kg body weight for 7 days. The D+E group ran on a treadmill for 60min/day(20minutes every 4 hours) at 10m/min and a 10degrees grade during dexamethasone injection. Body weight of the control group increased significantly from days of experiment, that of the dexa group decreased significantly from day 4 of the dexa group decrease significantly from day 4 of the experiment resulting in a 82.4% decrease compared to the first day of the experiment. Body weight of the D+E group decrease significantly from day 5 of experiment resulting in a 81.77% decrease compared to the first day of the experiment. Body weights, muscle weight and myofibrillar protein content of the plantaris and gastrocnemius decrease significantly and muscle weight of the sleys tended to decrease with dexamethasone injection. Glutamine synthetase activity of the hind-limb muscles increase significantly with the dexamethasone injection. The relative weight of the soleus was comparable to the control group and that of plantaris decrease significantly and that of gastrocnemius tended to decrease compared to that of the control in the dexa group. Body weight and muscle weight of the plantaris and gastronemius of the exercise group were comparable to the control group, and the muscle weight of soleus showed a tendency to increase. The relative weight of the soleus increased significantly and that of the plantaris and gastrocnemius were comparable to the control in the exercise group. Myofibrillar protein content of the soleus and plantaris increased significantly and there was no change of GS activity of the hind-limb muscles compared to the control in the exercise group. Body weight of the D+E group was comparable to the dexa group, muscle weight of the hind-limb muscles increased significantly. Myofibrillar protein content of the soleus and plantaris increase significantly and that of the gastrocnemius tendency to increase compared to the dexa group. Body weight and muscle weight of the plantaris and gastrocnemius of the D+E group did not recover to that of the control group. Muscle weight of the soleus recovered to that of the control group. The relative weight and myofibrillar protein content of the hind-limb muscles recovered to that of the control group. From these results, it is suggested that regular exercise during dexamethasone injection might attenuate the muscle atrophy of the hind-limb muscles.

Study on vasodilator nerve in rat hind limb / 中国药理学通报

AIM To investigate whether there is vasodilator nerve innervation in rat hind limb and what the nature of vasodilator nerve is. METHODS Wistar rats were treated with reserpine 1 mg?kg -1 ip at 24 h before experiment. The rats were pithed and the hind limb vascular bed was perfused with Krebs-Henseleite solution containing 1 mmol?L -1 phenylephrine at 2 ml?min -1 speed. The hind limb perfused pressure (HPP) as a main index was continuously recorded. Spinal cord electrical stimulation (SES) was repeatedly applied via an electrode at L 1～2 level of lumber vertebra. The various tool drugs were administered by iv or infusion by added to persusion solution. The data is expressed as decrease percentages of HPP increased by continuous infusion of phenylephrine. RESULTS HPP was increased from (5 7?1 5) to (21 6?3 7) kPa ( n =37) after phenylephrine perfusion. SES caused a fall of HPP in frequency dependent and voltage dependent manner. An optimum parameters of SES (10 Hz, 50 V and 1 msec) was selected to observe effects of various tool drugs on depressor response of HPP to SES. Tetrotodoxin (0 3 ?mol?L -1 ) abolished the effect completely. L NAME (10 ?mol?L -1 ), a NO synthase inhibitor, had no effect. Ganglion blocker arfonad (110 mg?kg -1 , iv, M R blocker atropine (10 ?mol?L -1 ), ? receptor blocker propranolol (1 ?mol?L -1 ) and P 1 receptor blocker aminophylline (10 ?mol?L -1 ) had also no effect. Glibenclamide (0 1 mmol?L -1 ), an ATP sensitive K + channel blocker, markedly abolished and slightly reversed the effect. CONCLUTION The nonadrenergic noncholinergic vasodilator nerve exists in rat hind limb and is not nitroxidergic or purinergic nerve. This nerve may be peptidergic nerve which releases some peptide such as CGRP and the major mechanism of vasodilatation probably activates the ATP sensitive K + channel.