[Effect of acupoint injection with bone marrow mesenchymal stem cells on the blood flow in rats with hind limb ischemia].
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To investigate the best injection method of the bone marrow mesenchymal stem cells (BM-MSCs) transplantation for the treatment of a rat model with hind limb ischemia.Twenty four SD rats with hind limb ischemia were randomly divided into four groups: control group, model group, acupoint BM-MSCs injection group (API group) and thigh muscle BM-MSCs injection group (TMI group). The acupoints of "Sanyinjiao" (SP 6), "Housanli" (ST 36), "Zhaohai" (KI 6), "Huantiao" (GB 30) and "Yanglingquan" (GB 34) were selected for API group, and five non-acupoints were selected on gastrocnemius and adductor of ischemic hind limb for TMI group. Both groups were accepted BM-MSCs transplantion. Model rat with hind limb ischemia was established with the method of blocking the femoral artery and its branches. The changes of blood flow (perfuse unit, PU) was monitored with laser Doppler flowmetry (LDF). In order to describe the visual changes in blood flow, the PU index (PUI) was determined as the ratio of ischemic to non-ischemic hind limb blood perfusion. And also, the levels of VEGF,bFGF in serum were tested to analyze the immunohistochemical expression quantity of VEGF and bFGF.Comparing with the model and the TMI groups, the PUI value on 3rd, 14th and 21th days after BM-MSCs transplantation were significantly increased in the API group (P < 0.05, P < 0.01). In contrast to the model group, the VEGF,bFGF levels in serum and the immunohistochemical expression quantity of VEGF and bFGF in the API and TMI groups were significantly increased (all P < 0.01).Transplantation of BM-MSCs through the acupoint can more significantly and quickly increase the blood flow and cause the greater improvement on hind limb ischemia than that of through the way of muscle injection.Keywords:
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Objective To study the therapeutic effect and mechanisms of Senhuang liquid in treatment of limb ischemias in rats. Methods The models of hind limbs ischemia were established by lauric acid of femoral arteries injection in 90 rats,and the 90 rats modelled successfully were randomly divided into control group( group A,n = 30),group B( n = 30) and group C( n = 30). Group A was left untreated,latero-hind limbs of models were respectively soaked into 75% Alcohol liquid in group B,and Senhuang liquid in group C( 15 min each time,in the morning and at night / 1 d). The ischemic degrees of hind limbs were observed on 21thd after the operation; muscle specimens of hind limbs were removed,microvessel densities( MVD) were detected,and the expressions of vascular endothelial growth factor( VEGF) in protein level were also detected by immunocytochemistry strept avidin-biotin complex method. Results Compared with those in group A and B,in group C the ischemic degrees of hind limbs were significantly relieved( P 0. 05,P 0. 01),and the expressions of MVD and VEGF were significantly increased( P 0. 05); the differences in latero-hind limbs of model ischemia degrees,expressions of MVD and VEGF were no statistically significant( P 0. 05). Conclusion Senhuang liquid can regulate the VEGF expression,promote capillary angiogenesis,effectively improve limb ischemias in rats and prevent the gangrene and secondary infection of the acra.
Hindlimb
Microvessel
Forelimb
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Objective To observe the effect of lumbar sympathetic ganglion blocking with high intensity focused ultrasound(HIFU)on substance P(SP)of spinal cord andβ-endorphine(β-EP)in blood plasma in arterial occlusion animal model.Methods 26 canis familiaris were randomly assigned to 3groups:HIFU therapy group(group A,n=12),non-therapy group(group B,n=12)and normao control group(group C,n=2).In group A and B,the arteria cruralis was liberated and all branches(including arteria profunda femoris)were cutted,then a helical tinsel was inserted into the arteria.In group A,the 2nd~ 6th ganglion sympatheticums were blocked with HIFU after postoperative 2weeks.In every time point after the therapy,2dogs were randomly selected from groups A and B every time forβ-EP examination,and then the spinal cord SP was detected,dogs in group C were directly detected.Results(1)Comparison with the group C,β-EP in the groups A and B was significantly decreased(P0.01),β-EP in the group A was higher than that in the group B after therapy(P0.05).(2)In the groups A and B,SP was located in the I,Ⅱlayers of dorsal horn in spinal cord,postoperative average optical density(OD)of SP was significantly higher than that in the group C(P0.01);the OD in the group A was less than that in the group B after therapy(P0.05).(3)In the model dog of arteria occlusion,blood plasmaβ-EP was negative correlated with the SP in the spinal cord.Conclusion Lumbar sympathetic ganglion blocking for hind limb arterial occlusion animal model by HIFU can increase blood plasmaβ-EP and decrease SP of spinal cord,and elevate the pain threshold value and then relieve pain.
Hindlimb
Lumbar Spinal Cord
Group B
Group A
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Objective To investigate effect of local injection of shh protein on ischemic hind limbs of elderly rats,and to explore the possible mechnism.Methods Firstly,to build the model of ischemic hind limbs of elderly rats,which were then divided into two groups the experimental group was injected shh proteins below the ligation level,while the controlled group was injected normal saline.After 14 days,angiographies of injected hind limbs were performed,immunohistochemical examination of ischemic limbs were conducted for measurement of von Willebrand factor(vWF),CD31and capillary density.Meanwhile,functional assessment and thermometry of hind limbs were conducted.Results Two weeks after injection,micro vascular density of the injected limbs in the experimental group(623.07±81.69)mm2 was considerably higher than in the controlled group(528.25±71.67)mm2.Angiography showed that the experimental group(2.46±0.37)mm2 had a denser collateral vessels than the controlled group(1.57± 0.39)mm2.All the results were statistically significant.Conclusions Injection of shh proteins into elderly rats' ichemic hind limbs could stimulate the proliferation of collaterals and enhance angiogenesis.
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Collateral circulation
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Objective To observe recombinant human granulocyte colony stimulating factor(rhG-CSF) in promotion of angiogenesis in different-gender adult rats with ischemia of hind limb. Methods Healthy adult male and female Wistar rats,20 for each gender,were enrolled.A hind-limp ischemic model was established by cutting off the left femoral arteries and their branches.The rats were equally randomized to two groups: control group and experimental group.Subcutaneous injection of rhG-CSF was offered to the experimental,and equal volume NS to the control.WBC was detected before and five days after the injection;adductor was collected four weeks later and sent for pathology.The density and number of blood vessels were detected by image analysis system;the VEGF of the adductor by immunohistochemistry. Results The white blood count,density and positive VEGF were significantly higher in the experimental than that in the control,the differences between the two groups were significant(t=4.318-4.981,P0.05);but no significant difference was noted in terms of the gender(P0.05). Conclusion Subcutaneous injection of rhG-CSF can facilitate angiogenesis of ischemic limb,improve ischemic status of the limb,the effects do not show association with the gender.
Limp
Arteriogenesis
Subcutaneous injection
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Objective: To compare the change of skin temperature of the hind limb,the microstructure of the nerve ganglion and the substance P(SP) in the spinal cord under the two disposals of lumbar sympathectomy with high intensity focused ultrasound(HIFU) and dehydrate alcohol on the hind limb arterial occlusive disease model in dogs.Methods: Hind limb ischemia models were made on 27 mongrel dogs.They were randomly assigned to 3 groups in 2 weeks after the surgery(n=9 each): control group(group A),HIFU therapy group(group B),alcohol therapy group(group C).In group A,no disposal was given to.In groups B and C,HIFU or the dehydrated alcohol were used to block the lumbar sympathetic ganglion.The change of the limb skin temperature,the microstructure of the nerve ganglion and the SP in the spinal cord were detected before the surgery,before the therapy(2 weeks after the surgery),1 week after the therapy.Results:(1)Hind limb skin temperature in the 3 groups decreased significantly(P0.05) after the surgery.The temperature returned after therapy in groups B and C.(2) All neurons were necrotized in group B,most of the neurons were necrotized in group C.(3) SP immunization positive reactant localized in the laminae I,Ⅱ in the dorsal horn in the 3 groups.After the surgery,the average optical density(OD) of SP in the lumbar segment increased significantly(P0.01).After the therapy,the OD in groups B and C decreased significantly(P0.05) and there was no significant difference between the two groups(P0.05).Conclusion: Lumbar sympathetic ganglion blocking with HIFU or dehydrated alcohol can block the lumbar sympathetic ganglion effectively.There was no significant difference in hightening the hind limb skin temperature and lowering SP in spinal cord between two groups.The necrosis of neurons was more severe in HIFV group than dehydrated alcohol group.
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Group B
Lumbar Spinal Cord
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The objective of the study was to experimentally substantiate a possibility of treating lower limb chronic ischaemia by means of administration of bone marrow cells. The study was performed on 100 laboratory 'Wistar' rats subdivided into four groups: intact, control, first and second study groups. The intact group consisted of 10 animals, with the remaining three containing 30 rats each. All animals, except the intact ones, were subjected to modelled critical ischaemia of a hind limb by means of excision of major vessels of the extremity. The rats from the first study group were given 'Myelopid' (calf bone marrow extract) at a dose of 50 μg/kg injected into the femoral muscles 3 hours after the operation, then after 1, 2 and 3 days. The second study group rats underwent cellular therapy with the mononuclear fraction of bone marrow autologous cells. Bone marrow was procured from the femoral bone of the contralateral limb. The exfusate was subjected to double centrifugation followed by isolation of the mononuclear fraction of the bone marrow according to the Boyum technique. The obtained mononuclear fraction of autologous cells of the bone marrow amounting to 4×106 cells in a volume of 200 μl was injected into the ischaemized limb of the animal. The control group received no treatment at all. In the first study group, the level of microcirculation as compared with the control group on day 10 increased by 48.3%, on day 21 by 50.6%, and on day 28 by 105.9%. In the second study group, the level of microcirculation as compared with the control group on day 10 increased by 67.4%, on day 21 by 85.7%, and on day 28 by 97% (the differences between the two study groups were statistically insignificant). Neither had the area of muscular necrosis statistically significant differences between the study groups, decreasing on day 10 averagely by 14.75%, on day 21 by 6.5% as compared with the control group animals, to become completely reduced on day 28. A conclusion was drawn that 'Myelopid' and the mononuclear fraction of autologous cells of bone marrow exerted a positive effect on the level of microcirculation, the scope of necrotic lesion of muscular tissue in the ischaemized extremity of laboratory animals and may potentially be used for treatment of patients suffering from obliterating diseases of lower-limb arteries.Цель исследования – экспериментальное обоснование возможности лечения хронической ишемии конечности введением клеток костного мозга. Исследование выполнено на 100 лабораторных крысах 'Wistar', которые были разделены на четыре группы: интактную, контрольную, первую и вторую опытные группы. В интактную группу вошло 10 крыс, в остальные – по 30 животных в каждую. У всех крыс, за исключением интактных, моделировалась критическая ишемия конечности путем иссечения магистральных сосудов конечности. В первой опытной группе крысам вводился препарат 'Миелопид' (экстракт костного мозга телят) по 50 мкг/кг в мышцы бедра через 3 часа после операции, затем через 1, 2 и 3 суток. Во второй опытной группе проводилась клеточная терапия мононуклеарной фракцией аутологичных клеток костного мозга. Забор костного мозга осуществлялся из бедренной кости контрлатеральной конечности животного. Эксфузат подвергался двойному центрифугированию с последующим выделением мононуклеарной фракции костного мозга по методу Boyum. Полученную мононуклеарную фракцию аутологичных клеток костного мозга в количестве 4х106 клеток в объеме 200 мкл вводили в ишемизированную конечность животного. В контрольной группе лечение не проводилось. В первой опытной группе уровень микроциркуляции по сравнению с контрольной группой на 10 сутки возрастал на 48,3%, на 21 сутки – на 50,6%, на 28 сутки – на 105,9%. Во второй опытной группе уровень микроциркуляции по сравнению с контрольной группой на 10 сутки возрастал на 67,4%, на 21 сутки – на 85,7%, на 28 сутки – на 97% (различия между двумя опытными группами статистически незначимы). Площадь некроза мышц также не имела статистически значимых различий между опытными группами и на 10 сутки уменьшилась в среднем на 14,75%, на 21 сутки – на 6,5% по сравнению с контрольной группой животных и полностью редуцировалась к 28 суткам. Сделан вывод, что препарат 'Миелопид' и мононуклеарная фракция аутологичных клеток костного мозга оказывают положительное влияние на уровень микроциркуляции, объем некротического поражения мышечной ткани в ишемизированной конечности экспериментальных животных и потенциально могут применяться для лечения больных, страдающих хроническими облитерирующими заболеваниями артерий нижних конечностей.
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Objective To investigate the effects of vasoactive substances and in the relief of rat hindlimb ischemia after meridian point injection of bone marrow mesenchymal stem cells(BM-MSCs).Methods SD rats(n=24) were randomly divided into the blank control group,model group,meridian point injection group(point group) and muscle injection group(muscle group).The model of hindlimb ischemia was established in rats by using apocope of femoral artery and branches.The acupoints of Sanyinjiao(SP 6),Housanli,Zhohai(K 16),Huantiao(GB 30) and Yanglingquan(GB 34) were chosen in the point group and random 5 points were chosen in the muscle group,and then injected BM-MSCs respectively.After 3 weeks the serum content of angiotensinⅡ(AngⅡ) and nitric oxide (NO) in abdominal aorta were detected,and AngⅡcontent and the expression of endothelin-1(ET-1) in hindlimb skeletal muscle were detected by using immunohistochemistry assay in all groups.Results The serum NO content increased significantly in the point group and muscle group than that in the blank group and model group(P0.01),while the difference in serum content of Angll was not statistically significant.The expression of ET-1 in hindlimb skeletal muscle increased significantly(P0.01) in the point group and muscle group,and that in the point group was higher than that in the muscle group(P0.01).The difference of AngⅡcontent in hindlimb skeletal muscle was not statistically significant. Conclusion The results showed that ET-1 and NO joined into the relieve of rat hindlimb ischemia after meridian point injection with BM-MSCs,and AngⅡdid not.
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Meridian (astronomy)
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Objective To explore and establish the model method of simple,stable,good repeatability about the diabetes rabbit's hind limbs ischemia.Methods Forty rabbits were randomly divided into experimental group(n=30) and control group(n=10).The experimental group were injected twice with alloxan(alloxan,ALX),the control group were injected with normal saline,to establish the model of diabetes rabbit.It was leaded to ischemia due to ligature and dividing the right hind limb's distal iliac artery and resection the superficial brachial artery of the diabetes rabbit's right hind limb.The degree of ischemia was assessed regularly food until 60 days of postoperative.Results In experimental group,30 rabbits were involved in modeling,two rabbits died after the 1st injection of ALX,three died after the 2nd injection of ALX,the rest of 25 rabbits were successful for making the diabetes model.Angiography showed that femoral wispy collateral vessels numeration rate increased from 0.54±0.26 to 1.57±0.28.Histologic appraisal showed there was partial atrophy and necrosis in rabbits' right hind limb muscle.Conclusion It was successful to make the model of stable and good repeatability about the diabetes rabbit's hind limbs ischemia,which was to provide the ideal model for the experimental study of Cell transplantation in the treatment of diabetes.
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Ligature
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Cnidium officinale Makino (COM) has been used traditionally to treat female sexual disorders, such as amenorrhea, hypomenorrhea and oligomenorrhea, by improving blood circulation.The present study aimed to investigate the alleviating effect of COM extracts on surgical injury-induced ischemia in the hind-limb of mice. In this study, female C57BL/6 mice were ovariectomized, and the vessels of the hind-limb were excised after ligation by surgical silk (6-0). The mice were orally administered with COM (150 or 300 mg/kg/day) for 3 weeks, and the blood flow rate of hind-limbs was evaluated by using a laser Doppler system after hind-limb ischemic surgery in an in vivo study. Additionally, the migration and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated in an in vitro study.The blood flow rate was synchronized to the nonischemic lesion of the hind-limb, and its elevation compared to the vehicle was observed at 14 and 21 days after hind-limb ischemic surgery in COM-treated groups. The number of capillaries increase in a dose-dependent manner in the COM-treated groups (150 and 300 mg/kg). In HUVECs, the activities of cell migration were significantly increased by 50 and 75 μg/mL for the COM-treated groups. In addition, the number of tubule branches and junctions was also increased by doses of COM (50 and/or 75 μg/mL).The results of our study suggested that the COM extract may have therapeutic application for the treatment of hind-limb ischemic damage, which is due to the improvement of the peripheral angiogenetic system.
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Therapeutic effect
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[Objective]Pneumatic tourniquet was used to make limb ischemia reperfusion of animal model to investigate the injury of peripheral nerve and the skeletal muscle.[Method]Thirty health 6-month-old New Zealand white rabbits(weighing 3.5±0.3 kg,male or female) were choosen,the left hind limb in rabbits was circled with inflatable tourniquet release at different times,resulting in the left hind limb ischemia-reperfusion injury model.Animals were randomly divided into 3 groups of 10 each,group A: a control group B: ischemia for 2 hours,group C: ischemia for 4 hours.Except group A all the groups were circled with inflatable tourniquet.Each group were measured with indicators of limb electrophysiology reperfusion after 1 hour,2 hours,3 hours,4 hours,5 hours,6 hours.The muscle of group A was observed by morphology after 6 hours.The muscle of group B and C were observed by morphology 6 hours after reperfusion.The walking function of left hind limb of every rabbits was evaluated 5 days after surgery.[Result] With the longing of ischemia-reperfusion time,the incubation period of the peripheral nerves extended,amplitude lowered and conduction velocity decreased in groups B,C compered with group A.The incubation,amplitude and conduction velocity among three groups had statistically significant difference(P0.05).Skeletal muscle under microscopy(group B and C) showed stripes disorders,muscle fiber rupture,interstitial vascular dilatation and congestion,and a large number of neutrophils infiltration.[Conclusion] After the interaction of ischemia and reperfusion,the physical damage cound further increase the functionality,or even lead to irreversible lesions.The animal model making has less damage to animals,and is similar to clinical practice.
Hindlimb
Limb ischemia
Inflatable
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