The effect of gradual or acute arterial occlusion on skeletal muscle blood flow, arteriogenesis, and inflammation in rat hindlimb ischemia

BACKGROUND: Current experimental models of critical limb ischemia are based on acute ischemia rather than on chronic ischemia. Human peripheral vascular disease is largely a result of chromic ischemia. We hypothesized that a model of chronic hindlimb ischemia would develop more collateral arteries, more blood flow, and less necrosis and inflammation than would acute hindlimb ischemia. We therefore developed a rat model of chronic hindlimb ischemia and compared the effects of chronic ischemia with those of acute ischemia on hindlimb skeletal muscle. METHODS: Acute or chronic ischemia was induced in 36 male Sprague-Dawley rats. Chronic ischemia caused blood flow, as measured by laser Doppler scanning and confirmed by muscle oxygen tension measurements, to gradually decrease over 1 to 2 weeks after operation. RESULTS: Histologic analysis showed chronic hindlimb ischemia better preserved muscle mass and architecture and stimulated capillary angiogenesis, while lacking the muscle necrosis and inflammatory cell infiltrate seen after acute ischemia. Surprisingly, the chronic ischemia group recovered dermal blood flow more slowly and less completely than did the acute ischemia group, as measured by laser Doppler (0.66 +/- 0.02 vs 0.76 +/- 0.04, P < .05) and tissue oxygen tension (0.61 +/- 0.06 vs 0.81 +/- 0.05, P < .05) at 40 days postoperatively. Consistent with poorer blood flow recovery, chronic ischemia resulted in smaller diameter collateral arteries (average diameter of the five largest collaterals on angiogram was 0.01 +/- 0.0003 mm vs 0.013 +/- 0.0007 mm for acute, P < .005 at 40 days postoperatively). Acute ischemia resulted in decreased tissue concentrations of vascular endothelial growth factor (VEGF) (0.96 +/- 0.23 pg/mg of muscle for acute vs 4.4 +/- 0.75 and 4.8 +/- 0.75 pg/mg of muscle for unoperated and chronic, respectively, P < .05 acute vs unoperated), and in increased tissue concentrations of interleukin (IL)-1beta (7.3 +/- 4.0 pg/mg of muscle for acute vs undetectable and 1.7 +/- 1.6 pg/mg of muscle for unoperated and chronic, respectively, P < 0.05 acute vs unoperated). CONCLUSIONS: We describe here the first model of chronic hindlimb ischemia in the rat. Restoration of blood flow after induction of hindlimb ischemia is dependent on the rate of arterial occlusion. This difference in blood flow recovery correlates with distinct patterns of muscle necrosis, inflammatory cell infiltration, and cytokine induction in the ischemic muscle. Differences between models of acute and chronic hindlimb ischemia may have important consequences for future studies of mechanisms regulating arteriogenesis and for therapeutic approaches aimed at promoting arteriogenesis in humans suffering from critical limb ischemia. CLINICAL RELEVANCE: Despite the substantial clinical differences between acute and chronic ischemia, researchers attempting to develop molecular therapies to treat critical limb ischemia have only tested those therapies in experimental models of acute hindlimb ischemia. We present here a novel model of chronic hindlimb ischemia in the rat. We further demonstrate that when hindlimb ischemia is developed chronically, collateral artery development is poorer than when hindlimb ischemia is developed acutely. These findings suggest that further tests of molecular therapies for critical limb ischemia should be performed in chronic hindlimb ischemia models rather than in acute hindlimb ischemia models.     

Nitrite Anion Therapy Protects Against Chronic Ischemic Tissue Injury in db/db Diabetic Mice in a NO/VEGF-Dependent Manner

Nitrite anion has been demonstrated to be a prodrug of nitric oxide (NO) with positive effects on tissue ischemia/reperfusion injury, cytoprotection, and vasodilation. However, effects of nitrite anion therapy for ischemic tissue vascular remodeling during diabetes remain unknown. We examined whether sodium nitrite therapy altered ischemic revascularization in BKS-Lepr^db/db mice subjected to permanent unilateral femoral artery ligation. Sodium nitrite therapy completely restored ischemic hind limb blood flow compared with nitrate or PBS therapy. Importantly, delayed nitrite therapy 5 days after ischemia restored ischemic limb blood flow in aged diabetic mice. Restoration of blood flow was associated with increases in ischemic tissue angiogenesis activity and cell proliferation. Moreover, nitrite but not nitrate therapy significantly prevented ischemia-mediated tissue necrosis in aged mice. Nitrite therapy significantly increased ischemic tissue vascular endothelial growth factor (VEGF) protein expression that was essential for nitrite-mediated reperfusion of ischemic hind limbs. Nitrite significantly increased ischemic tissue NO bioavailability along with concomitant reduction of superoxide formation. Lastly, nitrite treatment also significantly stimulated hypoxic endothelial cell proliferation and migration in the presence of high glucose in an NO/VEGF-dependent manner. These results demonstrate that nitrite therapy effectively stimulates ischemic tissue vascular remodeling in the setting of metabolic dysfunction that may be clinically useful.     

脉管复康片联合糖痹外洗方对糖尿病血管病变的疗效及其机制

目的：建立糖尿病大鼠下肢缺血模型,研究脉管复康片联合糖痹外洗方对糖尿病大鼠血管内皮损伤的疗效及其作用机制。方法：选取50只大鼠中的8只为正常对照组,余下大鼠采用高脂饮食联合腹腔注射链脲佐菌素法建立大鼠糖尿病模型,结扎大鼠左后肢股动脉,建立下肢缺血模型。取造模成功的32只大鼠,随机分为模型组和高、中、低剂量给药组,每组8只。给药21 d后处死大鼠,取其血样及左后肢腓肠肌组织,检测血清中血栓素A2(TXA2)、内皮素（ET）-1、一氧化氮（NO）、内皮型一氧化氮合成酶(eNOS)、血管内皮生长因子（VEGF）、血管细胞黏附分子-1(s VCAM-1)、C反应蛋白（CRP）含量;HE染色观察缺血后肢肌细胞形态;CD31免疫组织化学染色检测术侧腓肠肌微血管形成数量。结果：经给药治疗后,大鼠腓肠肌组织HE染色未见明显萎缩坏死,血管内皮功能趋于正常。与模型组相比,脉管复康片高、中、低剂量组大鼠血清中TXA2水平均显著降低（P <0.01）;eNOS水平均显著升高（P <0.001）;ET-1水平均有下降,高剂量组最显著（P <0.001）;高剂量组NO水平显著上升（P <0...     

An evaluation of resting arterial ischemia models in the rat hind limb

Techniques for using the rat hind limb as a model of pure arterial ischemia at rest have not been well defined. Because the rat has no profunda femoral artery, numerous collateral pathways exist to the hind limb, and femoral artery ligation is not an effective method of inducing arterial ischemia. After several anatomic studies, a two stage operation to produce arterial ischemia in the left hind limb was devised. The first stage involved surgical interruption of collateral and re-entrant vessels, and the second stage involved femoral artery ligation. Using Xenon 133 clearance as an estimate of blood flow, reduction in flow to 14, 24, and 37% of the simultaneously measured value in the right hind limb was obtained at 2 hours, 2 days, and 5 days post ligation. Oxygen extraction in the left hind limb doubled both at 2 hours and at 2 days post ligation. Histological evaluation of the anterior compartment musculature after 5 days demonstrated loss of nuclei, degenerating contractile elements, edema, and inflammatory infiltrate. Evaluation of rats that had undergone isolated femoral artery ligation showed a 66% reduction in flow 2 hours after ligation, but no reduction in flow at 5 days, no increase in oxygen extraction, and only nuclear changes on histological exam at five days.     

The effects of severe progressive hemodilution on regional blood flow and oxygen consumption.

Electrolyte solutions are effective in the immediate treatment of hemorrhagic shock, but the acceptable limits of hemodilution are not well defined. In this study oxygen consumption was measured in various tissues at rest and in maximally exercising skeletal muscle during progressive hemodilution. Twenty splenectomized, anesthetized dogs were studied (weight 22.6 +/- 2.0 kilograms). Measurements were made of cardiac output, capillary muscle blood flow in the hind limb, and renal and superior mesenteric arterial blood flow. Arteriovenous oxygen differences in the hind limb, kidney, gut, and in the whole body were calculated from the oxygen content of arterial and appropriate venous samples.     

Effect of chronic ischemia on constitutive and inducible nitric oxide synthase expression in erectile tissue

Arterial occlusive disease is one of the leading causes of organic erectile dysfunction (ED). Recent studies have shown that the incidence of cardiovascular disease closely correlates with the prevalence of ED. Also, ED is thought to be an early signal of impending cardiovascular problems. We previously found that the atherosclerosis of iliohypogastric arteries in the rabbit causes ED, down-regulates cavernosal neuronal nitric oxide synthase (nNOS) gene expression, and impairs NO synthesis. The goal of this study was to determine the effect of atherosclerosis-induced ischemia on cavernosal nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) expression and NO-mediated smooth muscle relaxation in the rabbit. Our study showed that iliac artery blood flow, intracavernosal blood flow, and intracavernosal oxygen tension were unchanged 4 weeks after the induction of arterial atherosclerosis, whereas they were significantly diminished at weeks 8 and 16. Erectile responses to nerve stimulation and cavernosal smooth muscle relaxation were unchanged at week 4 and were significantly diminished at weeks 8 and 16 after the induction of atherosclerosis. Western blotting showed that cavernosal nNOS and eNOS protein levels were unaffected at week 4 but were significantly decreased at weeks 8 and 16 after the induction of atherosclerosis. iNOS protein, however, markedly increased during the course of the induced arterial disease. Immunohistochemical staining showed no change in cavernosal eNOS or nNOS expression at week 4. A dramatic decrease in both was evident at 8 and 16 weeks. iNOS expression progressively increased between 4 and 16 weeks of atherosclerosis. Down-regulation of nNOS and eNOS, along with up-regulation of iNOS, may explain ischemic cavernosal smooth muscle relaxation impairment in the rabbit. Ischemically altered NOS expression may be of great pathophysiologic importance in atherosclerosis-induced ED. These data may provide further insight into the mechanism of arteriogenic ED.     

The induction of angiogenesis by the implantation of autologous bone marrow cells: a novel and simple therapeutic method.

BACKGROUND: Bone marrow contains many kinds of primitive cells that could differentiate to endothelial cells and secrete several growth factors. In the current study, we attempted to induce therapeutic angiogenesis by implanting autologous bone marrow cells (BMCs) and using a rat ischemic hind limb model. METHODS: BMCs were prepared by removing red blood cells. A rat ischemic hind limb model was made by the ligation of the left femoral artery and its branches. BMCs were injected into 7 points of the ischemic muscles. To assess angiogenesis, a microangiogram, laser Doppler, and histologic evaluation were performed after the surgical procedure. RESULTS: A microangiogram and histologic evaluation showed that angiogenesis was significantly induced in the ischemic hind limb by the implantation of BMCs. Laser Doppler imaging analysis showed that blood flow was significantly increased after implantation of BMCs. Some implanted BMCs were stained positively with CD31 and vascular endothelial-cadherin (VE-cadherin), which might have been incorporated into the vasculature. The condition of ischemia caused an elevation in the level of basic fibroblast growth factor in the ischemic muscle and also in interleukin-1beta derived from the implanted BMCs, which might contribute to angiogenesis. CONCLUSION: These findings indicate that autologous bone marrow implantation may be a novel and simple method for inducing therapeutic angiogenesis.     

TcPO2 values in limb ischemia: effects of blood flow and arterial oxygen tension

Transcutaneous oxygen tension (TcPO2) is determined by blood flow and arterial oxygen tension (PaO2) and has been advocated as a measurement of tissue perfusion in peripheral vascular disease. The purpose of this study was to define the relationship between regional blood flow, PaO2, and TcPO2. TcPO2 sensors were placed on the skin of the anterior tibial regions of the hind limbs of 15 dogs. After occluding collateral blood flow, an external flow probe was placed around the femoral artery and an adjustable clamp was used to produce graded ischemia. Progressive reductions in blood flow were correlated with TcPO2 values at inspired oxygen concentrations (FiO2) of 0.21, 0.50, and 1.00. TcPO2 measured at room air decreased nonlinearly in relation to flow with a marked drop occurring below 20% of baseline flow. TcPO2 measured at increased FiO2 was dependent primarily on PaO2 at flow rates greater than 50% of baseline. With reduction in flow below 25% of baseline, TcPO2 was dependent solely on flow and was not augmented by increases in PaO2. The data suggest that TcPO2 can accurately reflect changes in blood flow to an extremity when flow is severely restricted.     

Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model

BACKGROUND: We evaluated the effect of autologous marrow stromal cells (MSCs) on neovascularization and blood flow in an animal model of chronic limb ischemia. METHODS: Chronic hind limb ischemia was created by ligating the left common iliac artery of male Lewis rats. Three weeks after ligation, 5.0 million LacZ+MSCs (n = 10) or culture medium (n = 10) were injected into the anteromedial muscle compartment of the left thigh. At 4 and 6 weeks after injection, half the animals (n = 5) from each group underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtain a flow ratio. The animals also underwent angiography and measurements of blood vessel density and arteriolar density. Qualitative histologic assessment of the limb muscles was performed. RESULTS: LacZ+MSCs were found to differentiate into endothelium (F VIII+), vascular smooth muscle (positive a-smooth muscle actin), skeletal muscle (positive desmin), and adipocytes. Ischemic hind limbs where MSCs were implanted had greater vascular density and arteriolar density than control limbs (p < 0.001). Femoral artery flow index (left femoral artery flow/right femoral artery flow) was 0.89 +/- 0.12 and 0.90 +/- 0.06 for rats injected with MSCs measured at 4- and 6-weeks, respectively, compared with 0.50 +/- 0.15 and 0.50 +/- 0.10 for the control rats (p < 0.001). Angiography demonstrated reconstitution of the left femoral artery in rats that received MSC implantation through pelvic and abdominal wall collateral formation. CONCLUSIONS: Local MSC implantation induces a neovascular response resulting in a significant increase in blood flow to the ischemic limb. Marrow stromal cells are also capable of spontaneously regenerating the various components of muscular tissues.     

Vascular endothelial growth factor gene delivery by magnetic DNA nanospheres ameliorates limb ischemia in rabbits

BACKGROUND: Critical limb ischemia often leads to disability and limb loss. Vascular endothelial growth factor (VEGF), delivered either as recombinant protein or as gene therapy, has been shown to promote arteriogenesis and angiogenesis in animal models of limb ischemia. However, most of the studies used a nonspecific targeting system. MATERIALS AND METHODS: Magnetic DNA nanospheres containing expression plasmids encoding VEGF were synthesized, and their morphology, magnetropism, and stability were analyzed. The magnetic DNA nanospheres were administrated via an artery into a rabbit limb ischemia model. The expression of VEGF and vascularization were examined by immunohistochemistry. The angiography was taken to evaluate arteriogenesis. RESULTS: Magnetic DNA nanospheres were very stable and showed a high magnetropism. Gene delivery of such nanospheres via artery under a magnetic field led to the overexpression of VEGF in situ. The capillary density and capillary to muscle fiber ratio were doubled compared with those of the control animals. The arteriogenesis also was promoted in VEGF gene therapy group compared with controls but at later interval than capillary angiogenesis. CONCLUSIONS: Our results suggest that intra-arterial VEGF gene delivery by magnetic DNA nanosphere promotes angiogenesis and arteriogenesis and presents a potent therapeutic strategy for critical limb ischemia.     

H-Wave® effects on blood flow and angiogenesis in longitudinal studies in rats

Effects of repeated H-Wave? device stimulation (HWDS) on blood flow and angiogenesis in the rat hind limb were studied. The hypothesis tested was that HWDS acutely increases hind limb blood flow, and that repeated HWDS would elicit angiogenesis. Animals were HWDS-conditioned (``Conditioned') or sham-stimulated (``Sham') (n = 5/group) daily for 3 weeks. The contralateral limb in both groups served as the control. Each animal was injected with bromodeoxyuridine (BrDU). After 3 weeks, rats were anesthetized and iliac artery blood flow was measured bilaterally before, during, and after acute HWDS. HWDS of the Conditioned limbs elicited a 247% increase in blood flow above resting conditions compared to a 200% increase in control legs. Sham animals did not demonstrate between-leg differences in flow. Hindlimb musculature staining for BrDU revealed angiogenesis in Conditioned versus Sham groups. Flow changes accompanying HWDS corroborated earlier microvascular findings demonstrating a significant striated muscle arteriolar dilation with HWDS.     

Effect of electrical stimulation on arteriogenesis and angiogenesis after bilateral femoral artery excision in the rabbit hind-limb ischemia model

The effects of electrical stimulation (ES) on arteriogenesis (the opening of preexisting collaterals) and angiogenesis (formation of new capillaries) were studied after acute bilateral hind limb ischemia was induced via bilateral femoral artery excision in a rabbit model. The study evaluated the rabbit hind limbs' normal response to acute ischemia and to application of ES by calculating changes in arterial and capillary densities. Comparisons were made with our prior study, in which the femoral artery was unilaterally excised, as we attempted to expand on the topics of arteriogenesis and angiogenesis. Twelve adult New Zealand white rabbits were randomly assigned to 1 of 2 series. In Series 1, the control group, both femoral arteries were excised and no ES was applied. In Series 2, both femoral arteries were excised and ES was applied to the left limb. One lead was implanted into the left adductor muscle near the site of the excised left femoral artery (Series 2), and a stimulator (Thera, Medtronic, Inc, Minneapolis, MN) was implanted in a separate pocket. ES was applied at a rate of 3 V, 30 contractions per minute, beginning immediately after surgery and continuously for 1 month. Angiography was performed in all 12 rabbits 1 month after surgery to establish the anatomy of the collateral vessels and to demonstrate that the femoral artery stump continued to be an end artery. Contrast-opacified arteries (COAs) that crossed the grid's midline, and the total number of grid lines intersected by COAs, were tallied according to an established method. Capillary density was calculated as the number of capillaries per square millimeter of muscle. In Series 1, after 1 month, the number of COAs crossing the grid's midline was 4.5 +/-1.5 on the left and 4.8 +/-1.2 on the right side. In Series 2, the number of COAs crossing the grid's midline was 7.9 +/-1.8 on the left side (p<0.05 vs Series 1) and 5.9 +/-1.6 on the right side of the same rabbit (p=NS vs Series 1). In Series 1, 36.7 +/-5.4 and 30.5 +/-7.7 total intersections were crossed by COAs on the left and right sides, respectively. In Series 2, total grid intersections crossed by COAs were 48.4 +/-8.5 and 47.5 +/-9.1 in the left and right sides, respectively (p<0.001 vs series 1). Baseline capillary density before femoral artery excision was 180.2 +/-21.3/mm(2). The capillary densities on the left sides were 94.2 +/-19.1 and 264.5 +/-7.6 in Series 1 and 2, respectively (p<0.001). The right sides showed a similar pattern with capillary densities of 88.5 +/-37.2 and 135.8 +/-6.8 (p<0.05) in Series 1 and 2, respectively. When capillary density was compared on the left and right sides of the same rabbit in Series 2, a statistically significant increase was also found; 264.5 +/-7.6 vs 135.8 +/-6.8 (p<0.001) in the left and right sides, respectively. Comparisons of the effect of electrical stimulation and the body's normal physiologic response to acute ischemia revealed a significant increase in the opening of preexisting collaterals (arteriogenesis) and the promotion of capillary density (angiogenesis) with the use of electrical stimulation.     

Effect of a nitric oxide donor on microcirculation of acutely denervated skeletal muscle during reperfusion

The authors have shown that exogenous nitric oxide (NO) protects innervated skeletal muscle against reperfusion injury. This study further evaluated the effects of exogenous NO donor on denervated skeletal muscle. Forty-eight denervated rat cremaster muscles underwent 3 hr of ischemia, followed by 90 min of reperfusion, and received systemic infusion of 100 nmol/min s-nitroso-n-acetylcysteine (SNAC) or an equal amount of phosphate-buffered saline (PBS). Results showed that the average diameter in 10 to 20 microm arterioles was between 107 percent and 123 percent of baseline in the SNAC group, and between 55 percent and 84 percent in the PBS group during 90 min of reperfusion. These values in 21 to 40 microm and 41 to 70 microm arteries were between 100 percent and 110 percent in the SNAC group, and between 70 percent and 90 percent in the PBS group from 20 to 90 min of reperfusion. Compared to the PBS group, the SNAC group had a statistically significantly greater vessel diameter in both 10 to 20 microm (p<0.001) and 21 to 40 microm arterioles (p<0.01) during 90 min of reperfusion, and in 41 to 70 microm arteries (p<0.02) from 20 to 90 min of reperfusion. The overall blood flow of the muscle in the SNAC group increased from 37 percent of baseline at 10 min to 108 percent at 40 min of reperfusion, and remained above baseline thereafter. In contrast, this value in the PBS group was only between 27 percent and 68 percent of baseline during 90 min of reperfusion. The blood flow was statistically significantly (p<0.03) greater in the SNAC group than in the PBS group from 40 to 90 min of reperfusion. Among the conclusions were: (1) NO donor SNAC improves the microcirculation of denervated skeletal muscle during early reperfusion; and (2) this protection against reperfusion injury is independent of innervation in skeletal muscle.     

Angiogenesis induced by the injection of peripheral leukocytes and platelets

BACKGROUND: Peripheral leukocytes and platelets (LAPs) contain many kinds of cells with the ability to secrete several growth factors and cytokines. We attempted to induce therapeutic angiogenesis by injecting self-LAPs into a rat ischemic hindlimb model. MATERIALS AND METHODS: Supernatants from cultured LAPs were used for the endothelial cell (EC) proliferation assay, and LAPs were used in a cornea model to evaluate angiogenic potency. LAPs were injected directly into the male Dark Agouti rat ischemic hindlimb model, after which a microangiogram was done and the capillary/muscle fiber ratio was examined histologically. ELISA revealed the levels of contributing growth factors and cytokines present in the ischemic muscles. RESULTS: The EC proliferation assay showed that the supernatants of LAPs accelerated proliferation and that the LAPs induced angiogenesis in the cornea model. The microangiograms and histological evaluation revealed that angiogenesis was induced more effectively in the rats injected with LAPs (LAP group) than in the those injected with phosphate-buffered saline (PBS group). The levels of basic fibroblast growth factor (bFGF) in the ischemia, PBS, and LAP groups were significantly increased compared to those in the sham group. The level of interleukin-1beta (IL-1beta) in the LAP group was significantly more elevated than in the other groups. CONCLUSIONS: The injection of self-LAPs induced angiogenesis in a rat ischemic hindlimb model. Ischemia caused an elevation in the level of bFGF and also in IL-1beta derived from LAPs, which contributed to angiogenesis. This is a novel, yet simple and safe method of inducing therapeutic angiogenesis.     

Blood flow measurements in lower limb arteries using duplex ultrasound.

The increase in blood flow in exercise is to provide more oxygen to tissues. The kinetics of flow at the common femoral artery bifurcation were established in normal subjects together with its relationship with oxygen uptake. Furthermore, the changes in flow were evaluated in patients undergoing superficial femoral artery angioplasty. After a known anaerobic test (Wingate test), normal subjects underwent a preferential increase in profunda femoris artery flow (ninefold increase), compared with superficial femoral artery flow (fourfold increase), indicating predominantly thigh exercise. The relationship between oxygen uptake and lower limb blood flow was studied before, during and after moderate intensity exercise. Oxygen uptake was measured by mass spectrometry and assessed by breath-by-breath analysis. The rate of increase for limb blood flow, as indicated by the time constant, was faster (28.8 +/- 4.4 s; mean +/- sem) than oxygen uptake (41.5 +/- 7.2 s) at the onset of exercise. This implies that limb blood flow is in excess of the oxygen requirements of muscle and therefore not the critical determinant for oxygen uptake by muscle. Flow in the lower limb arteries was measured before and after superficial femoral artery angioplasty in 22 patients. In addition, collateral blood flow was estimated using a mathematical model. Follow-up was carried out to 1 year. At 1 month, a significant decrease in profunda femoris artery flow (from 224 +/- 84 to 98 +/- 43 ml min-1, P < 0.05, paired t test) and a marked diminution in collateral flow (from 186 +/- 34 to 18 +/- 8 ml min-1, P < 0.05) was noted with no change in total limb blood flow. As expected, a significant increase in superficial femoral artery blood flow was seen (148 +/- 79 to 312 +/- 94 ml min-1, P < 0.05). From the studies, it can be seen that non-invasive duplex ultrasound flow measurements can reliably be obtained in the lower limb, allowing the kinetics of flow after exercise and the changes in flow after surgical intervention to be evaluated. This work provides a foundation for the study of oxygen kinetics and limb blood flow in athletes, the elderly and patients with peripheral vascular disease.     

Appropriate control of ex vivo gene therapy delivering basic fibroblast growth factor promotes successful and safe development of collateral vessels in rabbit model of hind limb ischemia

PURPOSE: In our previous study, adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promoted significant collateral vessel development in a rabbit model of hind limb ischemia. The present study examined how to control the efficacy and safety of this gene therapy, and also evaluated the feasibility of repeat application of this procedure. METHODS: Modified hFGF gene with the secretory signal sequence was adenovirally transferred to cultured autologous fibroblasts, and various numbers of the cells (2 x 10(5), 1 x 10(6), 5 x 10(6), or 2.5 x 10(7)) or vehicle was injected through the left internal iliac artery in rabbits in whom the left femoral artery had been excised 21 days previously. Twenty-eight days after cell administration, calf blood pressure ratio, angiographic score, blood flow in the internal iliac artery, and capillary density of muscle tissue were measured to analyze collateral vessel development and tissue perfusion in the ischemic limb. To assess delivery efficiency and viral contamination, the distribution of injected cells and the time course of blood anti-adenovirus antibody titer were examined in rabbits treated with various numbers of gene-transduced cells. In addition, animals received two injections, 21 days apart, of fibroblasts infected with adenovirus vector containing the luciferase gene, and luciferase expression was measured to evaluate whether the present therapy is repeatable. RESULTS: At 28 days after cell administration, significant collateral vessel development without detectable side effects was observed in rabbits who received 5 x 10(6) or 2.5 x 10(7) cells, compared with those who received vehicle, and no significant development was detected in animals with fewer than 5 x 10(6) cells (P <.01 for calf blood pressure ratio and capillary density, P <.05 for angiographic score and maximum blood flow). There was no difference in collateral augmentation between rabbits with 5 x 10(6) and 2.5 x 10(7) cells. However, in animals with 2.5 x 10(7) cells a large number of injected cells accumulated in the lungs, anti-adenovirus antibody titer increased significantly, and calf blood pressure in the left hind limb of two rabbits decreased immediately after injection. Luciferase analysis showed very low gene expression after repeated administration. CONCLUSION: These findings suggest that 5 x 10(6) is a suitable number of cells to induce appropriate collateral vessel development and minimize potential side effects of this procedure. Despite use of ex vivo gene transfer, repeat administration of the cells was not feasible.Clinical relevance Since the present study determined the appropriate conditions for effective and safe stimulation of collateral vessels, the clinical relevance of the ex vivo therapy might be carried forward. However, the findings raised another issue that should be resolved before clinical application; that is, the number of gene-transduced cells able to be injected was strictly limited. To estimate the therapeutic range of cell number in humans, additional experiments using large animals are desirable.     

Correlation of a simple direct measurement of muscle pO(2) to a clinical ischemia index and histology in a rat model of chronic severe hindlimb ischemia

PURPOSE: The lack of suitable experimental models of chronic severe limb ischemia and deficiencies in the available methods that allow for direct intermittent measurement of regional limb perfusion are obstacles to the evaluation of recently developed molecular strategies to reverse severe limb ischemia. Our aim was to develop a model of clinically relevant severe limb ischemia and correlate a simple direct measurement of muscle pO(2) to a clinical ischemia index, muscle mass, and capillary density. METHODS: Severe hindlimb ischemia was induced in 44 adult rats with ligation of the left common iliac artery, the femoral artery, and their branches. The effect of ischemia on muscle pO(2) was measured in the left gastrocnemius with room air and with 100% oxygen at 3, 10, 24, and 40 days after ischemia was induced. Clinical ischemia index, muscle mass, cellular proliferation, and capillary density also were assessed. RESULTS: The clinical ischemia index of the left limb was most severe at day 10, with evidence of pressure sores, a pale and dusky limb, and abnormal gait. With the rats breathing room air, muscle pO(2) was significantly lower in the left limbs than in the right limbs at days 3, 10, 24, and 40. After an oxygen challenge (100% O(2)), muscle pO(2) was significantly lower at 3, 10, and 40 days. At 3 days, the fraction of muscle mass per total body weight of the left tibialis anterior (TA) was significantly greater than the right TA as a result of edema and inflammation. By days 10, 24, and 40, the left gastrocnemius and TA masses were significantly less than the right as a result of muscle atrophy. Histopathology showed severe necrosis in the left gastrocnemius and TA on day 3. Inflammation was greatest by day 10. Necrotic muscle regenerated but remained atrophic at 40 days. The TA was slower to recover than the gastrocnemius. Capillary densities and capillary-to-muscle fiber ratios were greater in the ischemic limb than in the normal limb at day 24. Cellular proliferation as determined with bromodeoxyuridine labeling reagent staining was maximal in the ischemic limb at day 3. CONCLUSION: We have developed a rat model of chronic severe hindlimb ischemia with persistent ischemia as shown with a simple direct measurement of muscle pO(2) for up to 40 days. This model of severe hindlimb ischemia may be applicable for future studies of molecular strategies to treat severe limb ischemia in humans.     

caveolin-1表达在糖尿病大鼠下肢缺血中的作用及其与eNOS/NO通路的关系

背景与目的：研究表明caveolin-1可促进血管新生,并与糖尿病病理过程密切相关。本研究推测caveolin-1可能在糖尿病下肢缺血中发挥作用,并在糖尿病大鼠急性下肢缺血模型实验中探讨其作用及机制。 方法：选用80只健康雄性SD大鼠,应用STZ法构建1型糖尿病大鼠模型,取建模成功的大鼠随机分为4组,分别行单纯股动脉游离（假手术组）、股动脉及分支离断（模型组）、股动脉及分支离断+尾静脉注射含caveolin-1的质粒（转染组）、股动脉及分支离断+尾静脉注射不含caveolin-1质粒脂质体溶液（空转组）。于术后14 d取各组大鼠腓肠肌组织标本,HE染色观察组织形态及炎性细胞浸润情况;ELISA法检测组织中NO水平;免疫组织化学染色检测组织中caveolin-1、eNOS和CD34标记的微血管密度（MVD）。 结果：与假手术组比较,模型组及空转组的腓肠肌组织萎缩明显,组织中有较多的炎症细胞浸润,转染组肌肉萎缩不明显,炎症细胞浸润较少;模型组和空转组的腓肠肌组织中NO表达明显降低,转染组NO表达明显增加（均P<0.01）;模型组、空转组、转染组caveolin-1表达均明显升高,转染组升高更为明显（均P<0.01）;模型组和空转组eNOS表达无明显变化（均P>0.05）,转染组eNOS表达明显升高（P<0.01）;模型组、空转组、转染组MVD均有增加,转染组升高更为明显（均P<0.01）;以上指标在模型组与空转组间的差异均无统计学意义（均P>0.05）。 结论：caveolin-1高表达对糖尿病下肢缺血有明显改善作用,其机制可能是通过活化eNOS/NO通路,从而促进NO生成有关。     

Enhanced brain angiogenesis in chronic cerebral hypoperfusion after administration of plasmid human vascular endothelial growth factor in combination with indirect vasoreconstructive surgery

OBJECT: Vascular endothelial growth factor (VEGF) is a secreted mitogen associated with angiogenesis. The conceptual basis for therapeutic angiogenesis after plasmid human VEGF gene (phVEGF) transfer has been established in patients presenting with limb ischemia and myocardial infarction. The authors hypothesized that overexpression of VEGF using a gene transfer method combined with indirect vasoreconstruction might induce effective brain angiogenesis in chronic cerebral hypoperfusion, leading to prevention of ischemic attacks. METHODS: A chronic cerebral hypoperfusion model induced by permanent ligation of both common carotid arteries in rats was used in this investigation. Seven days after induction of cerebral hypoperfusion, encephalomyosynangiosis (EMS) and phVEGF administration in the temporal muscle were performed. Fourteen days after treatment, the VEGF gene therapy group displayed numbers and areas of capillary vessels in temporal muscles that were 2.2 and 2.5 times greater, respectively, in comparison with the control group. In the brain, the number and area of capillary vessels in the group treated with the VEGF gene were 1.5 and 1.8 times greater, respectively, relative to the control group. CONCLUSIONS: In rat models of chronic cerebral hypoperfusion, administration of phVEGF combined with indirect vasoreconstructive surgery significantly increased capillary density in the brain. The authors' results indicate that administration of phVEGF may be an effective therapy in patients with chronic cerebral hypoperfusion, such as those with moyamoya disease.