The microvasculature in skeletal muscle. I. Arteriolar network in rat spinotrapezius muscle

A quantitative analysis of blood flow dynamics in skeletal muscle requires a detailed picture of the microvascular network. This report presents an analysis of the arteriolar network structure in the spinotrapezius muscle of the rat. The microvasculature is visualized by injection of a carbon suspension and recorded in the form of photomicrographs with a complete reconstruction of the microvasculature on transparent overlays. The spinotrapezius muscle has several major feeding arterioles which supply blood into an extensive meshwork of interconnecting or arcading arterioles spanning the entire muscle. The connections from the arcade arterioles to the capillaries are provided by transverse arterioles, which branch from the arcades at regular intervals. Each transverse arteriole forms a single asymmetric dichotomous tree and within each muscle there is a wide range in the size of transverse arterioles. A new branching schema is proposed to describe the arteriolar network. A set of network parameters is derived and typical values of these parameters in the spinotrapezius muscle of the rat are provided.     

Penetration of the systemic blood pressure into the microvasculature of rat skeletal muscle

A series of arterial micropressure measurements in different skeletal muscles of the Wistar-Kyoto and spontaneously hypertensive rat is presented. The micropunctures were carried out with minimal surgical intervention through small skin incisions and the micropressures were recorded simultaneously with femoral artery pressures. The measurement sites were located at the entry points into the muscles for the proximal and distal supply arteries and at the midpoint of the arteriolar arcade bridge which directly connects these two supply arteries in the center of the muscle parenchyma. In contrast to feed artery pressure values from exteriorized muscles, which in the past have been reported to be as low as 40 mm Hg, the current mean pressure values are substantially higher and in the range between 70 and 100 mm Hg, equivalent to 70 to 90% of the mean systemic pressure. Systolic and diastolic values exhibit comparable trends to the mean pressures and they are similar in muscles at different locations in the body. Although in spontaneously hypertensive rats the absolute pressures were significantly higher compared with their controls, the normalized pressures were virtually identical at the locations used in this study. These data indicate that the absolute pressure in the central arteries of spontaneously hypertensive animals is reduced to a greater degree than in Wistar-Kyoto rats, while in both strains the major pressure reduction in skeletal muscle still occurs in the microcirculation.     

Age-related alterations in the arterial microvasculature of skeletal muscle.

This study investigated the possibility that the aging process results in alterations in the structure and/or functional reactivity of the microvessels that could contribute to increased resistance to blood flow in working skeletal muscle. Initially, latex casts were made of the cremaster muscle microvasculature in adult (12 mo) and senescent (24 mo) male Fischer 344 rats. Although the average diameter was not different between age groups, segmental length (distance between adjacent branches) increased significantly (3rd order) during aging. Additionally, in vivo experiments were performed to determine the response of the vessels to the topical application of norepinephrine and adenosine. There was no increase in vasoconstriction produced by norepinephrine; however, the vasodilation in response to adenosine declined dramatically (1st and 2nd order) with advancing age. It can be concluded that the increase in skeletal muscle vascular resistance during contraction in aged male rats could be explained by morphological changes and/or the diminished vasodilation elicited by adenosine.     

吸烟减少大鼠骨骼肌细胞胰岛素受体基因表达

应用RT-PCR及免疫组化技术分别检测实验大鼠骨骼肌细胞胰岛素受体基因mRNA及蛋白表达.结果显示正常吸烟组、高脂饲养吸烟组及糖尿病吸烟组大鼠骨骼肌细胞胰岛索受体基因mRNA的表达显著低于各自对照组(0.50±0.06对0.84±0.09,0.38±0.01对0.59±0.05,0.37±0.05对0.55±0.05,均P<0.01),蛋白含量亦明显低于各自对照组(6.99±0.53对8.89±0.36,5.17±0.29对7.53±0.53,2.16±0.56对5.03±0.79,均P<0.01),这可能是长期吸烟导致胰岛素抵抗的分子机制之一.     

Effects of hemorrhagic shock on the microvasculature of skeletal muscle

The relationship between arteriolar and venular dimensions and the progressive failure of the homeostatic mechanisms leading to irreversibility in hemorrhagic shock was evaluated in mammalian skeletal muscle (rat cremaster). The small distribution arterioles (diameter = 17 μm) were observed to lose their tone and vasomotion at irreversibility although at 15 min after hemorrhage they exhibited enhanced vascular activity. Slowing of flow was seen to occur in the large venules (100 μm) and late in shock in smaller venules (25 μm). Venular dilatation was adjudged to be the vascular defect associated with the onset of irreversibility. Muscle surface pH and PO₂ followed a course similar to that seen in unanesthetized subjects. The red cell aggregation seen in the venules during the low flow state was generally reversed after reinfusion of the shed blood and restoration of arterial pressure.     

Effect of dietary salt on the skeletal muscle microvasculature in Dahl rats

The purpose of this study was to identify microvascular alterations that could contribute to increased peripheral vascular resistance in the Dahl salt-sensitive rat with salt-induced hypertension. Intravital microscopy was used to study the spinotrapezius muscle arteriolar network in anesthetized salt-sensitive rats fed either a high salt (7% sodium chloride) or low-normal salt (0.45% sodium chloride) diet for 4 weeks. Age-matched Dahl salt-resistant rats on high and low-normal salt diets served as controls. The high salt diet had no effect on arterial pressure in salt-resistant rats but increased arterial pressure in salt-sensitive rats. Mean resting diameter of arcade arterioles in salt-sensitive rats on high salt diet was reduced by 25% compared with salt-sensitive rats on low salt or salt-resistant rats on either diet. After abolition of vascular tone with 10(-3) M adenosine, arcade diameters were comparable in all groups. No difference among groups was found in either resting or passive diameter of the more distal transverse arterioles. Measurement of vessel lengths and numbers in cleared muscle specimens revealed no differences among groups in the anatomic density of either arcade or transverse arterioles. These data suggest that a reduction in the resting diameter of arcade, but not transverse, arterioles may increase spinotrapezius muscle vascular resistance in hypertensive salt-sensitive rats. The similarity in vascular densities among groups indicates that structural rarefaction of arterioles does not contribute to any increase in spinotrapezius muscle resistance at this stage of salt-induced hypertension.     

Endothelin-a receptor antagonist treatment improves the periosteal microcirculation after hindlimb ischemia and reperfusion in the rat

OBJECTIVES: To examine the microcirculatory changes in the rat tibial periosteum after hindlimb ischemia and reperfusion and to evaluate the effects of endothelin-A (ET-A) receptor antagonist therapy in this condition. The healing and functioning of vascularized bone autografts depend mainly on the patency of the microcirculation, and the activation of ET-A receptors may be an important component of the tissue response that occurs during ischemia-reoxygenation injuries. METHODS: Wistar rats were subjected to 1 hour of hindlimb ischemia and 3 hours of reperfusion. The periosteal microcirculation was visualized by intravital fluorescence microscopy. The leukocyte rolling and adherence in the postcapillary venules and the functional capillary density of the periosteum were determined. Two separate groups were treated with the selective ET-A receptor antagonist BQ 610 or the novel ET-A receptor antagonist ETR-p1/fl peptide at the onset of reperfusion. RESULTS: Reperfusion was accompanied by a significant decrease in functional capillary density and by an increase in the primary and secondary leukocyte-endothelial cell interactions. ET-A receptor inhibition reduced the leukocyte rolling and firm adherence and attenuated the decrease in functional capillary density in both treated groups. CONCLUSIONS: ET-1 plays a major role in microvascular dysfunction in the periosteum during reperfusion. The ET-1-ET-A receptor system might be an important target for tissue salvage therapy in transplantation surgery.     

The microvasculature in skeletal muscle. IV. A model of the capillary network

A reconstruction of the capillary network in the rat spinotrapezius muscle was carried out using carbon-filled specimens. A new branching schema for the capillaries, consisting of individual capillary bundles, is proposed. Each bundle is provided with blood through transverse arterioles and drained by collecting venules. A capillary bundle may be divided into capillary bundle elements consisting of a transverse arteriole, a collecting venule, and the intermediate capillary network. A set of seven independent network parameters are proposed and measurements are provided for two rat strains: Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). A comparison of network parameters indicates a multifaceted difference between the strains. In SHR the number of capillary cross-connections is reduced, individual capillary lengths are greater, and vessel diameters are slightly larger.     

胰岛素对大鼠骨骼肌细胞脂联素受体基因表达的影响

目的了解体外胰岛素对原代培养大鼠骨骼肌细胞脂联素受体1表达的影响。方法体外原代培养骨骼肌细胞,应用SYBRGreenⅠ染料建立一种快速、可靠的实时定量PCR,对其主要要素进行优化。观察不同胰岛素浓度不同作用时间下,大鼠骨骼肌细胞脂联素受体基因表达水平的动态变化。结果建立敏感、特异、快速检测脂联素受体1mRNA的实时定量PCR方法,随着胰岛素浓度的增加,脂联素受体1表达逐渐降低。在较低浓度（胰岛素浓度〈1nmol/L）时,脂联素受体1表达的降低无统计学意义,当胰岛素浓度增加到10nmol/L及以上时,骨骼肌细胞脂联素受体1表达的降低有统计学意义（P〈0.05）,这种抑制作用1h后出现,24h后达到高峰。结论成功地建立SYBRGreenⅠ实时定量PCR检测脂联素受体基因的表达方法,体外高胰岛素对骨骼肌细胞脂联素受体1mRNA表达有抑制作用,并呈时间和剂量依赖性。     

Dietary protein deprivation decreases the serine phosphorylation of insulin receptor substrate-1 in rat skeletal muscle

Evidence has shown that protein malnutrition tends to increase peripheral insulin sensitivity, but the molecular mechanism underlying this increase is not yet clear. Here we show that, in rat muscle, the state of insulin receptor (IR) substrate-1 (IRS-1), a pivotal component of the signaling pathway of the IR, changes drastically according to protein supply. After rats were fed a protein-free diet (PF) or a 12% casein diet for 1 week, their IR and IRS-1 states were analyzed by immunoblotting using various antibodies. PF slightly increased the amount of IR without affecting the state of IR tyrosine phosphorylation. In contrast, PF decreased the amount of IRS-1 and markedly increased phosphorylation of IRS-1 tyrosine residues after insulin injection. Moreover, IRS-1 in PF rats exhibited faster mobility in SDS-PAGE as well as far less phosphorylation of Ser612 and Ser307, indicating hypophosphorylation on its serine residues. Results of additional experiments using energy-restricted (pair-fed) rats and streptozotocin-induced diabetic rats suggest that dietary protein deficiency by itself alters serine phosphorylation of IRS-1, while the up-regulation of tyrosine phosphorylation requires other factors, such as a reduction in basal plasma insulin. The serine dephosphorylation followed by up-regulation of insulin-dependent IRS-1 tyrosine phosphorylation in skeletal muscle of PF rats in vivo is similar to a phenomenon observed in cultured cells under restriction of amino acids in the medium. With these findings, it could be inferred that the reduction of serine phosphorylation contributes to the sensitization of IRS-1 to IR tyrosine kinase under protein malnutrition.     

The microvasculature in skeletal muscle. II. Arteriolar network anatomy in normotensive and spontaneously hypertensive rats

A quantitative comparison of the anatomical arrangement of arterioles in the skeletal muscle of mature (16 to 20-week-old) Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) is provided. In both species several feeding arterioles supply blood to a network of arterioles covering the entire muscle, designated as arcade arterioles. The connections from the arcade arterioles to the capillary network are provided by the transverse arterioles. Comparison of the spinotrapezius muscle of the WKY and the SHR shows several types of rearrangement of the network. In both species there is a wide distribution of vessel size in the arcade and transverse arterioles. The length of the arcade arterioles per unit muscle volume is higher in the SHR, forming a denser network. There are almost twice as many transverse arteriolar trees per unit tissue volume in hypertensive animals although on the average each transverse tree has shorter branches. No evidence for significant anatomical rarefaction was found among arcade and transverse arterioles. Arcade arterioles following maximal dilation were found to be 25% narrower in the SHR, whereas for transverse arterioles no differences in diameter could be detected under these vasodilated conditions.     

饮酒对大鼠骨骼肌胰岛素受体及其底物表达和磷酸化的影响

观察摄入不同剂量的酒精5个月后,大鼠骨骼肌胰岛素刺激后葡萄糖摄取能力和胰岛素受体(IR)、IR底物(IRS)1及IRS-2的表达及胰岛素刺激后酪氨酸磷酸化水平的变化,发现饮酒可降低骨骼肌胰岛素刺激后糖摄取,同时伴有IR、IRS-1和IRS-2表达及酪氨酸磷酸化水平代偿性上调。     

Size and distribution of endothelial plasmalemmal vesicles in consecutive segments of the microvasculature in cat skeletal muscle.

Vessels belonging to five individual microvascular units (arteriole-capillary-venule sequence) in the tenuissimus muscle of cat were identified in the electron microscope by utilizing a serial sectioning technique. Morphometric analyses were perormed on the plasmalemmal vesicle population in four defined vessel segments: terminal arterioles, arteriolar fourth of capillaries, venular fourth of capillaries, and postcapillary venules. The sizes and numbers of vesicles, classified as luminal, abluminal, and free vesicles, were assessed. The abluminal vesicles were regularly more numerous than the luminal ones in the same segment. There were markedly different patterns of vesiculation along the five microvascular units. This finding was interpreted tentatively as indicating a reactivity of the endothelial transport function to factors of an unknown nature in the local microenvironment.     

Energy-dependent uptake of 3,5,3'-triiodo-L-thyronine in rat skeletal muscle

The uptake of [125I]T3 in rat skeletal muscle was investigated by incubating intact soleus muscles with a tracer amount of [125I]T3. At 37 C [125I]T3 uptake increased asymptotically; at 60 min the muscle contained 10% of the total [125I]T3 or 0.238 +/- 0.021% per mg wet tissue. At 0 C the [125I]T3 uptake was 1/5 of that at 37 C. The specific [125I]T3 uptake, determined by subtracting the uptake in the presence of 10 microM unlabeled T3 from the total [125I]T3 uptake, attained a plateau after 60 min. Washout experiments, done by first incubating the muscle for 60 min at 37 C or 0 C with [125I]T3 and then at 0 C for 3 h with unlabeled T3, showed that 21 +/- 2% or 58 +/- 4% of the radioactivity, respectively, was released, indicating an intracellular location of the hormone after incubation at 37 C. Addition of increasing concentrations of L-T3, D-T3 and L-T4 caused a progressive inhibition of the [125I]T3 uptake; the 50% inhibitory concentrations being 400 nM, 7 microM, and more than 15 microM, respectively. Preincubation of soleus muscles with metabolic inhibitors almost completely inhibited [125I]T3 specific uptake, with oligomycin and antimycin causing 98 +/- 4% and 81 +/- 3% reduction, respectively. Monodansylcadaverine and bacitracin, inhibitors of receptor-mediated endocytosis, reduced the specific [125I]T3 uptake in a dose-dependent manner up to 67 +/- 3% and 62 +/- 2%, respectively. These results indicate the presence of a saturable, stereospecific, and energy-dependent process responsible, at least in part, for T3 uptake in rat skeletal muscle. This specific T3 uptake may be a receptor-mediated endocytosis process.     

Bone marrow-derived cells do not engraft into skeletal muscle microvasculature but promote angiogenesis after acute injury

The skeletal muscle is supported by a vast network of microvessels with the capacity to regenerate in response to injury. However, the dynamics of microvascular repair and the origin of reconstituted endothelial cells in the skeletal muscle are poorly understood. A growing body of literature exists to indicate bone marrow (BM)-derived cells engraft into regenerating vascular endothelium and muscle macrovasculature. Therefore, we investigated the extent of BM contribution to skeletal muscle microvasculature after acute injury. Because reporters and markers commonly used to trace donor BM cells are not endothelial specific but are also expressed by leukocytes, we generated novel BM chimeras utilizing Tie2-green fluorescent protein BM cells transplanted into CD31 and Caveolin-1 knockout recipients. In turn, we surveyed BM vascular contribution, not just by the presence of green fluorescent protein, but also CD31 and Caveolin-1, respectively. After stable BM reconstitution, chimera limb muscles were cardiotoxin (CTX) injured and examined 21 days post-injury for the presence of green fluorescent protein, CD31, and Caveolin-1. Acute muscle injury by CTX is characterized by initial microvasculature death followed by rapid endothelial regeneration within 14 days post-damage. Histological analysis of injured and uninjured contralateral limb muscles revealed a complete absence of BM engraftment in the muscle vasculature of wild-type and CD31/Caveolin-1 knockout chimeras. In contrast, F4/80(+) cells isolated from CTX-injured muscle, expressed endothelial-related markers and promoted angiogenesis in?vitro. Therefore, despite the absence of BM engraftment to regenerated skeletal muscle microvasculature, macrophages recruited after injury promote angiogenesis and, in turn, vascular regeneration.