微灌技术在调查微血管渗透性的大鼠肠系膜条例
Summary
The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.
Abstract
实验测量的单独灌注微血管渗透性属性提供的调节中培养的内皮细胞单层的整个微血管床功能交换性能血管通透性分子和细胞机制调查之间的桥梁。 A到导管插入并灌注大鼠肠系膜小静脉的微血管，测量微血管壁的渗透系数方法。所需要的主设备包括一个活体显微镜用支持显微操作以定位三个不同的微型工具的大改性阶段：（1）一个斜面玻璃微量吸管到导管插入并灌注微血管; （2）的玻璃微封堵以瞬时方框灌注和使经血管水流运动的测量在测量流体静压力，以及（3）一个钝玻璃棒以稳定肠系膜组织在插管的部位。修改后的兰迪斯微闭塞techniqUE使用红细胞悬浮在人工灌注液的液体经血管运动的标志，同时也使这些流动的实验条件变化以及水压和胶体渗透压差穿过微血管的重复测量得到精心控制。第一使用控制灌注液，然后经过重新插管相同微血管与测试灌流液的水力传导率的测量，使成对的这些精心控制的条件下的微血管应答的比较。尝试的方法延伸到微血管小鼠预期修改血管通透性遗传修饰的肠系膜是因为不存在的长的直链和无支链的微血管在小鼠肠系膜严重的限制，但在大鼠相似遗传修饰的最近情况使用所述CRISPR / Cas9技术开料调查的，其中本文描述的方法可以应用的新领域。 </P>
Introduction
微灌在脉管嗣继承建立经由微量吸管在通常小于40微米的直径的血管受控流动的已知组合物的人工灌注液。灌注容器保持在其正常组织环境内，并灌注动物的血液到插管的时间。当结合的范围内的视频成象或荧光技术，原位微灌用于支持跨微血管的壁水和溶质的流动的条件下，其中为这些流动的驱动力是已知的并且在血管壁的渗透性属性可以是测量直接评估。此外，通过控制周围组织中的微血管（灌注液和灌流）的流体的组合物，微血管通透性和交换的调节可以通过使内皮细胞形成的微血管壁被暴露于各种电子商务调查xperimental条件（激动剂，修改灌注的条件下，荧光指标来衡量细胞内的成分和信令）的时间精确测量周期（秒小时）。此外，关键的细胞的分子结构调节屏障的超微结构或细胞化学评价所用的相同的微血管，其中渗透性直接测量进行调查。该方法因此形成的细胞和分子机制的研究之间的桥梁来修改内皮屏障功能中培养的内皮细胞单层和调查在完整微血管。请参阅下面的评论作进一步评估1-6。
微灌的一个限制是，它只能用于在微血管床是薄的，透明的，并具有足够的结构完整性，以使插管用玻璃微量吸管。虽然早期的研究中使用的青蛙微血管肠系膜和薄皮肤心绞痛米uscle 7,8，迄今为止最常用的制备在哺乳动物模型是大鼠肠系膜9-15。大多数研究都集中在研究过1-4小时的时期血管通透性的急性变化，但最近的调查已经扩展到测量个体船舶24-72小时的初始灌注12,16后。最近开发的CRISPR技术，这将让更多的转基因大鼠模型可用于研究血管通透性第 17条应启用该通信所描述的方法，在这些重要的新模型大鼠肠系膜小静脉的微血管被应用。
该方法要求一个倒置显微镜配备有定制的显微镜阶段足以容纳动物制备既和用于位置微型工具靠近灌注容器中并对准一个灌注微量与容器的至少三个微操作内腔。例如定制平台的xy显微镜阶段（约90×60厘米）能够从一个1厘米厚的钢板用防锈涂料来制造。该级被连接到一个工程索引表或两个燕尾滑动在水平面安装成直角并支撑在特氟隆支柱或球转移运动。一个典型的钻机（ 见图 2）有许多共同之处用于一系列的活体微循环实验，如测量单血管的血液血管平滑肌的流量和红细胞压积，局部氧输送血液灌注微血管，调节显微镜和微定位设备肌张力，并注入到整个循环荧光示踪剂的局部微血管积累。18-26
该技术的基本方面是体积流量（j v）的横跨微血管壁的规定表面面积（S）的测量。去完成这通过本文所述的改性兰迪斯技术简单的倒置显微镜是足够的。一个小的摄像机安装在图像端口和视频信号上，具有添加的时间的基础上，被显示在视频监视器上，要么在计算机上的数字形式或者作为在视频记录器的数字或模拟信号记录。一旦微血管插管微血管可见的摄像机的一部分可以通过移动台和操纵器作为一个单元而不中断插管被改变。
经血管流动的测量，也可以使用与适当的过滤器一复杂的荧光显微镜更详细的调查，如用于溶质渗透率，细胞质钙或其它细胞机制，以及共焦成像6,12,13的荧光比率监测测量钻机组合， 27。所有微灌方法的主要优点是使重复测量的能力，同船根据驱动力，如静水和肿胀压力，或引起的变化血管反应，炎症性疾病的控制变化。最常见的设计是测量水力传导率（L p）的对经由填充有控制灌注液和红细胞悬液微量第一灌注的容器相同的容器中的配对比较，以建立基线渗透性状态，然后用第二吸移管与测试试剂加入到灌注液。多插管是可能的再灌注控制吸管经过反复的恶性循环。
本协议演示了一个小静脉血管的插管和微灌大鼠肠系膜记录水通量穿过微血管壁并测量血管壁的部分 L P，公用路径的渗透性为整个完好水和溶质的一个有用的指标内皮屏障。该过程被称为改性兰迪斯techniqUE因为原来兰迪斯原理用红细胞的相对运动作为灌注被阻止被保留28后经血管的液体交换的量度的，但实验条件的范围 （例如，横跨微血管壁的静水和白蛋白肿胀压力差异）微灌后可用远比uncannulated血液灌注微血管8,29更大。
Protocol
伦理声明：所有的程序进行审查和批准的机构动物护理和使用委员会。 微量移液器，限位装置和拦截1.初步加工使用电子牵拉调整，使得，当拉动时，在管的拉伸部分的长度约为1厘米和两半偏于对称拉在半几个清洁硼硅玻璃毛细管。确保该锥度是与图1的尺寸相一致。使用两个一半为微量，限位和阻滞剂。 使用斜角的气动砂轮30具有0.5微米的…
Representative Results
图4示出了结果从测量变化期L p的时间过程在大鼠小静脉微血管具有四个灌流液依次插管33以恒定的压力计算部分 L P的大小被用作变化微血管壁渗透性的量度，第一在用含有1％牛血清白蛋白那么当容器暴露使用含有10nM的浅滩的第二微量的抗炎剂缓激肽（BK）灌注液中的控制状态。缓激肽引起的对控制值返回在随后的10-15分钟一过性升高的部分 L P。然后…
Discussion
部分 L P的计算的详细情况。虽然经血管的流体运动发生同时容器自由地灌注，例如交换是太小期间自由灌注被测定，因为它通常是在容器的灌注率小于0.01％。然而，当灌注瞬时通过堵塞微血管，经血管流动停止（即，过滤）从标记红细胞在管腔作为流体的标记物红细胞和闭塞的位点之间的列中的运动测量缩短所示图3。根据实验方案的非支链容器600〜1000微米的?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是由卫生部资助HL44485和HL28607国家机构的支持。
Materials
| MICROSCOPE, TABLE AND STAGE | |||
| inverted microscope (metallurgical type) with trinocular head for video: example | Olympus | CK-40 | try to place eyepieces higher relative to stage–you have to look through eyepieces while reaching around to top of stage over intervening micromanipulators |
| inverted microscope (metallurgical type) with trinocular head for video: example | Leica | DMIL | try to place eyepieces higher relative to stage–you have to look through eyepieces while reaching around to top of stage over intervening micromanipulators |
| narrow diameter, long working distance objective: example | Nikon | Nikon E Plan 10×/0.25 LWD | |
| stage platform–1/2 inch or 1 cm sheet steel | welding shop | this should be heavy to reduce vibration | |
| Unislide x-y table: dove tail slides | Velmex | AXY4006W1 | |
| VIDEO | |||
| CCD video camera: example | Pulnix | TM-7CN (no longer available) | no color needed |
| video capture system with audio–generic | |||
| video playback system (completely still frame, single frame motion) | |||
| small microphone | |||
| MICROMANIPULATORS, HOLDERS | |||
| micromanipulator, XYZ (3) | Prior/Stoelting (no longer available) | look for fine Z, and larger range of travel in coarse drives for ease of positioning | |
| hydraulic probe drive, one way | FHC | 50-12-1C | need to buy either manual drive or electronic drive |
| manual drum drive | FHC | 50-12-9-02 | |
| or hydraulic drive, 3 way | Siskiyou Corporation | MX610 (1-way) or MX630 (3-way) | great for short arms, water filled and must be sent back for refill ~every 2 years |
| connectors/rods/holders | Siskiyou Corporation | MXC-2.5, MXB etc. | |
| pin vise | Starrett | 162C | to hold restrainer |
| pipette holder | World Prescision Instruments | MPH3 | |
| water manometer ~120 cm | |||
| MICROSCOPE TRAY | |||
| clear Plexiglas for microscope tray for animal | |||
| 3/4 inch polished quartz disc ~1/4 inch tall | Quartz Scientific Inc. | custom | (or polished plexiglass, glass); make sure the height is less than working distance of objective |
| Plexiglas glue (Weld-on 4: CAUTION CARCINOGEN) | |||
| medical adhesive for tissue well | NuSil | MED-1037 | |
| All-purpose silicone rubber heat mat, 5" L x 2" W | Cole Parmer | EW-03125-20 | heater for microscope tray–needs cord and controller–240V version available |
| Power Cord Adapter for Kapton Heaters and Kits, 6 ft, 120 VAC | Cole Parmer | EW-03122-75 | |
| STACO 3PN1010B Variable-Voltage Controller, 10 A; 120 V In, 0-140 V Out | Cole Parmer | EW-01575-00 | |
| PIPET MANUFACTURE | |||
| vertical pipette puller | Sutter Instrument Company | P-30 with nichrome filament | |
| 1.5 mm OD thin wall capillary tubing | Sutter Instrument Company | B150-110-10 | |
| pipette grinder air stone and dissection microscope–see reference in text | or purchase a package from Sutter Instruments or World Precision Instruments | ||
| RX Honing Machine, System II | RX Honing Machine Corporation | MAC-10700 Rx System II Machine | alternative for air stone, use with a dissecting microscope mounted at an angle |
| with ceramic sharpening disc | RX Honing Machine Corporation | use "as is" or attach lapping film | |
| lapping film sheets, 0.3 or 0.5 um | 3M | part no. 051144 80827 | 268X Imperial lapping film sheets with adhesive back–can be purchased from Amazon |
Riferimenti
- Curry, F. R. Permeability measurements in an individually perfused capillary: the ‘squid axon’ of the microcirculation. Experimental physiology. 93, 444-446 (2008).
- Curry, F. R., Adamson, R. H. Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge. Cardiovasc Res. 87, 218-229 (2010).
- Curry, F. R., Adamson, R. H. Tonic regulation of vascular permeability. Acta physiologica. 207, 628-649 (2013).
- Michel, C. C. Fluid exchange in the microcirculation. The Journal of physiology. 557, 701-702 (2004).
- Tarbell, J. M., Simon, S. I., Curry, F. R. Mechanosensing at the vascular interface. Annual review of biomedical engineering. 16, 505-532 (2014).
- Sarelius, I. H., Kuebel, J. M., Wang, J., Huxley, V. H. Macromolecule permeability of in situ and excised rodent skeletal muscle arterioles and venules. American journal of physiology. Heart and circulatory physiology. 290, H474-H480 (2006).
- Curry, F. E., Frokjaer-Jensen, J. Water flow across the walls of single muscle capillaries in the frog, Rana pipiens. The Journal of physiology. 350, 293-307 (1984).
- Michel, C. C., Mason, J. C., Curry, F. E., Tooke, J. E., Hunter, P. J. A development of the Landis technique for measuring the filtration coefficient of individual capillaries in the frog mesentery. Q J Exp Physiol Cogn Med Sci. 59, 283-309 (1974).
- Adamson, R. H., Zeng, M., Adamson, G. N., Lenz, J. F., Curry, F. E. PAF- and bradykinin-induced hyperpermeability of rat venules is independent of actin-myosin contraction. American journal of physiology, Heart and circulatory physiology. 285, H406-H417 (2003).
- Huxley, V. H., Rumbaut, R. E. The microvasculature as a dynamic regulator of volume and solute exchange. Clinical and experimental pharmacology, & physiology. 27, 847-854 (2000).
- Rumbaut, R. E., Wang, J., Huxley, V. H. Differential effects of L-NAME on rat venular hydraulic conductivity. American journal of physiology, Heart and circulatory physiology. , 279-H2023 (2000).
- Yuan, D., He, P. Vascular remodeling alters adhesion protein and cytoskeleton reactions to inflammatory stimuli resulting in enhanced permeability increases in rat venules. Journal of applied physiology. 113, 1110-1120 (2012).
- Zhou, X., He, P. Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca(2)(+)]i, nitric oxide, and gap formation in intact venules. American journal of physiology, Heart and circulatory physiology. 301, H1788-H1797 (2011).
- Adamson, R. H., et al. Oncotic pressures opposing filtration across non-fenestrated rat microvessels. The Journal of physiology. 557, 889-907 (2004).
- Adamson, R. H., et al. Epac/Rap1 pathway regulates microvascular hyperpermeability induced by PAF in rat mesentery. American journal of physiology, Heart and circulatory physiology. 294, H1188-H1196 (2008).
- Curry, F. E., Zeng, M., Adamson, R. H. Thrombin increases permeability only in venules exposed to inflammatory conditions. American journal of physiology, Heart and circulatory physiology. 294, H1188-H1196 (2003).
- Sander, J. D., Joung, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature. 32, 347-355 (2014).
- Bagher, P., Davis, M. J., Segal, S. S. Intravital macrozoom imaging and automated analysis of endothelial cell calcium signals coincident with arteriolar dilation in Cx40(BAC) -GCaMP2 transgenic mice. Microcirculation. 18, 331-338 (2011).
- Duza, T., Sarelius, I. H. Increase in endothelial cell Ca(2+) in response to mouse cremaster muscle contraction. The Journal of physiology. 555, 459-469 (2004).
- Oshiro, H., et al. L-type calcium channel blockers modulate the microvascular hyperpermeability induced by platelet-activating factor in vivo. Journal of vascular surgery. 22, 732-739 (1995).
- Chen, W., et al. Atrial natriuretic peptide-mediated inhibition of microcirculatory endothelial Ca2+ and permeability response to histamine involves cGMP-dependent protein kinase I and TRPC6 channels. Arteriosclerosis, thrombosis, and vascular biology. 33, 2121-2129 (2013).
- Harris, N. R., Whitt, S. P., Zilberberg, J., Alexander, J. S., Rumbaut, R. E. Extravascular transport of fluorescently labeled albumins in the rat mesentery. Microcirculation. 9, 177-187 (2002).
- Yuan, W., Li, G., Zeng, M., Fu, B. M. Modulation of the blood-brain barrier permeability by plasma glycoprotein orosomucoid. Microvascular research. 80, 148-157 (2010).
- Sugiura, Y., Morikawa, T., Takenouchi, T., Suematsu, M., Kajimura, M. Cilostazol strengthens the endothelial barrier of postcapillary venules from the rat mesentery in situ. Phlebology / Venous Forum of the Royal Society of Medicine. 29, 594-599 (2014).
- Guo, M., et al. Fibrinogen-gamma C-terminal fragments induce endothelial barrier dysfunction and microvascular leak via integrin-mediated and RhoA-dependent mechanism. Arteriosclerosis, thrombosis, and vascular biology. 29, 394-400 (2009).
- Dewar, A. M., Clark, R. A., Singer, A. J., Frame, M. D. Curcumin mediates both dilation and constriction of peripheral arterioles via adrenergic receptors. The Journal of investigative dermatology. 131, 1754-1760 (2011).
- Lee, J. F., et al. Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature. American journal of physiology, Heart and circulatory physiology. 296, H33-H42 (2009).
- Landis, E. M. Microinjection studies of capillary permeability. II. The relation between capillary pressure and the rate at which fluid passes through the walls of single capillaries. Am J Physiol. 82, 217-238 (1927).
- Curry, F. E., Huxley, V. H., Sarelius, I. H., Linden, R. J. . Techniques in cardiovascular physiology Part 1. P3/1, 1-34 (1983).
- Vurek, G. G., Bennett, C. M., Jamison, R. L., Troy, J. L. An air-driven micropipette sharpener). J Appl Physiol. 22, 191-192 (1967).
- Curry, F. E., Clark, J. F., Adamson, R. H. Erythrocyte-derived sphingosine-1-phosphate stabilizes basal hydraulic conductivity and solute permeability in rat microvessels. American journal of physiology, Heart and circulatory physiology. 303, H825-H834 (2012).
- Bagher, P., Polo-Parada, L., Segal, S. S. Microiontophoresis and micromanipulation for intravital fluorescence imaging of the microcirculation. Journal of visualized experiments : JoVE. , (2011).
- Adamson, R. H., et al. Attenuation by sphingosine-1-phosphate of rat microvessel acute permeability response to bradykinin is rapidly reversible. American journal of physiology, Heart and circulatory physiology. 302, H1929-H1935 (2012).
- Bates, D. O. Vascular endothelial growth factors and vascular permeability. Cardiovasc Res. 87, 262-271 (2010).
- Adamson, R. H., et al. Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice. The Journal of physiology. 539, 295-308 (2002).
- Curry, F. R., et al. Atrial natriuretic peptide modulation of albumin clearance and contrast agent permeability in mouse skeletal muscle and skin: role in regulation of plasma volume. The Journal of physiology. 588, 325-339 (2010).
- Neal, C. R., Bates, D. O. Measurement of hydraulic conductivity of single perfused Rana mesenteric microvessels between periods of controlled shear stress. The Journal of physiology. 543, 947-957 (2002).
- Adamson, R. H., et al. Albumin modulates S1P delivery from red blood cells in perfused microvessels: mechanism of the protein effect. American journal of physiology, Heart and circulatory physiology. 306, H1011-H1017 (2014).
- Huxley, V. H., Wang, J. J., Sarelius, I. H. Adaptation of coronary microvascular exchange in arterioles and venules to exercise training and a role for sex in determining permeability responses. American journal of physiology, Heart and circulatory physiology. 293, H1196-H1205 (2007).
- Huxley, V. H., Williams, D. A. Basal and adenosine-mediated protein flux from isolated coronary arterioles. Am J Physiol. 271, H1099-H1108 (1996).
- Davis, M. J., Gore, R. W. Double-barrel pipette system for microinjection. Am J Physiol. 253, H965-H967 (1987).
- Adamson, R. H., et al. Sphingosine-1-phosphate modulation of basal permeability and acute inflammatory responses in rat venular microvessels. Cardiovasc Res. 88, 344-351 (2010).
- Zeng, Y., Adamson, R. H., Curry, F. R., Tarbell, J. M. Sphingosine-1-phosphate protects endothelial glycocalyx by inhibiting syndecan-1 shedding. American journal of physiology, Heart and circulatory physiology. , H306-H363 (2014).
Citazione di questo articolo
Curry, F. E., Clark, J. F., Adamson, R. H. Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery. J. Vis. Exp. (103), e53210, doi:10.3791/53210 (2015).