肢体缺血再灌注后微循环变化及其发生机制的实验研究——1.兔足背肌腱表面微循环活体观察法的建立

我们对兔后肢趾长伸肌腱的血供系统进行了解剖观察,发现其环韧带以远接近跖趾关节的一段是制作微循环活体观察标本的理想部位。该处肌腱表面有丰富的微血管,且血管的背景为白色的肌腱,反衬明显;肌腱上方仅有皮肤和少量疏松组织覆盖,因此容易解剖。在此基础上,我们成功地创立了足背肌腱表面微循环活体观察法,为利用兔后肢缺血模型研究肢体缺血再灌注后的活体微循环变化开辟了一条新途径。     

肢体缺血再灌注后微循环变化及其发生机制的实验研究：1.兔…

我们对兔后肢趾长伸肌腱的供系统进行了解剖观察，发现其环韧带以远接近跖趾关节的一段是制作微循环活体观察标本的理想部位。该处肌键表面有丰富的微血管，且血管的背景为白色的肌腱，反衬明显；肌腱上方仅有皮肤和少量疏松组织覆盖，因此容易解剖。在此基础上，我们成功地创立了足背肌键表面微循环活体观察法，为利用兔后肢缺血模型研究肢体缺血再灌注后的活体微循环变化开辟了一条新途径。     

兔后肢缺血再灌注后足背肌腱表面微循环变化

应用活体显微镜技术，观察了兔后肢常温止血带缺血２ｈ（ｎ＝８）及５（ｎ＝８）再灌注后最初１ｈ期间足背肌腱表面微循环动态变化，尤其是白细胞内皮粘附及微血管灌注状况的变化，旨在探讨缺血再灌注损伤的发生机制，从而指导临床治疗。结果表明：①肢体缺血再灌注后缺血组织微静脉内皮上粘附的白细胞数显著增加，而且缺血时间越长，增加越显著。②肢体缺血５ｈ再灌注后，缺血组织的微循环并不能均匀恢复，部分区域发生＂无复流现象＂，包括原发性无复流和继发性毛细血管灌注衰竭两种形式。提示：①白细胞内皮粘附参与了缺血再灌注损伤的病理生理过程。②肢体缺血５ｈ再灌注后发生的局部组织损伤并非皆属缺血再灌注损伤，部分区域可能系单纯缺血性损伤，部分区域则可能属缺血再灌注一继发性缺血损伤。     

缺血再灌注对骨骼肌微血管及微循环的影响

为了研究缺血再灌注对骨骨各肌微血管和微循环的影响，采用气囊止血带，在常温下使兔后肢缺血２小时（ｎ＝８）和５小时（ｎ＝８）。在解除止血带后，通过活体显微镜观察再灌注最初１小时期间足背肌腱表面微循环变化，并于再灌注后１小时和７２小时，自胫前肌切取电镜和光镜标本，观察骨骨各肌微血管超微结构和组织形态变化。表现：①肢体缺血２小时再灌注后，虽然少数细静脉内可见轻度红细胞聚集和白细胞贴壁数增加，但腱外膜微循环可迅速恢复，分布也较均匀；骨骨各肌微血管仅有轻微损伤，肌纤维可存活。②肢体缺血５小时再灌注后，腱外膜微循环不能迅速而均匀地恢复，大部分区域发生无复流，细静脉内有大量白细胞粘附和聚集；骨骨各肌微血管内皮细胞显著肿胀，基底膜断裂，间质高度水肿，最终大部分肌纤维坏死。结果证明：缺血再灌注可导致微血管损伤和微循环紊乱，其严重程度取决于缺血时间的长短，并是决定缺血组织实质细胞转归的重要因素之一。     

兔后肢缺血再灌注后足背肌腱表面微循环变化

应用活体显微镜技术,观察了兔后肢常温止血带缺血2h(n=8)及5h(n=8)再灌注后最初1h 期间足背肌腱表面微循环动态变化,尤其是白细胞内皮粘附及微血管灌注状况的变化,旨在探讨缺血再灌注损伤的发生机制,从而指导临床治疗。结果表明:①肢体缺血再灌注后缺血组织微静脉内皮上粘附的白细胞数显著增加,而且缺血时间越长,增加越显著。②肢体缺血5h 再灌注后,缺血组织的微循环并不能均匀恢复,部分区域发生“无复流现象”,包括原发性无复流和继发性毛细血管灌注衰竭两种形式。提示:①白细胞内皮粘附参与了缺血再灌注损伤的病理生理过程。②肢体缺血5h 再灌注后发生的局部组织损伤并非皆属缺血再灌注损伤,部分区域可能系单纯缺血性损伤,部分区域则可能属缺血再灌注—继发性缺血损伤。     

肢体缺血再灌注后微动脉对去甲肾上腺素、乙酰胆碱及硝酸甘油反应性变化

目的 探讨肢体缺血再灌注后向血管痉挛的发生机制。方法 应用兔后肢止血带常温缺血模型及足背肌腱表面微循环观察法,观察肢体缺血２小时及５小时再灌注后微动脉对局部滴注血管滴注血管收缩剂、内皮依赖性血管舒张剂及内皮非依赖性血管舒张剂的反应性变化。结果 与缺血前比较,肢体缺血２小时再灌注后,微动脉对上述三种血管活性物质的及硝酸甘油的无显著性变化;缺血５小时再灌注后,微动脉对去甲肾上腺素的收缩反应仍无显著性变     

动静脉转流后微循环的研究

将兔双后肢制成缺血坏死模型，然后于股浅动，静脉间建立分期动静脉转流，通过微循环显微电视仪器观察发现，术后３周微循环中已到经静脉－静脉吻合支的正常血流，其中的流速和流量，亦在术后３～８周，逐步恢复至正常数值，经股浅动脉近侧段注入墨汁后，取患肢末端皮肤和肌肉作活组织检查，也可见到均匀分布于毛细血管中的碳微粒。     

碱性成纤维细胞生长因子促进兔缺血后肢血管新生的实验研究

目的:了解局部应用碱性成纤维细胞生长因子(bFGF)对促进兔缺血后肢血管新生的作用。方法:25只日本大耳白兔随机分2组,外科结扎切断各兔股动脉及其分支,制作兔后肢缺血模型。试验组各兔在缺血后肢肌肉内多次注射重组人bFGF蛋白(n=15);对照组给予等剂量生理盐水(n=10)。术后4周,各兔行腹主动脉造影观察侧支血管形成情况,取内收肌和腓肠肌肌肉行病理切片HE染色应用图像分析系统统计血管密度,并用免疫组织化学方法检测内收肌和腓肠肌中VEGF阳性表达的血管数。结果:试验组兔侧支循环血管条数、血管密度及VEGF阳性表达的血管数均大于对照组(P<0.01),缺血状态得到改善。结论:在兔缺血后肢肌肉中注射bFGF蛋白可促进血管新生,增加兔缺血后肢血液灌注,改善肢体缺血状态。     

几丁糖预防兔屈肌腱粘连的实验研究

以兔为实验模型，将后肢第二、三足趾ＩＩ区屈肌腱造成实验性损伤。实验组鞘内滴入２％几丁糖溶液０．２ｍｌ，对照组不放。下肢石膏管形固定。分别于３、６周后处死，行大体标本粘连等级分级，肌腱滑动距离，末节趾间关节活动角度和完成上述两项所耗的功生物力学测试，光镜和透射电镜观察。结果证明几了糖能促进组织正常修复，抑制成纤维细胞活动，具有预防肌腱粘连的作用。     

皮管透明窗微循环观察法

我们自行设计兔皮管透明窗微循环观察法，该透明窗具有体积小，密封，组织刺激性小，透光良好等优点。窗内血管清晰，微循环血流状态清楚，可辨别新生的微动静脉，可录相进行微机图像处理，特别是可在无麻醉影响下长时间进行活体微循环变化观察。可经窗在体视显微镜电视放大系统下连续观察植入皮管组织内的血管束的微循环重建过程，获得满意的实验结果。此方法也可用于不同动物，不同部位，植入不同组织的微循环连续观察。     

Effect of granulocytes on the microcirculation in free-flap surgery

Introduction: Impaired capillary perfusion may result in flap failure. Platelet emboli, vasospasm and/or polymorphonuclear leukocytes (PMNs) have been identified as possible causes. This study investigates the role of PMNs in causing impaired capillary perfusion in a free-flap model. Methods: Sprague-Dawley rats were injected with either anti-neutrophil serum or saline. Their cremaster muscle was isolated on its pedicle. After arterial repair and reperfusion, capillary perfusion was counted each hour for 6 h. Results: The number of PMNs was significantly reduced in the animals treated with anti-neutrophil serum. However, capillary perfusion did not improve in this group. Conclusions: These results demonstrate that depleting circulating PMNs does not counterbalance the reduction of perfused capillaries, i.e., does not increase their number. It is suggested that reduced capillary perfusion downstream from an anastomotic repair is not mediated by the presence of PMNs in the microcirculation. Received: 19 February 1998 / Accepted: 3 July 1998     

Innervation of the microcirculation

Neurogenic control of the peripheral circulation is accomplished by alterations in nerve discharge to the pre- and postcapillary vascular network in the various organs. The postganglionic sympathetic adrenergic nerves constitute the most important efferent pathway for neural control. The physiologic response of the microvasculature to neural influences depends on a number of factors: the pattern of distribution of the innervation to the microvessels is one of the more important determinants. In addition to its influence on the contractile state of vascular smooth muscle, the adrenergic nerves also have a trophic influence on the smooth muscle cells. Following surgical denervation of a vascular bed, the adrenergic nerve terminals degenerate, and subsequently reinnervate, the vasculature. During the period following denervation, a number of functional and morphologic changes occur in the smooth muscle. This review emphasizes those aspects of the structure and function of adrenergic nerves that may have particular relevance for microsurgery.     

The effect of photodynamic therapy on the microcirculation

Photodynamic therapy (PDT) is a new form of cancer therapy involving tumor localization by photosensitizing drugs such as dihematoporphyrin ether (DHE). Light irradiation of drug-sensitized tissue results in photoactivation of DHE and tumor necrosis. The mechanism of action is incompletely understood but involves the generation of singlet oxygen which produces cytotoxic effects on tissues containing the compound. In addition, microcirculatory aberrations have been described during PDT. We have studied the acute effects of PDT on the microcirculation using in vivo television microscopy of the rat cremaster. This has enabled us to observe the effects of PDT on both paired and unpaired arterioles and venules using two different wavelengths of activating light (blue, 450-490 nm, and green, 530-560 nm). For both wavelengths of activating light, significant reduction in blood flow of all vessels was seen during PDT. This, in combination with the formation and embolization of platelet thrombi, resulted in stasis of blood flow in 80% of arterioles and 90% of venules. Observation for 2 hr after the completion of photoactivation revealed reperfusion in 20% of the arterioles but none of the venules. Blood flow was reduced by a combination of vasoconstriction and platelet thrombus formation. Reducing the total activating energy from 120J/cm2 to 24J/cm2 significantly reduced the response in venules and the incidence of stasis in both arterioles and venules. We conclude that the photoactivation of DHE results in significant vasoconstriction and thrombosis of normal vessels and that if these effects are seen at later times after DHE administration and in tumor neovasculature they may contribute to the mechanism by which PDT results in tumor necrosis.     

Direct studies on the control of the renal microcirculation

Recent advances in videomicroscopy combined with the development of several innovative in vivo and in vitro preparations now allow for direct study of the renal microcirculation. Blood flow in vasa recta capillaries of the rat can be studied by using video-microscopy, and changes in the flow of red blood cells in the renal cortex and papilla can be continuously monitored by using laser-Doppler flowmetry. All elements of the renal microcirculation can be visualized with the in vitro perfused juxtamedullary nephron or hydronephrotic kidney preparations. Pressures, blood flows, and vascular diameters in individual vessels can be directly measured in these preparations. Renal arterioles can be microdissected from the kidneys of several species and cannulated and pressurized in vitro for the study of pressure-diameter relationships and vascular responses to different agonists. In addition, membrane potentials and intracellular ion concentrations can now be measured in isolated renal arterioles by using microelectrodes and fluorescence microscopy. Thus, it is possible to study the control of renal vascular tone at all levels of integration from signal transduction in isolated cells to direct analysis of the regulation of hemodynamics in different regions of the microcirculation of the kidney in intact animals.     

Aspects of the microcirculation.

The microcirculation is an important but little understood part of the cardiovascular system. As new techniques have been developed, more accurate information has become available concerning the changes in the microcirculation in both health and disease. A review has been made of some of the more important facets of the microcirculation of particular interest to surgeons. Reference is made to basic physiology, especially the mechanisms controlling flow in the microcirculation. The significance of changes in small blood vessels in hypovolaemic, septic, and progressive shock is also discussed, and the role of platelet aggregation in shock states is explored.