Asai Akihiro, Sahani Nita, Ouchi Yasuyoshi, Martyn Jeevendra, Yasuhara Shingo
Department of Anesthesiology and Critical Care, Shriners Hospital for Children, Massachusetts General Hospital, and Harvard Medical School, USA.
J Vis Exp. 2007(4):210. doi: 10.3791/210. Epub 2007 May 28.
Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy.
We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized, ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.
RESULT & CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.
在发明了许多不同的成像方式之后，体内微循环的调节因素和详细生理学仍未完全阐明。虽然许多宏观血流参数反映了流速，但在微观观察中，血流速度和红细胞（RBC）通量的变化并不呈线性关系。有报道称，在微观观察中，红细胞速度与红细胞通量、红细胞通量与血浆流量之间存在差异，并且外周组织中血流调节存在空间和时间异质性，这为使用活体显微镜对微循环调节进行更详细研究的需求提供了科学依据。
我们改进了Jeff Lichtman对小鼠胸锁乳突肌进行体内显微镜观察的方法。将小鼠麻醉、通气，并注射PKH26L荧光标记的红细胞用于显微镜观察。
荧光标记的红细胞可被宽视野显微镜很好地检测和区分。电刺激引起的肌肉收缩导致红细胞通量增加。对包括红细胞速度和毛细血管密度在内的其他参数进行量化是可行的。小鼠对手术、注射染色红细胞、显微镜观察和电刺激耐受性良好。未观察到肌肉或血管损伤，这表明我们的方法侵入性相对较小，适合长期观察。
