Funk Kristina, Scheerer Nina, Verhaegh Rabea, Pütter Carolin, Fandrey Joachim, de Groot Herbert
University of Duisburg-Essen, Institute of Physiological Chemistry, University Hospital Essen, Essen, Germany.
University of Duisburg-Essen, Institute of Physiology, University Hospital Essen, Essen, Germany.
PLoS One. 2014 Oct 31;9(10):e111151. doi: 10.1371/journal.pone.0111151. eCollection 2014.
After severe muscle trauma, hypoxia due to microvascular perfusion failure is generally believed to further increase local injury and to impair healing. However, detailed analysis of hypoxia at the cellular level is missing. Therefore, in the present work, spectroscopic measurements of microvascular blood flow and O2 supply were combined with immunological detection of hypoxic cells to estimate O2 conditions within the injured muscle area.
Severe blunt muscle trauma was induced in the right Musculus gastrocnemius of male Wistar rats by a standardized "weight-drop" device. Microvascular blood flow, relative hemoglobin amount, and hemoglobin O2 saturation were determined by laser Doppler and white-light spectroscopy. Hypoxic cells were detected by histologic evaluation of covalent binding of pimonidazole and expression of HIF-1α.
Directly after trauma and until the end of experiment (480 minutes), microvascular blood flow and relative hemoglobin amount were clearly increased. In contrast to blood flow and relative hemoglobin amount, there was no immediate but a delayed increase of microvascular hemoglobin O2 saturation. Pimonidazole immunostaining revealed a hypoxic fraction (percentage area of pimonidazole-labelled muscle cells within the injured area) between 8 to 3%. There was almost no HIF-1α expression detectable in the muscle cells under each condition studied.
In the early phase (up to 8 hours) after severe blunt muscle trauma, the overall microvascular perfusion of the injured area and thus its O2 supply is clearly increased. This increased O2 supply is obviously sufficient to ensure normoxic (or even hyperoxic) conditions in the vast majority of the cells.
严重肌肉创伤后，一般认为微血管灌注衰竭导致的缺氧会进一步加重局部损伤并阻碍愈合。然而，目前尚缺乏细胞水平上缺氧情况的详细分析。因此，在本研究中，我们将微血管血流和氧气供应的光谱测量与缺氧细胞的免疫检测相结合，以评估损伤肌肉区域内的氧气状况。
使用标准化的“重物下落”装置对雄性Wistar大鼠右腓肠肌造成严重钝性肌肉创伤。通过激光多普勒和白光光谱法测定微血管血流、相对血红蛋白量和血红蛋白氧饱和度。通过对匹莫硝唑共价结合的组织学评估和HIF-1α的表达来检测缺氧细胞。
创伤后即刻直至实验结束（480分钟），微血管血流和相对血红蛋白量明显增加。与血流和相对血红蛋白量不同，微血管血红蛋白氧饱和度没有即刻升高，而是出现延迟升高。匹莫硝唑免疫染色显示缺氧分数（损伤区域内匹莫硝唑标记的肌肉细胞所占面积百分比）在8%至3%之间。在所研究的每种条件下，肌肉细胞中几乎检测不到HIF-1α表达。
在严重钝性肌肉创伤后的早期阶段（长达8小时），损伤区域的整体微血管灌注及其氧气供应明显增加。这种增加的氧气供应显然足以确保绝大多数细胞处于常氧（甚至高氧）状态。
