Intravital microscopy of the murine pulmonary microcirculation

Abstract

Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated BALB/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7- to 10-mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with alpha-cyanoacrylate. Removal of intrathoracic air via a trans-diaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min, with mean arterial blood pressure above 70 mmHg, and mean arterial Po(2) and arterial Pco(2) in the range of 90-100 Torr and 30-40 Torr, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50-microm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O(2)) or infusion of the thromboxane analog U-46619 were prominent in medium-sized arterioles (30- to 50-microm diameter), minor in small arterioles <30 microm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.

Intravital microscopy of the lung: minimizing invasiveness

“…However, physiological motion of the chest from breathing and a beating heart greatly impairs image acquisition and thus prevents the extensive quantitative analysis of dynamic phenomena observed in the [30] 1925 Lung clamping Light microscopy Cat, rabbit [22] 1930 Manual tracking of lung movements using a mobile microscope Light microscopy Dog, frog, alligator [31] 1933 Lung clamping Light microscopy Rabbit, cat, dog [21] 1934 Curare Light microscopy Cat [48] 1969 Suction Light microscopy Dog [23] 1993 Implanted thoracic window Videomicroscopy Rabbit [58] 1998 Implanted thoracic window Videomicroscopy Rabbit [37] 1999 Apnea Confocal Rat [36] 1999 Apnea Confocal Rat [24] 2000 Suction Videomicroscopy Rat [50] 2000 Suction Confocal Mouse [38] 2001 Apnea Videomicroscopy Rat [45] 2004 Suction Videomicroscopy Mouse [25] 2008 Apnea Videomicroscopy Mouse [26] 2009 Slow breathing; apnea; implanted thoracic window lungs of living animals. For this reason, and for several centuries, the investigation of dynamic phenomena at a microscopic scale in the lungs of a living animal has been uniquely based on the subjective interpretation of the experimenter's observation.…”

“…The animal preparation prior to imaging is performed according to this consensual protocol [11,18,[23][24][25][26][27][28][29]:…”

“…The use of this strategy allows investigation of vascular and circulatory disorders [25,[36][37][38], study of the microvascular endothelial activation in response to oxidative stress inducers (Figure 2c) [28], measurement of the pulmonary endothelial surface layer of mice [35,39], and the phagocytic capacity of pulmonary immune cells [40][41][42].…”

“…Adapted to lung IVM, this technique is generally used in association with other stabilization techniques [23,25,28,45,56]. To our knowledge, this technique has never been used for free-moving lung IVM of mice without slowing the respiratory rate (see section 'Oversampling of free-moving lung') because of the hardly-reproducible motion coming from the heartbeat-breathing combination.…”

“…While mouse physiology requires a breathing frequency of 106 bpm to 230 bpm, depending upon other factors such as strain and weight [57], it is possible to modulate this frequency to 130 bpm [28], 120 bpm [11,27,29], 100 bpm [25], or even 60 bpm for the recording period after which ventilation is returned to 100 bpm [26]. This decrease of the breathing frequency noticeably increases stability of the images.…”