Poster P12 - Age-related increases in vascular resistance measured at the capillary level within the murine lung using a crystal ribcage

Introduction: The mechanical, biological, and immunological properties of the lung undergo progressive remodeling throughout normal aging, including changes to alveolar and capillary functions [1]. While histology or atomic force microscopy (AFM) quantify these changes at static endpoints [1,2], quantification of the dynamic changes that occur in response to mechanical or biological stimuli remains elusive, particularly over a well-controlled dynamic range in a functioning lung. Vascular distention – the change in diameter of capillaries and major vessels in response to pressure changes – is one such dynamic process. Mathematical models have predicted changes in vascular distension in response to exercise [3], and AFM studies have characterized age-related increases in stiffness [2], yet the direct experimental quantification of age-related changes in vascular resistance over a controlled pressure range remains unanswered.
We overcome these limitations by utilizing the recently developed crystal ribcage [4] to image the vasculature of a functional mouse lung across a range of ages ( < 1–80+ weeks). Using a range of biologically relevant vascular (0–15cmH2O) and alveolar (0–18cmH2O) pressures, we directly quantify vascular distention in arterioles/venules and capillaries of the functional lung. The optically transparent nature of the crystal ribcage – a polystyrene shell that follows the geometry of the chest cavity – enables real-time, high-resolution imaging of the vasculature response as the alveolar and circulatory conditions (e.g., pressure and flow) are precisely adjusted. Our approach presents a valuable measure of age-related biomechanical changes in respiration-circulation coupling with direct impact on trafficking and activities of circulating cells, including immune and cancer cells.

Materials and

Methods: Following tracheal intubation, the lung-heart bloc is removed from C57/BL6 mice. All ages except very old (80+ weeks) are from a transgenic B6.129 line with an intrinsic tdTomato membrane reporter. Closed-loop media perfusion is established via cannulas inserted into the pulmonary artery and left atrium. Pre-sacrifice, Evans Blue Dye is delivered intracardiac to label the vasculature lumen; media perfusate is additionally supplemented with Evans Blue Dye. The lung is placed inside a crystal ribcage imaging device [4] (Fig. 1A, Fig. 1B) and held under quasi-static inflation before vascular (perfusate) pressure is varied systematically from 0 cmH2O to 15 cmH2O. At each vascular pressure, alveolar (inflation) pressures are varied from 0 cmH2O to 18 cmH2O. 3D image stacks acquired via fluorescent confocal microscopy at each pressure capture the changes in vasculature at the varying pressures, before quantification is carried out using ImageJ software and custom-written Matlab scripts.

Results, Conclusions, and Discussions: Using the crystal ribcage, we image the vasculature of the functional mouse lung at high resolution in response to a range of alveolar and vascular pressures (Fig. 1C). We probe the various responses to varying pressures, including changes to septal wall and lumen thickness. Vessel response is heavily dictated by vascular pressure: as vascular pressure increases, the diameter of vessels increases in response (1D). In quantifying the changes in lumen thicknesses in both arterioles/venules and capillaries (Fig. 1E, Fig. 1F), we see notable aging effects emerging at the capillary level (Fig. 1F). These increases in diameter likely correspond to substantial decreases in vascular resistance as described by Poiseuille’s Equation: R=8ηl/πr^4, where resistance R is dictated by fluid viscosity η, vessel length l, and vessel radius r, as well as notable age-related increases in stiffness, particularly at the capillary level. Indeed, the 27% reduction in diameter from 12.86µm to 10.08µm shown here would result in an over 200% increase in vascular resistance. Such a dramatic increase would have considerable effects on mechanotransduction, signaling pathways, and fate of endothelial cells as well as circulating cells such as immune and cancer cells.

The crystal ribcage allows for the direct quantification of dynamic mechanical processes that occur in the lung over a biologically relevant and well controlled range. We have applied this technology to determine vascular distention in a functional lung across a range of ages ( < 7 days – 80+ weeks) for the first time, revealing notable differences in this property with aging. Our results hold valuable implications for the study of age-related degeneration of lung functional properties, and the subsequent age-related disease vulnerabilities and progression.

Acknowledgements (Optional): [1]: Schulte, H., et al., Front. Physiol., 2019; 10:1466
[2]: Sicard, D., et al., Am. J. Physiol. Lung Cell Mol Physiol., 2018, 314(6):L946-l955
[3]: Malhorta, R., et al., Circ. Heart Fail., 2016, 9(6)
[4] Banerji, R., et al., Nat. Methods, 2023; 20:1790-1801.