Neurovascular proximity in the diaphragm muscle of adult mice

Regional blood flow to the diaphragm muscle varies with the workload of inspiration. To provide anatomical insight into coupling between muscle fiber recruitment and oxygen supply, we tested whether arterioles are physically associated with motor nerve branches of the diaphragm.

Following vascular casting, intact diaphragm muscles of C57BL/6 and CD-1 mice were stained for motor innervation. Arteriolar networks and nerve networks were mapped (~2 microm resolution) to evaluate their physical proximity.

Neurovascular proximity was similar between muscle regions and mouse strains. Of total mapped nerve lengths (C57BL/6, 70 ± 15 mm; CD-1, 87 ± 13 mm), 80 ± 14% and 67 ± 10% were ≤250 microm from the nearest arteriole and associated predominantly with arterioles ≤45 microm in diameter. Distances to the nearest arteriole encompassing 50% of total nerve length (D(50)) were consistently within 200 microm. With nerve networks repositioned randomly within muscle borders, D(50) values nearly doubled (p < 0.05). Reference lines within anatomical boundaries reduced proximity to arterioles (p < 0.05) as they deviated from the original location of motor nerves.

Across two strains of mice, motor nerves and arterioles of the diaphragm muscle are more closely associated than can be explained by chance. We hypothesize that neurovascular proximity facilitates local perfusion upon muscle fiber recruitment.