Inhibition of NOS Does Not Affect Oxygen Dependent Capillary Blood Flow Response In Vivo
Authors: Fraser Graham Mathew , Milkovich Stephanie , Ellis Christopher Gerald 
Published: November 2023

Objective

To determine if nitric oxide (NO) plays a significant role in regulating blood flow, in response to changing oxygen (O2) conditions within skeletal muscle capillaries.

Methods

Five male Sprague‐Dawley rats, 140 – 190g rats were anaesthetized with pentobarbital, tracheotomized, ventilated, and maintained in a normotensive state. A laparotomy was performed and the left common iliac was catheterized with a cannula advanced retrograde to the distal abdominal aorta. The extensor digitorum longus muscle was blunt dissected and reflected onto a glass cover slip (dose response), or permeable membrane above a gas exchange chamber (O2 response) set within a microscope stage. The muscle was bathed in warm saline and isolated from the air via polyvinylidene chloride film and a glass coverslip. Dose response was determined in 3 animals by injecting a nitric oxide synthase inhibitor, L‐NMMA, in logarithmic increasing concentrations ranging from 10−5 to 1 mg/kg into the iliac artery. O2 dependent blood flow responses were determined in 2 animals. Gas concentrations (N2, CO2, and O2) within the exchange chamber were dynamically controlled using mass flow valves connected to a computer. The muscle was allowed to equilibrate for 30 minutes at 5% CO2, 5% O2 and 90% N2. O2 concentrations at the surface of the muscle were oscillated from 7%–12%‐2%–7% in a square‐wave with each level maintained for 60s. CO2 was held at a constant 5% with N2 composing the balance of gas within the exchange chamber. Video sequences of capillary blood flow at 10× magnification were recorded in multiple fields in each animal. Videos were analyzed offline using custom software written in MATLAB yielding frame‐by‐frame measurements of capillary velocity, hematocrit and red blood cell supply rate (SR).

Results

Capillary blood flow dose response for L‐NMMA was measured in 178 capillaries under baseline, and increasing concentrations of L‐NMMA. All L‐NMMA concentrations caused a significant decrease in SR compared to vehicle (Figure 1) with a 42% reduction at 10−2 mg/kg; higher doses did not result in a further decrease of SR. The time dependent O2 blood flow response was measured in 58 capillaries under baseline conditions and after infusion of 10−2 mg/kg L‐NMMA (Figure 1). An average phase lag of 20 s was observed in the SR response to O2 concentration changes in the chamber under both conditions. Phase lag was partially due to the intrinsic time delay of the gas delivery system. Average SR under 7% O2 concentration was 45% lower with L‐NMMA compared to control. High O2 caused a decrease in SR of 53% for control vs. 60% under L‐NMMA, normalized to the baseline mean SR at 7% O2. Low O2 caused an increase in SR of 14% and 47% with control and L‐NMMA respectively. Post oscillation, 7% O2 restored average SR to 93% and 100% of baseline in control and L‐NMMA respectively.

Conclusion

Alteration of tissue surface O2 causes an O2 dependent capillary blood flow response to both low and high O2 challenges that is preserved following the introduction of a nitric oxide synthase inhibitor. This suggests that NO does not play a significant role in regulating blood flow, in response to changing O2 conditions within skeletal muscle capillaries.