Microvascular Blood Flow Response to Hyperinsulinemic‐Euglycemic Clamp in Zucker Diabetic Sprague Dawley Rat Model of Type 2 Diabetes

Abstract

ObjectiveQuantify and characterize the skeletal muscle microvascular blood flow in Zucker Diabetic Sprague Dawley (ZDSD) rat model of T2D at rest and during a hyperinsulinemic‐euglycemic clamp.HypothesisChronic hyperglycemia in the ZDSD rat model of T2D causes blunted capillary hemodynamic response to hyperinsulinemic‐euglycemic clamp compared to the age and diet matched control Sprague Dawley (SD) rats.MethodsBiweekly blood glucose and conscious blood pressure measurements were collected from 7‐ to 25‐week‐old male SD (n=12) and ZDSD (n=18) rats. At 27 weeks of age, animals were fasted overnight prior to intravital video microscopy experiments. On the day of experiment animals were anaesthetized with an intraperitoneal injection of pentobarbital, tracheotomized, and carotid and external jugular cannulas were inserted to monitor physiological status and for administration of fluids. The extensor digitorum longus (EDL) muscle of the lower hind limb was isolated, blunt dissected and reflected over a glass coverslip on the stage of an inverted microscope. Recordings of the EDL were completed at baseline and during hyperinsulinemic‐euglycemic clamp. Hyperinsulinemic‐euglycemic clamp was achieved by continuous intravenous infusion of insulin (Humulin R 2U/hr/kg) and a 50% glucose mixture to titrate blood glucose to euglycemia (5‐7 mM). Mixed blood samples were collected from a tail clip to determine systemic blood glucose. Analysis of intravital videos were completed offline using custom MATLAB software. ZDSD rats that had fasting blood glucose of less than 20 mM at 19 weeks of age were excluded from the hyperglycemic group. Animals exhibiting aberrant microvascular blood flow at baseline or during euglycemic clamp were excluded from the data set. All animal protocols were approved by Memorial University of Newfoundland’s Animal Care and Use Committee.ResultsHyperglycemic ZDSD rats had a significantly higher blood glucose than SD controls from 17 to 25 weeks old and at the beginning of euglycemic clamp (26.13 ± 0.97 and 5.95 ± 0.37, p < 0.0001, Fig 1A). SD rats had a significantly higher glucose infusion rate than ZDSD rats (22.58 ± 1.81 and 13.19 ± 2.76, p < 0.0001, Fig 1B). SD rats had a significant increase in red blood cell (RBC) velocity (149.8 ± 56.71 and 210.0 ± 62.14 µm/s, p = 0.0051, Fig 2A), RBC supply rate (SR) (12.49 ± 8.23 and 19.91 ± 12.25 cells/s, p = 0.004, Fig 2B) and RBC oxygen saturation(sO2) (38.05 ± 11.65 and 45.70 ± 10.13, p = 0.014, Fig 2D) between baseline and euglycemia. However, ZDSD animals had no significant difference in RBC velocity (178.1 ± 63.91 and 194.7 ± 37.69 µm/s, p = 0.3683, Fig 2A), RBC supply rate (14.63 ± 7.55 and 20.03 ± 7.46 cells/s, p = 0.0704, Fig 2B), hematocrit (31.03 ± 3.23 and 33.02 ± 6.22 %, p = 0.4309, Fig 2C), and RBC sO2 (61.91 ± 11.40 and 64.00 ± 11.59, p = 0.5324 Fig 2D). The RBC sO2 was significantly higher in ZDSD than SD at baseline (61.91 ± 11.40 compared to 38.05 ± 11.65, p = 0.0166, Fig 2D).ConclusionSD rats had a robust increase in capillary hemodynamics during hyperinsulinemic‐euglycemic clamp. However, the skeletal muscle capillary hemodynamics of ZDSD rats did not significantly change between baseline and during euglycemia. These finding suggests a potential impairment in skeletal muscle microvascular blood flow response in this type 2 diabetic rodent mode. The elevated RBC sO2 in ZDSD rats may indicate a dysregulation of capillary blood flow in the ZDSD model.