Drag-Reducing Polymers improve Vascular Hemodynamics and Tissue Oxygen Supply in Mouse Model of Diabetes Mellitus

Abstract

Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia and glucose intolerance caused by impaired insulin action and/or defective insulin secretion. Long-term hyperglycemia leads to various structural and functional microvascular changes within multiple tissues, including the brain, which involves blood-brain barrier alteration, inflammation, and neuronal dysfunction. We have shown previously that Drag-Reducing Polymers (DRP) improve microcirculation and tissue oxygen supply, thereby reducing neurologic impairment in different rat models of brain injury. We hypothesized that DRP could improve cerebral and skin microcirculation in the situation of progressive microangiopathies associated with diabetes using a mouse model of diabetes mellitus. Diabetes was induced in C57BL/6 J mice with five daily consecutive intraperitoneal injections of streptozotocin (50 mg/kg/day). Animals with plasma glucose concentrations greater than 250 mg/dL were considered diabetic and were used in the study following four months of diabetes. DRP (2 ppm) was injected biweekly during the last two weeks of the experiment. Cortical and skin (ear) microvascular cerebral blood flow (mCBF) and tissue oxygen supply (NADH) were measured by two-photon laser scanning microscopy (2PLSM). Cerebrovascular reactivity (CVR) was evaluated by measuring changes in arteriolar diameters and NADH (tissue oxygen supply) during the hypercapnia test. Transient hypercapnia was induced by a 60-second increase of CO₂ concentration in the inhalation mixture from 0 to 10%. Compared to non-diabetic animals, diabetic mice had a significant reduction in the density of functioning capillaries per mm³ (787±52 vs. 449±25), the linear velocity of blood flow (1.2±0.31 vs. 0.54±0.21 mm/sec), and the tissue oxygen supply (p<0.05) in both, brain and skin. DRP treatment was associated with a 50% increase in all three parameters (p<0.05). According to the hypercapnia test, CVR was impaired in both diabetic groups but more preserved in DRP mice (p<0.05). Our study in a diabetic mouse model has demonstrated the efficacy of hemorheological modulation of blood flow by DRP to achieve increased microcirculatory flows and tissue oxygen supply.

1. Introduction

Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia and glucose intolerance caused by impaired insulin action and/or defective insulin secretion. Long-term hyperglycemia leads to various structural and functional microvascular changes within multiple tissues, including the brain, which involves blood-brain barrier alteration, inflammation, and neuronal dysfunction [1]. Functional cerebral and peripheral microcirculation, such as decreased baseline regional cerebral blood flow (CBF) and impaired vasoreactivity to CO2, have been shown in diabetic patients [2, 3]. We have demonstrated previously that Drag-Reducing Polymers (DRP) improve microcirculation and tissue oxygen supply, thereby reducing neurologic impairment in different rat models of brain injury [4, 5]. We hypothesized that DRP could improve cerebral and skin microcirculation and tissue oxygen supply in the situation of progressive microangiopathies associated with diabetes using a mouse model of diabetes mellitus.

2. Materials and Methods

Animal procedures were approved by the Institutional Animal Care and Use Committee of the Lovelace Biomedical Research Institute under the Animal Protocol #FY20-067. Diabetes was induced in C57BL/6J mice ((The Jackson Laboratory, Bar Harbor, ME, USA) with five daily consecutive intraperitoneal injections of streptozotocin (50 mg/kg) as previously described [6]. Animals with plasma glucose concentrations greater than 250 mg/dL were considered diabetic and were used in the study following four months of diabetes. DRP (2 ppm) or saline was injected biweekly during the last two weeks of the experiment. A biweekly injection regimen was chosen as it has been shown previously that DRP concentration in the blood falls to 50% within 48 hours [7]. Cortical and skin (ear) microvascular cerebral blood flow (mCBF) and tissue oxygen supply (NADH) were measured by two-photon laser scanning microscopy (2PLSM). Cerebrovascular reactivity (CVR) was evaluated by measuring changes in arteriolar diameters and NADH (tissue oxygen supply) during the hypercapnia test. Transient hypercapnia was induced by a 60-second increase of CO2 concentration in the inhalation mixture from 0 to 10%.

DRP preparation.

Polyethylene oxide (PEO, MW ~4000 kDa) was dissolved in saline to 0.1% (1000 ppm), dialyzed against saline using a 50 kD cutoff membrane, diluted in saline to 50 ppm, slow rocked for ~2 hours, and then sterilized using a 0.22 μm filter [4].

Two-Photon Laser Scanning Microscopy.

For long-term in-vivo imaging of the mouse cortex, we used an optical clearing skull window using two clearing solutions without performing a craniotomy [8]. For skin imaging, the ear was shaved and treated with a skin-clearing solution [9] before each imaging. The number of perfused capillaries, microcirculation, and tissue oxygen supply were visualized using Olympus BX 51WI upright microscope and water-immersion XLUMPlan FI 20x/0.95W objective as previously described [4]. Excitation was provided by a Prairie View Ultima multiphoton laser scan unit powered by a Millennia Prime 10 W diode laser source pumping a Tsunami Ti: sapphire laser (Spectra-Physics, Mountain View, CA). Red blood cell flow velocity was measured in microvessels ranging from 3-50 μm diameter up to 500 μm below the surface of the parietal cortex and ear skin. NADH autofluorescence measurement was used to evaluate mitochondrial activity (metabolic status) and tissue oxygenation [10]. In offline analyses using NIH ImageJ software, three-dimensional anatomy of the vasculature in areas of interest was reconstructed from two-dimensional (planar) scans of the fluorescence intensity obtained at successive focal depths in the cortex (XYZ stack).

Optical clearing.

For skull optical clearing, solution 1 (saturated supernatant solution of 75% (ethanol and urea) was applied to the exposed skull for about 10 min to allow the skull to turn transparent [8]. Then, Solution 1 was removed, and Solution 2 (sodium dodecylbenzenesulfonate) was added to the same area for further clearing within 5 min. For skin imaging, the ear was shaved and treated with skin clearing solution (PEG-400 + Thiazone + Sucrose) before each imaging [9].

Cerebrovascular reactivity testing by hypercapnia challenge.

Changes in arteriolar diameters and NADH were measured during the hypercapnia test as previously described [11]. Transient hypercapnia was induced by a 60-second increase in CO₂ concentration to 10% in the inhalation mixture through the face mask. Each trial consisted of 3 minutes of baseline data acquisition, followed by 1 minute of the hypercapnia challenge, and 3 minutes of the post-hypercapnic surveillance period.

Statistical analyses were done using GraphPad Prism software (La Jolla, CA, USA by independent Student’s t-test or Kolmogorov–Smirnov tests where appropriate. Differences between groups and time were determined using a two-way repeated measures ANOVA analysis for multiple comparisons and post hoc testing using the Mann-Whitney U test. Variables are expressed as mean ± standard error. The level of significance was set at 0.05.

3. Results

At the baseline, the number of functioning capillaries per mm3 was 1020 ± 98 in the brain cortex and 954 ± 76 in the ear skin (Fig 1). Just before the treatment (14 weeks of diabetes), the number of functioning capillaries fell to 524 ± 69 in the brain cortex and 520 ± 58 in the ear skin without difference between groups (Fig 1).

Fig. 1.

Number of functional capillaries per mm³ of tissue. Left: Brain; Right: Ear skin. N=10 mice per group, (*p < 0.05).

Two-week DRP treatment led to a significant increase in the density of functioning capillaries per mm3 (787 ± 52), compared to vehicle-treated 449 ± 25 (Fig 1, p < 0.05). Similarly, the linear capillary blood flow velocity dropped from 1.38 ± 0.18 mm/s in the brain and 1.2 ± 0.19 mm/s in the skin to 0.71 ± 0.21 and 0.76 ± 0.2 in the brain and skin, respectively (Fig 2). After DRP treatment, capillary blood flow velocity re-stored to 1.2 ± 0.31 mm/sec and 1.0 ± 0.21 in the brain and ear, respectively, compared to 0.54 ± 0.21 and 0.62 ± 0.2 in the saline-treated mice (Fig 2, p < 0.05).

Fig. 2.

Capillary flow velocity (mm/s) Left: Brain; Right: Ear skin. N=10 mice per group, (*P<0.05).

Microvascular flow impairment led to tissue hypoxia as reflected by increased NADH autofluorescence in both, brain cortex and skin (Fig 3). Capillary perfusion improvement in DRP-treated mice led to enhancement of tissue oxygen supply 1.05 ± 0.04 and 1.04 ± 0.03 in the brain cortex and skin, respectively, comparing to 1.32 ± 0.06 and 1.22 ± 0.04 in the saline group (Fig 3, p < 005).

Fig. 3.

NADH autofluorescence (inversely reflecting tissue oxygen supply). Left: Brain; Right: Ear Skin. N=10 mice per group, (*P<0.05).

Baseline hypercapnia test showed that cerebral arterioles dilated during inhalation of 10% CO2 (by 44.0 ± 3.5%) and constricted to the pre-hypercapnia diameter after hypercapnia termination reflecting intact CVR (Fig 4). Diabetes led to the impairment of CVR that was improved by DRP (Fig 4, p<0.05). Simultaneous NADH autofluorescence imaging did not show any significant changes in tissue oxygen supply during hypercapnia at a baseline due to arteriolar dilatation and increased RBC traffic, reflecting intact microvascular CBF regulation. In diabetic animals, NADH autofluorescence during hypercapnia increased, reflecting decreased tissue oxygen supply due to impaired CVR (Fig 4). DRP mitigated the reduction in tissue oxygen supply during hypercapnia by improving CVR (Fig 4, p < 0.05).

Fig. 4.

DRP improve cerebrovascular reactivity as reflected by change in the diameter of the arterioles during hypercapnia test (left); and NADH autofluorescence dynamics during the hypercapnia test. N=10 mice per group, (*P<0.05).

4. Conclusions

Hemorheological modulation of blood flow by DRP increases the number of functioning capillaries, restores capillary perfusion, and improves tissue oxygen supply in the brain and skin in a mouse model of diabetes mellitus and can be potentially used as a supportive therapy.