Glycemic control prevents neovascularization in the GK model of diabetes.

<p>(A) Vascular density is significantly increased in both cortex and striatum of GK rats and this was prevented by metformin treatment started at the onset of diabetes. (B) Vascular volume and (C) vascular surface area are also increased in diabetes. (D) Both micro and macrovascular volume as well as surface area (E) are increased in GK rats as compared to control. (F) Diabetic vascular correlations are extremely disproportionate and the slopes of the two lines are significantly different. (G, H) Immature cerebral microvessels are more abundant in diabetes. Representative images of cerebral striatal vasculature showing perfused (green-FITC dextran) and non-perfused vessels (Red-Isolectin). GK rats exhibit increased immature vasculature and glycemic control with metformin reduced the immature vasculature. F values are indicated under each ANOVA analysis group. *p<0.05 vs treatment, **p<0.01 vs control or treatment, # p<0.05 vs control, ***p<0.001 vs control or treatment. Mean ± SEM, n = 6−8.</p

Astrocytic structural alterations are prominent in the cortex of Lepr^db/db mice.

<p>(A) Representative images showing GFAP stained astrocytes (in red) wrapping around the vessels perfused with FITC (in green). (B) Diabetic mice show increased astrocytic surface density in the cortex compared to the control group. t values are indicated under each analysis group. *p<0.005 vs control. Mean ± SEM, n = 3−4.</p

Cerebral Neovascularization and Remodeling Patterns in Two Different Models of Type 2 Diabetes

<div><p>We previously reported intense pial cerebral collateralization and arteriogenesis in a mild and lean model of type 2 diabetes (T2D), Goto-Kakizaki (GK) rats. Increased cerebral neovascularization differed regionally and was associated with poor vessel wall maturity. Building upon these findings, the goals of this study were to determine whether a) glycemic control prevents this erratic cerebral neovascularization in the GK model, and b) this pathological neovascularization pattern occurs in Lepr^db/db model, which is the most commonly used model of T2D for studies involving cerebral complications of diabetes. Vascular volume, surface area and structural parameters including microvessel/macrovessel ratio, non-FITC (fluorescein) perfusing vessel abundance, vessel tortuosity, and branch density were measured by 3D reconstruction of FITC stained vasculature in GK rats or Lepr^db/db mice. GK rats exhibited an increase in all of these parameters, which were prevented by glycemic control with metformin. In Lepr^db/db mice, microvascular density was increased but there was no change in nonFITC-perfusing vessels. Increased PA branch density was associated with reduced branch diameter. These results suggest that T2D leads to cerebral neovascularization and remodeling but some structural characteristics of newly formed vessels differ between these models of T2D. The prevention of dysfunctional cerebral neovascularization by early glucose control suggests that hyperglycemia is a mediator of this response.</p> </div

Peripheral vascularization is severely impaired in Lepr^db/db mice.

<p>Representative images of blood vessels in gastrocnemius and soleus muscles as well as in the retina are given on top (A) and cumulative bar graphs are shown at the bottom (B). t values are indicated under each analysis group. *p<0.05 vs control. Mean ± SEM, n = 5.</p

Diabetes increases branch density, lumen diameter and tortuosity of PAs and its subsequent branches in the GK model of T2D.

<p>Representative images showing (A) vascular branching on PAs and surface cortical vessels taken under 10X, (B) inner vessel walls outlined using the Fiji software (Red arrows represent the Penetrating arterioles (PA) and the blue arrows depict the immediate branched from the PA (PA¹), outlined yellow), and (C) tortuous cortical vessels imaged under 25X objective. GK rats exhibit profound increase in branch density, diameter and tortuosity of PA and PA¹. While there was no significant change in branching of PAs with glycemic control, lumen diameter and tortuosity was reduced. F values are indicated under each ANOVA analysis group. *p<0.01 vs control, **p<0.001 vs control or treatment, ***p<0.0001 vs control or treatment. Mean ± SEM, n = 6−8.</p

Metabolic parameters of both the diabetic models.

*<p>p<0.001 vs control.</p

Glycemic control prevents impaired neovascularization in the skeletal muscle and retina in the GK model of diabetes.

<p>Representative images of blood vessels in gastrocnemius and soleus muscles as well as in the retina are given on top (A) and cumulative bar graphs are shown at the bottom (B). Glycemic control with metformin restores the peripheral blood vessels and prevents increased retinal vascularization. F values are indicated under each ANOVA analysis group. *p<0.05 vs control or treatment. Mean ± SEM, n = 5−7.</p

Evidence for neovascularization in the Lepr^db/db model of diabetes.

<p>(A) Not vascular density but (B) vascular volume and (C) vascular surface area are significantly increased in the cortex of Lepr^db/db mice. (D) Cortical microvascular volume and surface area are also increased. (F) Linear regression graph depicts the correlation between the vascular volume and surface area of the vasculature (G, H) There was no difference in the perfused/nonperfused vessel ratio in the Lepr^db/db mice in cortex. t values are indicated under each analysis group. *p<0.05 vs control, Mean ± SEM, n = 5.</p

Contribution of cannabinoid receptors activation to the vascular effects of anandamide.

<p>In <b>A</b>, the vessels were pretreated with a CB₁ antagonist (AM 251, 1 µM) and in <b>B</b> with a CB₂ antagonist (AM 630, 1 µM) for 30 minutes. The relaxation to anandamide was evaluated in U46619-precontracted mesenteric arteries from lean Zucker rats (LZRs) and obese Zucker rats (OZRs). Each point represents the mean ± SEM. *, P<0.05 vs. LZR. #, P<0.05 vs. respective group in the absence of blockade. N = 6/group.</p

Effect of obesity on cannabinoid receptors protein expression in mesenteric arteries.

<p>Panels show densitometric analysis of the Western blots for CB₁ and CB₂ protein expression in vessels from lean Zucker rats (LZRs) and obese Zucker rats (OZRs). In <b>A</b> and <b>B</b>, Western blots for CB₁ and CB₂ receptors, respectively. In <b>C</b>, Western blots for TRPV1 receptors. Results were normalized to β-actin expression and expressed as units of change from the control. Data are expressed as mean ± SEM. *, P<0.05 vs. LZR. N = 5/group.</p