Ozagrel attenuates early streptozotocin-induced constriction of arterioles in the mouse retina

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Abstract

Retinal blood flow in human diabetics has been reported to follow a biphasic time course in which an initial period of reduced flow and ischemia is often followed by a hyperemic and angiogenic phase in which flow can exceed normal levels. The purpose of the present study is to investigate the mechanisms of the initial decrease in flow, since early interventions could provide the most effective treatment strategies. C57BL/6 mice were injected with streptozotocin (STZ) at 12 weeks of age and remained hyperglycemic until data were gathered 4 or 8 weeks later. Experimental measurements included retinal arteriolar red blood cell velocity and arteriolar diameters, with the diameters measured prior to and following an intravenous injection of the thromboxane synthase inhibitor ozagrel (100 mg/kg). Arterioles leading out of the optic disk constricted significantly at 4 weeks post-STZ (p < 0.001) compared to age-matched controls, but not at 8 weeks post-STZ. Calculations of retinal blood flow indicated a 45% decrease at 4 weeks post-STZ, but only a 26% decrease by 8 weeks. Not all arterioles constricted equally in response to STZ; the most substantial constrictions were present in arterioles that were more closely arranged with countercurrent venules leading back into the optic disk. Injection of ozagrel provided significant dilation of constricted retinal arterioles. In addition, the pattern of dilation was consistent with the sites of the most severe constriction, i.e., ozagrel-induced dilation in the STZ mice occurred to the greatest extent in the arterioles more closely paired with the venules draining the microvascular bed. In summary, STZ induces a biphasic alteration in retinal blood flow in mice, in which thromboxane contributes to the initial reduction in blood flow at 4 weeks. Moreover, the thromboxane-induced arteriolar constriction is dependent on the proximity of the retinal arterioles to countercurrent venules.

Introduction

Diabetic retinopathy (DR) affects hundreds of thousands of Americans over the age of 18 with Type 1 diabetes mellitus (Roy et al., 2004). Human DR often proceeds to proliferative diabetic retinopathy in which new blood vessel growth occurs on the surface of the retina (Fong et al., 2003, Yam and Kwok, 2007), which can interfere with vision. Few symptoms appear prior to the proliferative phase (Yam and Kwok, 2007); however, early treatment is critical to slowing the progression of DR (DCCT Group, 1995, Yam and Kwok, 2007). In early stages of DR, retinal blood flow is reduced substantially (Clermont et al., 1997), and areas of ischemia develop. The contribution of reduced blood flow and ischemia to the eventual disease progression needs further investigation, with the possibility that ischemia could lead to the production of potentially pathological mediators such as vascular endothelial growth factor. As the disease progresses, retinal blood flow increases toward control levels and even exceeds controls when the severity of retinopathy extends beyond microaneurysms only (Clermont et al., 1997).

Animal models of diabetes are used to study the early retinal consequences of hyperglycemia. Diabetic retinopathy is known as a microvascular pathology, and therefore several investigations in diabetic animals have focused on events such as microvascular accumulation of leukocytes and platelets, capillary dropout, altered retinal perfusion, and hypoxia (de Gooyer et al., 2006, De La Cruz et al., 1998, De La Cruz et al., 2000, Joussen et al., 2001, Linsenmeier et al., 1998, Moreno et al., 1995, Yamashiro et al., 2003). However, the mechanisms of early reductions in blood flow in animal models have not established the molecular mediators involved. One such potential mediator is the potent vasoconstrictor thromboxane, which has been implicated in the reduced capillary density found in the streptozotocin-induced rat model of Type I diabetes (De La Cruz et al., 1998, De La Cruz et al., 2000, De La Cruz et al., 2002, Moreno et al., 1995).

Thromboxane-induced vasoconstriction has been investigated in other animal models of inflammation, including ischemia–reperfusion (Mazolewski et al., 1999) and dextran sodium sulfate-induced intestinal inflammation (Harris et al., 2005). In these two models, the vasoactive influence of the constrictor appears to be dependent on the physical arrangement of arterioles and venules in the microvascular bed: thromboxane-induced vasoconstriction of arterioles depends on the proximity of the arteriole to the inflamed venules in which platelets and leukocytes accumulate.

In most microvascular beds of the body, arterioles and venules are found in a close, countercurrent pairing, which can be utilized in feedback regulation of blood flow. This regulation can take the form of venule-induced dilation, for example, in the functional hyperemia that delivers more blood flow upon demand (Hester and Hammer, 2002). In contrast, venule-dependent arteriolar constriction has been reported to occur with inflammatory conditions such as ischemia–reperfusion and hypercholesterolemia (Harris, 1999, Kim et al., 2007, Zamboni et al., 1993). In the retina, alternating arterioles and venules extend from (and into, respectively) the optic disk, and, therefore, it could be expected that venule-dependent modulation of arteriolar flow could be enhanced in the arterioles that are more closely paired with the draining venules.

Therefore, the aims of the present study were to: (1) investigate the extent of arteriolar constriction and retinal blood flow at early time points following induction of hyperglycemia (4 and 8 weeks); (2) determine whether the constriction of arterioles is mediated by thromboxane; and if so, (3) determine whether the thromboxane-dependent arteriolar constriction is more severe in arterioles that are more closely paired with draining venules.

Section snippets

Animals

Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 mice (Jackson Laboratories), 12 weeks old, were randomly chosen for intraperitoneal (i.p.) injection of streptozotocin (STZ; Sigma, St. Louis, MO; 200 mg/kg dissolved in pH 4.5 sodium citrate buffer) or sodium citrate buffer alone. Animals were injected within 15 min of mixing STZ into solution. Separate experimental groups were used for measurements of red blood cell

Animal data

On the day of STZ injection, there was no difference in body weight between the four groups of mice (Table 1). After 4 weeks and 8 weeks of hyperglycemia, body weight was statistically lower in STZ-injected animals compared with age-matched control animals on the day of experimental measurements. Streptozotocin induced an approximate 2–3 fold increase in non-fasting plasma glucose levels. The measurement range of the glucometer was limited to ≤600 mg/dl, and therefore we report median values (

Discussion

Retinal blood flow in human diabetics follows a biphasic time course of a substantial reduction followed by a later restoration and even increase (Bursell et al., 1996, Clermont et al., 1997). The mechanisms of the initial decrease in flow need to be elucidated, inasmuch as the resulting ischemia could lead to the production of inflammatory and angiogenic mediators such as vascular endothelial growth factor (de Gooyer et al., 2006). The present study demonstrates a biphasic time course of

Acknowledgments

This study was funded by the Juvenile Diabetes Research Foundation (1-2003-159) and by the National Institutes of Health (EY017599).

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