【摘要】目的：研究两组异构体 (R, R)XY1 到 (R, R)XY12 以及 (S, S)XY1 到 (S, S)XY12对兔眼血流的影响。方法：用彩色微球技术测量兔眼在高眼压下(40mmHg)各组织的血流情况。结果：12个 (R, R)XY 化合物以10g/L，50μL的剂量滴入兔眼，其中4个化合物可提高脉络膜血流。所有 (S, S)XY 系列的化合物对兔眼血流没有影响。结论： (R, R)XY 系列中一些化合物可以增加兔眼血流，这些可能对眼部血流降低相关疾病的防治有作用。在24个化合物中， (R, R)XY1 和 (R, R)XY9 最有药效。
【关键词】 眼血流；缺血；异构体
AbstractAIM: To evaluate the effects of two series of enantiomers [(R, R)XY1 through (R, R)XY12 and (S, S)XY1 through (S, S)XY12] on ocular blood flow in rabbits.
METHODS: Colored microsphere technique was used for in vivo experiments to determine the ocular blood flow in various tissues of ocular hypertensive (40mmHg) rabbit eyes.RESULTS: Of the twelve compounds of (R, R)XY series examined, four increased choroidal blood flow at 10g/L, 50μL instilled into eyes. All compounds of (S, S)XY series were not effective on ocular blood flow.CONCLUSION: Some compounds of (R, R)XY series increased the ocular blood flow, which might be useful for the prevention and treatment of ocular blood flow related eye diseases. Among all twentyfour compounds, (R, R)XY1 and (R, R)XY9 seem to be the most potent ones.
KEYWORDS: ocular blood flow; ischemia; enantiomer
INTRODUCTION
Because of a partial or complete loss of blood flow, there is an imbalance between the energy demands of the tissue in question and the supply of energy substrates in ischemia [1]. The malfunction of ocular circulation/blood flow is closely related to numerous eye diseases, including glaucoma, ischemic retinopathy, and agerelated macular degeneration (AMD), which results in irreversible morphological and functional changes[13]. Therefore, the search for drugs that can improve ocular blood flow is crucial. It is well known that nitric oxide (NO) raises CGMP via stimulation of guanylate cyclase which leadsto vasodilation and an increase in blood flow [49]. Application of NO donors for the improvement of ocular blood flow isparticularly important because it could be used for the treatment or prevention of the aforementioned eye diseases. It has been reported that ZX5, one of the numerous NO donors synthesized, could increase choroid blood flow stereospecifically in rabbit eyes [9]. In this study the compounds of (R, R)XY and (S, S)XY, as analogues of enantiomers (R, R)ZX5 and (S, S)ZX5 respectively, have been investigated to find out whether they are involved in the improvement of ocular blood flow (Figure 1). It is hoped that this study may lead to the discovery of some drugs that could be used for the prevention or treatment of aforementioned eye diseases.
MATERIALS AND METHODS
Materials All the compounds of (R, R)XY and (S, S)XY used in this study were synthesized by N1position modification of (R, R)ZX5 and (S, S)ZX5. The structures of (R, R)XY and (S, S)XY are shown in Table 1. The design, synthesis of these compounds will be reported elsewhere. Dimethyl sulfoxide (DMSO) was purchased from Sigma Chemical (St. Louis, MO).Ocular Blood Flow in Rabbits New Zealand white rabbits, weighing 2.53.0kg, were anesthetized with 35mg/kg ketamine and 5mg/kg xylazine intramuscularly. Half of the initial dose was given hourly to maintain anesthesia. An ocular hypertensive model was created by raising the intraocular pressure (IOP) of the left eye to 40mmHg which reduced the ocular blood flow to approximately 1/3 of the normal values. The left ventricle was cannulated through the right carotid artery for the injection of colored microspheres, and the femoral artery was cannulated for blood sampling. One percent drug solution (50μL) or vehicle (50μL) was instilled topically to the left eye, and the ocular blood flow of the ocular hypertensive rabbits was measured with colored microspheres at 0, 30, 60, and 120 minutes thereafter. At each time point, 2 million microspheres in 0.2mL were injected and blood samples were taken from the femoral artery for exactly one minute immediately following the injection of microspheres as a reference. The blood sample was collected in a heparinized tube, and the volume was recorded. The rabbits were euthanized with an injection of 100mg/kg pentobarbital sodium after the last blood sampling. The left eyes were enucleated and dissected into the iris, ciliary body, retina, and choroid. The tissue samples were weighed.
The details of sample processing and microsphere counting were provided by EZ Trac. In brief, hemolysis reagent was added to the microfuge tubes with the blood sample, then vortexed and centrifuged for 30 minutes at 6000r/min. The supernatant was removed, and tissue/blood digest reagents I and II were added. The tubes were capped, vortexed and centrifuged for 15 minutes at the same revolution as above. The supernatant was removed, and the microspheres were resuspended in a precise volume of the counting reagent. The number of microspheres was counted with a hemocytometer.
The tissue/blood digest reagent I was added to the microfuge tubes with the tissue samples, sealed, and heated at 95℃ for 15 minutes. The tubes were vortexed for 30 seconds, then reheated and revortexed until all tissue samples were dissolved. Reagent II was then added while the tissue samples were still hot. Then the tubes were capped, vortexed, and centrifuged for 30 minutes. The protocol thereafter, was the same as that used to process the blood samples, and the microspheres were counted. The blood flow of each tissue at a certain time point was calculated from the following equation: Qm = (Cm×Qr)/Cr, where Qm is the blood flow of a tissue in terms of μL/min/mg, Cm is the microsphere count per mg of tissue, Qr is the flow rate of blood sample in terms of μL/min, and Cr is the total microsphere count in the referenced blood sample.
Statistical Analysis All data were presented as mean±standard deviation (SD). Nonpaired Students ttest was performed to analyze the significance between two means at a certain time point. The differences were considered significant at P≤0.05.
RESULTS
When the IOP was raised from normal values around 1820mmHg to 40mmHg, the ocular blood flow reduced to 1/3 of the original values. The blood flow continued to reduce gradually over the time period of 2 hours during the experiments (Table 24, Figure 2,3).
The effects of (R, R)XY and (S, S)XY compounds on ocular blood flow were tested with (R, R)XY1 through (R, R)XY12, and (S, S)XY1 through (S, S)XY12. When 10g/L, 50μL of compounds were instilled to the eyes, the choroidal blood flow was significantly increased by (R, R)XY1, (R, R)XY3, (R, R)XY8 and (R, R)XY9 at all time points (30, 60, 120 minutes after drug instillation) as compared with corresponding controls (Table 2, Figure 2).
As for the blood flow in ciliary body, three out of the four compounds showed significant increase. (R, R)XY1 increased the blood flow significantly at 30 minutes and 60 minutes after drug instillation; (R, R)XY8 at 120 minutes thereafter; and (R, R)XY9 at all time points after drug instillation, whereas (R, R)XY3 did not affect the blood flow in ciliary body significantly (Table 3, Figure 3).
In case of the blood flow in iris, three out of the four compounds showed significant increase. (R, R)XY1 increased the blood flow significantly at 30 minutes and 60 minutes after drug instillation; (R, R)XY8 at all time points thereafter; and (R, R)XY9 at 60 minutes and 120 minutes after drug instillation, whereas (R, R)XY3 did not affect the blood flow in iris significantly (Table 4, Figure 3). Since the retina of rabbit contains very little vasculature, it is hard to show any drug effects on it.
Compared with (R, R)XY1, (R, R)XY3, (R, R)XY8 and (R, R)XY9, the corresponding enantiomers (S, S)XY1, (S, S)XY3, (S, S)XY8 and (S, S)XY9 did not show any effect on the blood flow at any time point in all tissues of the eyes after drug instillation (Figure 2).
Furthermore, other (R, R)XY and all (S, S)XY compounds did not show any effect on the blood flow at any time point in all tissues of the eyes after drug instillation.
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