Retinal Oxygenation in Diabetic Retinopathy

描述(申请人提供)：目前治疗糖尿病视网膜病变的方法并不完全成功。我们的理由是，如果患者在病程早期就能预测到患者发生视网膜病变的风险，那么治疗方法的开发和评估可能会比目前更快。我们开发了一种独特的功能磁共振成像(FMRI)技术，可以测量受试者呼吸Carbogen(95%O2：5%CO2)时玻璃体体液中氧分压(DeltaPO2)的变化。使用这一急性视网膜“压力”测试，我们已经表明，在糖尿病3-4个月(视网膜病变出现之前)的大鼠和小鼠中，Carbogen呼吸产生的视网膜DeltaPO2与正常相比显著降低。低于正常的DeltaPO2与缺氧的存在是一致的，但它还不能明确地解释为缺氧的一种衡量标准。此外，在3个月糖尿病大鼠中，氨基胍(AMG)治疗阻止了视网膜DeltaPO2的下降。已知AMG可以防止糖尿病大鼠视网膜病变的形成，并抑制诱导型一氧化氮合酶(INOS)和蛋白激酶C(PKC)的活性(除其他作用外)。假设：实验性糖尿病视网膜DeltaPO2低于正常与视网膜缺氧有关(目标1)，iNOS和PKC活性升高(目标2和3)，但先于组织病理学的出现。具体目的1)比较糖尿病大鼠糖尿病前、糖尿病后2周、糖尿病后3个月和9个月的视网膜PO2和DeltaPO2。将使用功能磁共振成像和Carbogen挑战来测量视网膜DeltaPO2。为了测量PO2，将一滴全氟碳液滴注入视网膜前玻璃体中，通过视网膜上方或下方，并进行19F磁共振波谱分析。具体目的2)比较选择性诱导型一氧化氮合酶抑制剂(L-NIL(L-N(6)-(1-亚氨基乙基)赖氨酸)治疗前后正常和糖尿病大鼠3个月和15个月时视网膜组织学变化，以及正常、糖尿病和糖尿病诱导型一氧化氮合酶基因敲除小鼠视网膜组织学改变。具体目的3)比较选择性PKC抑制剂(LY333531)治疗前后正常和糖尿病大鼠视网膜DeltaPO2(3个月时)和组织学(3个月和15个月时)。此外，我们还将比较正常小鼠、过表达PKCβII的转基因小鼠、糖尿病小鼠和糖尿病PKCβII基因敲除小鼠的视网膜DeltaPO2。拟议的研究结果将证明视网膜DeltaPO2的变化是否与视网膜缺氧或多种生化异常的发展有关，这些与糖尿病视网膜病变有关，但现有技术无法在体内测量。这些研究的结果可能导致开发一种非侵入性的实时方法来早期评估糖尿病视网膜病变及其治疗。

DESCRIPTION (provided by applicant): Current treatments for diabetic retinopathy are not entirely successful. We reason that therapies could be more rapidly developed and evaluated than is currently possible if a patient's risk of developing retinopathy could be predicted early in the course of the disease. We have developed a unique functional magnetic resonance imaging (fMRI) technique that measures the change in partial pressure of oxygen (deltaPO2) in the vitreous humor while the subject breathes carbogen (95% O2: 5% CO2). Using this acute retinal "stress" test, we have shown that carbogen breathing in rats and mice at 3-4 months of diabetes (before the appearance of retinal lesions) produces a significantly reduced retinal deltaPO2 compared to normals. A subnormal deltaPO2 is consistent with the presence of hypoxia but it cannot yet be unambiguously interpreted as a measure of hypoxia. In addition, in 3 month diabetic rats, aminoguanidine (AMG) treatment prevented the decrease in retinal deltaPO2. AMG is known to prevent retinal lesion formation in diabetic rats and inhibit the activity of both the inducible form of nitric oxide synthase (iNOS) and protein kinase C (PKC) (among other actions). Hypothesis: Subnormal retinal deltaPO2 in experimental diabetes is correlated with retinal hypoxia (Aim 1), and increased iNOS and PKC activity (Aims 2 and 3) but precedes the appearance of histopathology. Specific Aim 1) To compare retinal PO2 and deltaPO2 before, and at 2 wks, and 3 and 9 months of diabetes in rats. fMRI and a carbogen challenge will be used to measure the retinal deltaPO2. To measure PO2, a perfluorocarbon droplet will be injected into the preretinal vitreous over superior or inferior retina and 19F magnetic resonance spectroscopy performed. Specific Aim 2) To compare the retinal deltaPO2 (at 3 months) and histology (at 3 and 15 months) in normal and diabetic rats with and without treatment with a selective iNOS inhibitor (L-NIL (L-N(6)-(1-iminoethyl)lysine)), and in normal, diabetic, and diabetic iNOS knockout mice. Specific Aim 3) To compare the retinal deltaPO2 (at 3 months) and histology (at 3 and 15 months) in normal and diabetic rats with and without treatment with a selective PKC inhibitor (LY333531). In addition, we will compare retinal deltaPO2 in normal mice, transgenic mice that overexpress PKC beta II, diabetic mice, and diabetic PKC beta II-knockout mice. The results of the proposed studies will prove whether or not changes in retinal deltaPO2 correlate with the development of retinal hypoxia or multiple biochemical abnormalities that are associated with diabetic retinopathy but cannot be measured in vivo by existing techniques. The results of these studies could lead to the development of a non-invasive real time method for the early evaluation of diabetic retinopathy and its treatment.