Previous studies have shown that neutrophils, the NLR, and DR have a certain relationship with features of DR [10, 11]. Still, no studies are available for UWFA results. Therefore, this study aimed to explore the correlation between the different manifestations of retinopathy under UWFA and the systemic indicators of white blood cells. This study retrospectively analyzed the different features of UWFA images in DR patients and observed retinal microvascular lesions more extensively and deeply, reflecting the process of disease progression. Each image feature was graded and compared with the peripheral blood leukocyte system. The results suggest that the features of UWFA imaging in patients with DR are correlated with white blood cell population indexes. The study results further confirmed the inflammatory injury mechanism of DR and provided some insight for us to predict the degree of DR microvascular disease from the level of circulating white blood cells.
Many DR lesions are related to the peripheral retina, and a broader observation of the peripheral retina is very important for the screening, diagnosis, treatment, and prognosis of the disease . However, conventional angiography can only show 30°-50° of the retinal surface in a single view. For patients with DR, UWFA provides a better view of the peripheral retina than conventional fundus angiography . A retrospective study showed that UWFA could display an angiographic sign associated with DR known as peripheral vascular leakage (PVL), which is a late leakage of dye in the retinal arteries and veins, reflecting the breakdown of the blood-retinal barrier (BRB) in active retinopathy. In this hospital and during the study period, a Heidelberg Spectralis ultrawide field lens (102°)  was used for UWFA to observe a wider range of peripheral retinal changes and obtain a broader understanding of DR. On this basis, the four main imaging features (microvascular leakage, nonperfusion, neovascularization, and proliferative membranes) were analyzed. The dye leakage of retinal arteries and veins suggested the destruction of the BRB. When the nonperfusion of the retina is greater than 7 DD, new blood vessels can be generated and are proportional to the size of the nonperfusion area . Therefore, in this study, 7 DD was used as the basis for classifying nonperfusion areas in UWFA images. The progression of DR involves a long period of nonperfusion and hypoxia that induces new blood vessels and fibroproliferative membranes.
After tissue migration and activation of peripheral blood neutrophils under pathological conditions, cytokines, chemokines, matrix metalloproteinases, and other substances can be secreted, leading to tissue damage and immune cell infiltration . Some phenotypes also inhibit lymphocytes , which can promote the occurrence of chronic inflammation. To a certain extent, the NLR can reflect the dynamic balance between inflammation and the immune response [9, 11]. Previous studies have suggested that neutrophils impair the integrity of the retinal pigment epithelial barrier . In this study, microvascular dye leakage in UWFA images suggested BRB destruction and vascular endothelial function damage. The vascular endothelium performs important functions, including managing microvascular permeability, coagulation, inflammation, vascular tension, and neovascularization . Diabetes mellitus, hypercholesterolemia, and hypertension lead to a dysregulation of vascular endothelial L-arginine/nitric oxide synthase (eNOS) and resulting in vascular endothelial dysfunction . HbA1c is strongly associated with increased levels of circulating adhesion molecules (ICAM, VCAM, etc.), which are considered to be indicators of endothelial cell injury and are correlated with the severity of DR . This study showed that microvascular dye leakage was correlated with the proportion of neutrophils, monocytes, and the NLR in peripheral blood leukocytes. The results were therefore consistent with previous studies.
Damaged capillary endothelial cells, reduced tissue perfusion and oxygenation, and aggravated vascular damage occlusion resulted in no perfusion on UWFA. Schroder et al.  proposed for the first time in 1991 the concept that activated white blood cells lead to capillary obstruction and proved that granulocytes and monocytes were trapped in retinal capillaries. Compared with normal cells, white blood cells in diabetic patients, especially those in capillaries, release more reactive oxygen species, damage endothelial cells, and pericytes, and lead to retinopathy through oxidative stress [25, 26]. Elevated levels of inflammatory mediators caused by the accumulation of advanced glycation end products can also lead to persistent chronic inflammation of the retina, resulting in the activation of white blood cells, adherence to vascular endothelium, and extravasation [27, 28]. In addition, there is evidence that elevated white blood cell count within the normal range is associated with the occurrence of microvascular and macrovascular complications of diabetes . The results showed that the degree of capillary occlusion was correlated with the proportion of monocytes and eosinophils in peripheral blood. These conditions indicate that the retina progresses from vascular endothelial cell damage in the early stage to capillary occlusion in the later stage, and the ratio of eosinophils and monocytes in peripheral blood might reflect the course of the disease.
Monocytes are one of the major leukocyte subtypes and are also considered markers of inflammation . Animal studies have shown that when many monocytes enter the retinal tissue, the retinal pigment epithelium acts as a channel for monocytes to transport to the retina [31, 32]. Studies have shown that plasma levels of monocyte chemotactic protein-1 (MCP-1), which regulates monocyte chemotactic and inflammatory processes, are significantly increased in patients with DR . There is also an association between peripheral blood monocyte levels and the prevalence of DR . In this study, the range of retinal neovascularization and the presence of fibroproliferative membrane in UWFA images were significantly correlated with the proportion of peripheral blood mononuclear cells. Studies have shown that monocytes and macrophages might be involved in the process of angiogenesis in atherosclerosis [35, 36]. Monocytes in circulating blood are considered precursors to tissue macrophages, whose function is to transform into a variety of tissue-sensing macrophages during normal homeostasis and inflammation. After tissue injury, inflammation and macrophage aggregation are induced, and macrophage proliferation in situ and synthesis of vascular endothelial growth factors (VEGFs) are promoted. Subsequently, it promotes the growth of new blood vessels and activates fibroblasts to produce collagen . During DR progression, chronic inflammation leads to microvascular injury, which goes through the damage repair process involving macrophages. This process explains the results of this study well. The ratio of monocytes in peripheral blood is correlated with the degree of retinal neovascularization and proliferative fibrous membrane.
PDR is the final stage in the progression of DR and will lead to blindness [1, 2]. In the present study, only the eosinophil ratio was independently associated with PDR. Diabetes involves a dysregulated immunity, including eosinophils . Elevated eosinophil counts have been observed in both men and women with PDR . PDR might involve eosinophil-specific chemokines that play important roles in the inflammation observed in DR .
This study has some limitations. The sample size was small, and the patients were from a single center, leading to the possibility of regional differences. In this study, leukocyte-related indicators were studied, but other inflammatory markers in peripheral blood were not studied. In addition, the white blood cells could not be used to evaluate the damage of other diabetic target organs completely. The traditional direct evaluation of intraocular fluid is more valuable, but detecting inflammatory factors in intraocular fluid such as aqueous humor was not done for comparison. The extraction of intraocular fluid for detection is invasive, and a more safe, economical, and valuable detection method is necessary.