Discussion
While endocannabinoids receptors have been shown to be expressed in various tissues of the eye, this study investigated aqueous concentrations of endocannabinoids in individuals with or without diabetes. We observed differences in the aqueous concentrations among individuals undergoing routine cataract surgery according to gender and diabetes status. In women without diabetes, higher levels of aqueous PEA and the sum of endocannabinoid were found compared with men without diabetes, and AEA was lower in women with diabetes compared with those without diabetes. The levels of AEA and OEA were lowest in women with diabetes with more significant retinopathy. When comparisons were made by severity of retinopathy, aqueous 2-AG levels were greatest in women with retinopathy, lower in men with no retinopathy and lowest in patients without diabetes. Diabetes status and ocular comorbidity suggested trends in lower aqueous levels of AEA. While the underlying cause and role of the differences in ocular concentration of these endocannabinoids remain speculative, we contend that this observation supports the need for further studies to investigate the potential role of ECS in the pathogenesis of diabetic retinopathy.
In the whole group, prior to gender stratification, we observed the aqueous concentration of 2-AG was slightly higher in patients with diabetes compared with those without diabetes, while the concentration of AEA was slightly lower in those with diabetes compared with those without diabetes, with PEA and OEA similar between patients with and without diabetes. A similar observation was noted when comparing those with and without ocular comorbidity. However, following gender stratification, the aqueous concentrations of AEA in women without diabetes were significantly higher than in both men and women without diabetes, with higher PEA and sum of endocannabinoid in women without diabetes compared with men without diabetes. Men without diabetes had the lowest values for each endocannabinoid, which were higher in men with diabetes. Our observation of the reduced aqueous AEA concentrations in diabetes is in contrast to a previous study which showed increased tissue levels of AEA in the retina, ciliary body and cornea.16 The reason for this is unclear, but the higher levels in women without diabetes may have produced this paradox and the gender of the samples was not specified. It is not known if non-steroidal anti-inflammatory drugs provided periprocedure may play some role in influencing the results as endocannabinoids are metabolised by cyclo-oxygenase 2 (COX-2).24 The ethanolamides AEA, OEA and PEA are hydrolysed by fatty acid amide hydrolase, which is inhibited by COX-2 inhibitors, and 2-AG is oxidised by COX-2 to active metabolites. COX-2 is increased in human diabetic and ischaemic retina as well as in retinal astrocytes of animal models of proliferative retinopathy and in cell lines grown under hypoxic conditions.25 Prostanoids are angiogenic and induce VEGF-A, and COX-2 inhibition can reduce angiogenesis26; therefore, the use of nepafenac drops preoperatively may have had an influence on both proinflammatory and anti-inflammatory mediators. 2-AG, which has a different metabolic pathway, was increased in more severe retinopathy in women, which compares with previous findings.16 A trial of nepafenac drops in patients without diabetes prior to cataract surgery would be informative.
Gender differences in circulating endocannabinoid have previously been reported.19 23 While the reason for gender differences seen in circulating endocannabinoid, and now in ocular endocannabinoid, is unknown, it is thought to be due to the interaction between sex hormones and the ECS. Previous studies have shown that oestrogen modulates the endocannabinoid activity.27 28 Thus, future studies to determine the role of endocannabinoid-based therapy in modulating the pathogenesis of diabetic retinopathy should include gender stratification. Unfortunately, we did not have clinical information on the levels of sex hormones, or any documentation of use of hormone replacement therapy or oral contraceptive pills.
The aetiological role of ECS in the pathogenesis of diabetic retinopathy is unclear. A previous study has reported the beneficial effects of CB1 receptor inhibition or genetic deletion27 in the development of diabetic retinopathy. In addition, CB1 receptor inhibition was shown to limit the vascular inflammation and cell death in a mouse model of diabetic retinopathy and in human retinal cell line exposed to high glucose,29 and attenuates hyperglycaemia-induced apoptosis in retinal pigment epithelial cells.30 Since diabetic nephropathy shares some pathogenic features of diabetic retinopathy which characterised diabetic microvascular diseases, it is important to note that the CB1 receptor is overexpressed by renal podocytes in animal models in both type 1 and type 2 diabetes.31 32 In contrast to our observation in the present study, reduction of 2-AG, which acts on both CB1 and CB2, was observed in the renal cortex from mice with early streptozotocin (STZ)-induced diabetes, and podocyte CB2 receptor expression is markedly downregulated in human biopsies from patients with advanced diabetic nephropathy.33 These previous data on diabetic nephropathy indicate that the protective CB2 receptor signalling is impaired while the detrimental CB1 receptor signalling is enhanced in diabetic microvascular diseases. It is likely that both hyperglycaemia and hypertension are important drivers of these changes since alterations as in cultured podocytes exposed to high glucose, were shown to increase CB1 receptor expression,34 while mechanical stress, mimicking glomerular capillary hypertension, downregulates CB2 receptors.33 35 By extrapolation, these differences may have an impact on the endocannabinoid ocular fluid concentration observed in the present study.
Some limitations to our study which may affect the interpretation of our study should be acknowledged. First, we do not have detailed metabolic and clinical parameters of study participants, such as haemoglobin A1c levels, 24-hour blood pressure levels, duration of diabetes, or concurrent glucose-lowering and blood pressure-lowering therapies. The latter is also relevant since therapies which reduce activation of the renin angiotensin system have been shown to attenuate diabetic retinopathy progression.36 Furthermore, active maculopathy and proliferative retinopathy must be controlled prior to cataract surgery, and all patients with diabetic maculopathy, preproliferative or stable treated proliferative diabetic retinopathy were commenced on the anti-inflammatory nepafenac drops a day before surgery to reduce the risk of pseudophakic macular oedema. The potential effects of these routine presurgery protocols to reduce inflammation on the ocular concentration of endocannabinoid are not known. A further limitation is the relatively small number of patients and the unbalanced higher number of female patients, which limit our full interpretation of the observed ocular endocannabinoid levels as well as stratification of patients according to degree of diabetic retinopathy and gender status. Nonetheless, we believe that the current study adds to the available literature regarding the role of endocannabinoid fluid in the pathogenesis of diabetic retinopathy. While previous intervention studies with CB1 receptor antagonists have been performed in animal models of diabetic nephropathy,31 34 37 we believe evidence derived from this study as well as from others should form a basis for future studies to investigate the effects of modulating the ECS in models of diabetic retinopathy.