Introduction
Diabetic retinopathy, which occurs in both type 1 and type 2 diabetes, is fast becoming a worldwide epidemic. The global prevalence of type 2 diabetes is rapidly increasing, and diabetic retinopathy continues to be one of the leading causes of visual loss in adults aged 20–74 years in developed countries [
1]. Most individuals with type 1 diabetes develop diabetic retinopathy, and a growing number of those with type 2 diabetes now manifest it, with high blood sugar, insulin resistance and a relative lack of insulin for which limited therapies are currently available.
There are urgent needs to develop both prevention and intervention strategies. One major impediment to this is the lack of a well-characterised model of diabetic retinopathy in type 2 diabetes in which obesity is one of the main traits; such a model could drive a new mechanistic understanding and allow the validation of new therapeutic targets. The BTBR
ob/ob mouse is a well-established, robust model of diabetic neuropathy [
2] and diabetic nephropathy [
3] in type 2 diabetes. Published data on these mice at 22 weeks of age suggest that they also develop retinal thinning [
4]. We initiated an in-depth characterisation of the retinal phenotype of this model, aiming to describe the earliest pathology and define useful endpoints for pharmacology.
The obese gene (
ob; also known as
Lep) encodes the peptide hormone leptin, which is produced mainly from adipocytes to induce satiety. Preclinical studies on rodent models of type 2 diabetes have shown how leptin treatment can regulate glucose homeostasis [
5,
6] and reduce body weight and food intake [
7]. However, leptin treatment in obese human participants with type 2 diabetes did not have any weight loss effects and only marginally reduced blood glucose [
8,
9]. When the
ob allele of the leptin gene was crossed into the BTBR mouse strain, this BTBR
ob/ob mouse developed obesity due to a lack of appetite control [
10,
11] and manifested characteristics of type 2 diabetes such as progressive insulin resistance, hyperglycaemia and glucose intolerance [
12,
13].
The first clinical features of diabetic retinopathy to be recognised are retinal microvascular abnormalities. The initial signs are non-proliferative, such as rupture of blood vessels, capillary dilation dysfunction and microaneurysms. In addition, increased vascular permeability and degeneration are important in the development of retinopathy and visual impairment in diabetes. Furthermore, progression of the retinopathy leads to rosary-like or beading abnormalities of retinal veins [
1]. The retinal microvessel network in BTBR
ob/ob mice has previously been studied using in vivo optical coherence tomography (OCT)/microangiography [
4], revealing some similarities with human diabetic retinopathy. These authors concluded that, although the capillary density did not differ from that of wild-type mice and no microaneurysms occurred, retinal blood flow was significantly lower. They also reported that the thickness of the nerve fibre layer/inner plexiform layer (NFL/IPL) was reduced in the same adult mice. This conclusion was supported by another study as an event prior to a reduction in retinal function in the diabetic
db/db mouse [
14]. The retinal thinning occurs as a result of progressive neuronal alterations such as loss of synaptic activity and dendrites, apoptosis of neurons in the IPL and ganglion cells, and activation of microglial cells [
15,
16].
The purpose of the current study is to provide an in-depth characterisation of the progression of diabetic retinopathy in BTBR ob/ob mice in terms of its vascular, neurodegenerative and inflammatory manifestations. This should prove useful for both elucidating early neurodegenerative disease mechanisms in the retina and providing a platform for preclinical drug discovery.
Discussion
We describe here a detailed analysis of retinopathy in a mouse model of type 2 diabetes and obesity, which has shown pathological features consistent with human diabetic retinopathy. These included retinal microvascular changes together with preceding early retinal neurodegeneration and inflammation.
Until now, there has not been a thorough documentation and characterisation of the progression of retinal pathology in type 2 diabetes associated with chronic obesity and insulin insensitivity. This is due in part to the lack of rodent models that can reflect the chronic pathology of human diabetic disease in a time frame suitable for reproduction in laboratory studies. The mouse model of type 2 diabetes and diabetic retinopathy that we describe here shows a relatively early onset of diabetes; in our model retinal functional deficit and inflammatory changes occur at 6 weeks of age. In comparison, other genetic mouse models of diabetic retinopathy, such as Ins2
Akita and NOD mice, which are models of type 1 diabetes, show an onset of disease at 8 and 12 weeks of age, respectively [
24‐
26]. Our model, therefore, presents an opportunity to study aspects of early retinal changes and progression of retinopathy that appear to be consistent with human diabetic retinopathy.
Obese mice lacking appetite control due to a genetic deficiency related to the hormone leptin (whether in the receptor or the ligand), which therefore affects its role in the insulin–glucose axis, have been studied with regard to the underlying pathology in diabetic tissue [
2], including the retina [
4,
14], revealing some of the pathological features of human diabetic retinopathy. However, although leptin receptor-deficient (
db/
db) mice have been evaluated for diabetic retinopathy, an extensive characterisation of retinal disease in BTBR
ob/ob leptin (ligand)-deficient obese mice has not yet been carried out. In comparison with
db/db mice,
ob/ob mice exhibit more than fourfold higher blood glucose levels than non-diabetic controls. Similar to
db/db mice,
ob/ob mice also show retinal function deficit, loss of ganglion cells, increased apoptotic cells in the same layer and upregulation of GFAP in Müller cells [
14]. In terms of measurement of retinal thickness using SD-OCT, our findings support those of other groups using the same animals but at an older, 22 week, time point [
4]. The pathological features we report here, such as neuronal dysfunction and loss, gliosis and para-inflammation with retinal leucostasis, microvascular changes and leakage, not only support already-published findings from other animal models of diabetes [
4,
14], but can also be considered to resemble many changes that are common to human pathology [
1,
27,
28].
A particularly important consideration with regard to correctly interpreting the mechanisms of diabetic retinopathy underlying mouse models of diabetic retinopathy is the combination, in our study, of the BTBR genetic background and leptin deficiency. Although it is known that the BTBR background leads to diabetes in part because it harbours alleles that promote insulin resistance and restrict hepatic lipogenic capacity [
12], the BTBR strain is also known to have a heightened, more reactive immune profile [
29]. This may explain the loss of retinal function and inflammatory changes we observed from an early stage of the development of diabetes. This manifests as an increased baseline expression of proinflammatory cytokine in the brain and an increased proportion of activated brain microglia [
29,
30]. This may explain our observation of a shift in the pattern or phenotype of IBA-1-labelled cells in the inner retina. Other groups have shown that dipeptidyl peptidase-4 (DPP4) inhibitors, which inhibit the degradation of endogenous glucagon-like peptide-1 (GLP-1), attenuate the production of proinflammatory cytokines and lower blood glucose in these mice, suggesting that the inflammatory process could be a promising therapeutic target [
31]. In other models of diabetic retinopathy, retinal leucostasis has been observed within days of the onset of diabetes, and was associated with damaged endothelial cells [
17]. This is supported in our study by the early Rho-Con A counts, leucostasis being shown to be twofold higher in diabetic mice than controls. The impact of this heightened inflammatory state on neuronal loss following increased glucose levels in the retina requires investigation.
In addition, it is known that the loss of presynaptic protein hinders synaptic function [
32], leading to impaired neuron-to-neuron transmission, which could explain the deficit in retinal function and axon abnormalities that we observed in the inner retina. This could be related to elevated GFAP expression, as reported here and previously [
33], but the trigger for neurodegeneration could be complex and stem from the malfunction of many pathways.
A key feature of diabetic retinopathy in BTBR
ob/ob mice is the microvascular changes, including vascular leakage and capillary endothelial cell loss—both hallmarks of human diabetic retinopathy [
28]. This aspect is significant given the long-standing evidence, from models of diabetic retinopathy in type 1 diabetes, that retinal vascular changes may be independent of neuronal damage [
33]. The BTBR
ob/ob model of type 2 diabetes develops the same changes observed in models of type 1 diabetes. The similarity of lesions observed in this model to those previously reported in models of type 1 diabetes is consistent with clinical evidence that there is a similar pathogenesis underlying the retinopathy in both type 1 and 2 diabetes [
34]. As glycaemic control has little effect on the development and progression of neurodegenerative changes in these animals [
35], the presence of both neuronal and vascular features may, by way of a targeted therapeutic manipulation, represent a robust platform allowing the interdependence or interaction of these distinct features to be understood. This has obvious implications for the evaluation of novel therapies.
We believe that BTBR ob/ob mice have an important role to play in future diabetic retinopathy research, including evaluation of the mechanisms that drive early neuronal and later retinal vascular changes. Our detailed characterisation of this mouse model should facilitate further examination of pathological mechanisms of type 2 diabetes diabetic retinopathy and the development of novel therapies for human diabetic retinopathy.