BTBR ob/ob mouse model of type 2 diabetes exhibits early loss of retinal function and retinal inflammation followed by late vascular changes
Diabetic retinopathy is increasing in prevalence worldwide and is fast becoming a global epidemic and a leading cause of visual loss. Current therapies are limited, and the development of effective treatments for diabetic retinopathy requires a greater in-depth knowledge of disease progression and suitable modelling of diabetic retinopathy in animals. The aim of this study was to assess the early pathological changes in retinal morphology and neuronal, inflammatory and vascular features consistent with diabetic retinopathy in the ob/ob mouse model of type 2 diabetes, to investigate whether features similar to those in human diabetic retinopathy were present.
Male and female wild-type (+/+), heterozygous (+/−) and homozygous (−/−) BTBR ob/ob mice were examined at 6, 10, 15 and 20 weeks of age. Animals were weighed and blood glucose was measured. TUNEL and brain-specific homeobox/POU domain protein 3A (BRN3A) markers were used to examine retinal ganglion cells. We used immunostaining (collagen IV and platelet endothelial cell adhesion molecule [PECAM]/CD31) to reveal retinal vessel degeneration. Spectral domain optical coherence tomography was used to reveal changes in the thickness and structure of the retinal layer. Vitreous fluorophotometry was used to investigate vascular permeability. A-waves, b-waves and oscillatory potentials were measured under photopic and scotopic conditions. Concanavalin A leucostasis and immunostaining with glial fibrillary acidic protein (GFAP) and ionised calcium-binding adapter molecule 1 (IBA-1) identified differences in inflammatory status. Paraffin sections and transmission electron microscopy were used to reveal changes in the thickness and structure of the retinal layer.
Following the development of obesity and hyperglycaemia in 2-week-old and 3-week-old ob−/ob− mice, respectively (p < 0.001), early functional deficits (p < 0.001) and thinning of the inner retina (p < 0.001) were identified. Glial activation, leucostasis (p < 0.05) and a shift in microglia/macrophage phenotype were observed before microvascular degeneration (p < 0.05) and elevated vascular permeability occurred (p < 0.05).
The present characterisation of the development of diabetic retinopathy in the ob/ob mouse represents a platform that will enable the development of new therapies, particularly for the early stages of disease.
KeywordsDiabetes Diabetic retinopathy Eye Obesity Retina Type 2 diabetes mellitus
Brain-specific homeobox/POU domain protein 3A
Ganglion cell layer
Glial fibrillary acidic protein
Ionised calcium-binding adapter molecule 1
Industrial denatured alcohol
Inner nuclear layer
Inner plexiform layer
Nerve fibre layer
Optical coherence tomography
Outer nuclear layer
Outer plexiform layer
Platelet endothelial cell adhesion molecule
Retinal ganglion cell
- Rho-Con A
Rhodamine-conjugated concanavalin A
Spectral domain optical coherence tomography
Transmission electron microscopy
Triton X-100 in PBS
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 . 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  and diabetic nephropathy  in type 2 diabetes. Published data on these mice at 22 weeks of age suggest that they also develop retinal thinning . 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 . 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 . The retinal microvessel network in BTBR ob/ob mice has previously been studied using in vivo optical coherence tomography (OCT)/microangiography , 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 . 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.
Male and female wild-type (+/+) and heterozygous (+/−) BTBR ob/ob (obese) mice were obtained from the Jackson laboratory (BTBR.Cg-Lepob/WiscJ; Bar Harbor, ME, USA). These two genotypes and homozygous (−/−) BTBR ob/ob mice were bred at the UCL Institute of Ophthalmology. The Jackson Laboratory website reports no correlations between any known retinal degeneration genes and the BTBR mouse strain (www.jax.org/research-and-faculty/tools/eye-mutant-resource/retinal-degeneration-genes, last updated January 2018). Mice received food and water ad libitum, in a 12 h day/night cycle, temperature-controlled, clean environment.
All experiments were conducted according to UK Home Office Guidelines (/www.gov.uk/guidance/research-and-testing-using-animals, accessed Feb 2017) and Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research guidelines (www.arvo.org/About/policies/statement-for-the-use-of-animals-in-ophthalmic-and-vision-research/, last revised September 2016).
In vivo assays and recovery from anaesthesia
When required, mice were anaesthetised with an intraperitoneal injection of midazolam (Hypnovel; Roche, Welwyn Garden City, UK) 5 mg/kg, medetomidine (Domitor; Orion Pharma, Newbury, UK) 0.5 mg/kg and fentanyl (Sublimaze; Janssen, High Wycombe, UK) 0.05 mg/kg in water. Their pupils were dilated with phenylephrine hydrochloride 2.5% wt/vol. and tropicamide 1% wt/vol. (Bausch and Lomb, Kingston upon Thames, UK) before in vivo imaging or electroretinography was undertaken. Afterwards, mice were brought round with naloxone (Hameln, Gloucester, UK) 1.2 mg/kg, atipamezole (Antisedan; Orion Pharma) 2.5 mg/kg and flumazenil (Hameln) 2.5 mg/kg in saline (154 mmol/l NaCl).
The three genotypes were evaluated at 6, 10, 15 and 20 weeks of age. The animals were weighed, and blood glucose concentrations were measured (AlphaTRAK glucometer; Alameda, Pompano, FL, USA) at the same time of the day for all the animals in the study, at each of the ages. All in vivo studies (electroretinograms, leucostasis and OCT) except for fluorophotometry were carried out at these ages. Photographs of the physical appearances of mice were captured at 6 and 20 weeks of age.
List of antibodies for immunohistochemistry
Corresponding secondary antibodiesa
Type and dilution
Rabbit anti-collagen IV
Bio-Rad, Hercules, CA, USA
Donkey anti-rabbit conjugated to Alexa Fluor 488 nm
BD Biosciences, San Jose,
Donkey anti-rat conjugated to Alexa Fluor 647 nm
Abcam, Cambridge, UK
Donkey anti-goat conjugated to Alexa Fluor 488 nm
Dako, Stockport, UK
Donkey anti-rabbit conjugated to Alexa Fluor 594 nm/488 nm
Millipore, Watford, UK
Donkey anti-goat conjugated to Alexa Fluor 488 nm
For retinal whole-mounts, animals were euthanised and their retinas were dissected out and fixed as described above. Whole-mount preparations were blocked for 2 h in 5% wt/vol. donkey serum and permeabilised in 3% wt/vol. T-PBS for 2 h at room temperature. They were then incubated with primary antibodies (Table 1) in blocking solution overnight at room temperature. Secondary antibodies were applied for 2 h at room temperature and sections prepared as described above.
The antibodies were used as per the manufacturers’ instructions. The negative control procedures involved no primary antibody and relevant IgG isotypes. Images of sections or retinas were taken on Zeiss 700 or 710 confocal microscopes (Carl Zeiss, Cambridge, UK).
Concanavalin A leucostasis was performed as described by Joussen et al . After inducing deep anaesthesia with 200 mg/kg pentobarbital sodium (Euthatal, Merial, Harlow, UK), the mice were cardiac perfused with rhodamine-conjugated concanavalin A (Rho-Con A; Vector Laboratories), followed by PBS. Eyes were fixed in 4% wt/vol. paraformaldehyde for 1 h at room temperature, and the retinas were then mounted whole in Vectashield. Leucocytes inside vessels over the entire retina were counted under an epifluorescence Olympus BX51 microscope with a Retiga 2000R camera (QImaging, Surrey, BC, Canada).
Preparations of semi-thin optic nerve sections
Optic nerves were processed as previously described by Foxton et al . Briefly, the tissues were fixed overnight in Karnovsky’s solution at 4°C. Specimens were osmicated for 2 h in 1% wt/vol. osmium tetroxide and then dehydrated in 100% ethanol. Next, the optic nerves were incubated in propylene oxide for 30 min and placed in a 50:50 mixture of propylene oxide:Araldite (Electron Microscopy Sciences, Hatfield, PA, USA) overnight. This solution was changed to 100% Araldite wt/vol. and incubated overnight at 60°C. Semi-thin (0.75 μm) sections were cut and stained with 1% wt/vol. toluidine blue/borax in 50% ethanol before examination by light microscopy. For quantification, three non-overlapping images were taken at ×60 magnification, at the centre, middle and periphery of the optic nerve, using an Olympus BX51 microscope with a Retiga 2000R camera (QImaging). The number of axons in each image was counted, averaged for the mean for each optic nerve, and then expressed as axons density per image area (4500 mm2).
Image-guided large-field scotopic and large-field photopic electroretinograms were recorded from dark- (12 h) and light-adapted mice (Micron IV; Phoenix Research Laboratories, Pleasanton, CA, USA). Scotopic recordings were performed under dim red light. Series of 5 ms single-flash recordings were obtained at increasing light intensities from −2.5 to 3.0 log(cd × s/m2), where the logarithmic base is 10. Twenty responses per intensity were averaged for the mean with an interstimulus interval of 20 s. The a-wave, b-wave and oscillatory potential amplitudes were evaluated (LabScribe; iWorx System, Dover, NH, USA).
Paraffin sections and H&E staining
Paraffin section processing
Neutral buffered formalin
1.5 h each
1 h each
Paraffin wax (Leica)
1.5 h each
Samples were orientated and embedded in paraffin wax using a Leica Histocenter (Leica, Milton Keynes UK). Once cooled and solidified, they were cut at a thickness of 4 μm and heated for 20 min at 60°C prior to dewaxing. Sections were dewaxed twice for 1 min in xylene and rehydrated by two 1 min exposures to industrial denatured alcohol (IDA; Genta, Rudgate, UK). Following a 30 s wash in running tap water, the slides were stained with H&E using standard protocols.
Transmission electron microscopy
Retinal morphology was examined using transmission electron microscopy (TEM). Eyeballs were removed and processed as described above, and cut into ultrathin (75 nm) sections. They were then stained with Reynold’s lead citrate and washed in distilled water. The images were captured on a Jeol 1010 microscope (JEOL, Peabody, MA, USA) at 80 kV using DigitalMicrograph software version 3 (Gatan, Abingdon, UK).
The protocol used for the TUNEL assay has previously been described in Foxton et al . TUNEL staining was carried out on whole-mounted tissue. Retinas were dissected out and permeabilised in 3% wt/vol. T-PBS for 2 h at room temperature. The TUNEL protocol was performed according to the manufacturer’s instructions (Promega, Southampton, UK), and the retinas were then stained with 1:500 biotinylated isolectin B4 (IB4; Sigma-Aldrich, Dorset, UK) overnight at 4°C, followed by 1:500 streptavidin labelled with Alexa Fluor 594 for 2 h at room temperature. The tissues were washed in 0.3% wt/vol. T-PBS with 5 mmol/l DAPI and flat-mounted in Vectashield.
To quantify TUNEL-positive neurons, we used a Zeiss 710 confocal microscope (Zeiss, Oberkochen, Germany), generating tiled scans of the entire retina, with 30 μm Z-stacks through the ganglion cell layer (GCL) using a ×10 magnification water-immersion objective. IB4 staining and morphological criteria discriminated non-neuronal (endothelial and glial) cells from neuronal cells. The total number of TUNEL-positive cells in the GCL was counted.
Spectral domain OCT
The spectral domain OCT (SD-OCT) studies were performed using a Bioptigen Envisu R2200 SD-OCT imaging system (Bioptigen, Morrisville, NC, USA). Mice were anaesthetised and their pupils dilated as described above. Rectangular scans were performed, consisting of a 1.4 mm × 1.4 mm perimeter with 1000 A-scans per B-scan, with a total of 100 B-scans. Scans were obtained after localising the optic nerve. InVivoVue Reader version 2 (Bioptigen) was used to analyse the scans, and measurements were performed 500 μm away from the optic disc. The thicknesses of the following layers were measured: ONL, outer plexiform layer (OPL), inner nuclear layer (INL), a complex comprising the IPL, GCL and NFL, which we called IPL+GCL+NFL, and all the inner retinal layers together, which included the OPL, INL and IPL+GCL+NFL.
For this we used an ocular fluorophotometer, the Fluorotron Master Research Mouse Edition, which was kindly provided by Ocumetrics (Mountain View, CA, USA) for a short-term trial. Mice were anaesthetised and their pupils dilated as described above. For each animal, 50 μl of 1% wt/vol. (500 μg) fluorescein (Martindale, Romford, UK) was administered intravenously into a tail vein. After 10 min of administration, both eyes were scanned at four steps per millimetre. The fluorogram peak corresponding to the vitreous body was used. The readings were corrected against measurements of plasma fluorescein using cuvettes with the same fluorophotometer.
Randomisation was not carried out on the three genotypes during in vivo experiments because of the nature of animal breeding. Where possible, the experimenters were masked during the assessment and analysis, for example in the ex vivo experiments. Data are expressed as means and error bars are SEM. No criteria were applied to exclude data from the statistical analysis. Statistical analyses were performed using GraphPad Prism software version 6 (GraphPad Software, La Jolla, CA, USA). For comparisons between two groups, such as the 6- and 20-week-old animals, or between control and diabetic animals, the Mann–Whitney U test was used; the Shapiro–Wilk test was also used to test whether the data were normally distributed. The leucostasis, INL/ONL nuclear count, TUNEL assay, brain-specific homeobox/POU domain protein 3A (BRN3A) cell count, platelet endothelial cell adhesion molecule (PECAM, also known as CD31) and collagen IV count and fluorophotometry data were analysed in this way. When comparing longitudinal data (weight, blood glucose, electroretinogram and SD-OCT data), one-/two-way ANOVA and Newman–Keuls post hoc test were used.
Obesity and hyperglycaemia
We considered the mice to be hyperglycaemic when their blood glucose level increased above 13 mmol/l. The ob−/ob− mice reached this level at 3 weeks of age (Fig. 1b). At 20 weeks of age, the blood glucose level was two- to fivefold greater than that of ob+/ob+ and ob+/ob− mice. Thus, the onset of hyperglycaemia occurred approximately 1 week after the onset of obesity. The onsets of these changes were similar between sexes and the two conditions persisted with age; therefore the effect of sex on the following endpoints was not differentiated. The physical appearance of the mice differed according to weight (Fig. 1c).
Retinal vessel degeneration
Vitreous fluorophotometry has been shown to reveal early signs of blood–retinal barrier breakdown [20, 21, 22]. Measurements from the vitreous body can highlight vascular leakage in the retina . Vascular permeability determined using fluorescein fluorophotometry indicated that the aged ob−/ob− animals in particular had abnormal, exudative blood vessels (Fig. 6c). There were significant changes between the young and old diabetic mice, but no changes in the ob+/ob+ mice. Retinal vascular leakage in older diabetic mice was significantly greater than in aged-matched controls.
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 Ins2Akita and NOD mice, which are models of type 1 diabetes, show an onset of disease at 8 and 12 weeks of age, respectively [24, 25, 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 , 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 . 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 . 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 , the BTBR strain is also known to have a heightened, more reactive immune profile . 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 . 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 . 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 , 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 , 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 . 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 . 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 . As glycaemic control has little effect on the development and progression of neurodegenerative changes in these animals , 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.
The TEM data were processed by M. J. Hayes (Department of Electron Microscopy, UCL Institute of Ophthalmology, UK). The paraffin sections were prepared by D. Essex (Department of Pathology, UCL Institute of Ophthalmology, UK). The Bioptigen Envisu R2200 SD-OCT imaging system was provided by a multiuser equipment grant from the Wellcome Trust [099173/Z/12/Z].
All authors participated in the conception and design of the study, acquisition of data, or analysis and interpretation of the data; drafting the article or revising it critically for important intellectual content; and final approval of the version to be published. VKL and DTS are responsible for the integrity of the work as a whole.
This work was supported by the charity Diabetes UK.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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