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Diabetologia

pp 1–11 | Cite as

BTBR ob/ob mouse model of type 2 diabetes exhibits early loss of retinal function and retinal inflammation followed by late vascular changes

  • Vivian K. Lee
  • Brett M. Hosking
  • Joanna Holeniewska
  • Ewa C. Kubala
  • Peter Lundh von Leithner
  • Peter J. Gardner
  • Richard H. Foxton
  • David T. Shima
Open Access
Article

Abstract

Aims/hypothesis

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.

Methods

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.

Results

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).

Conclusions/interpretation

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.

Keywords

Diabetes Diabetic retinopathy Eye Obesity Retina Type 2 diabetes mellitus 

Abbreviations

BRN3A

Brain-specific homeobox/POU domain protein 3A

GCL

Ganglion cell layer

GFAP

Glial fibrillary acidic protein

IB4

Isolectin B4

IBA-1

Ionised calcium-binding adapter molecule 1

IDA

Industrial denatured alcohol

INL

Inner nuclear layer

IPL

Inner plexiform layer

NFL

Nerve fibre layer

OCT

Optical coherence tomography

ONL

Outer nuclear layer

OPL

Outer plexiform layer

PECAM

Platelet endothelial cell adhesion molecule

RGC

Retinal ganglion cell

Rho-Con A

Rhodamine-conjugated concanavalin A

SD-OCT

Spectral domain optical coherence tomography

TEM

Transmission electron microscopy

T-PBS

Triton X-100 in PBS

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.

Methods

Animals

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.

Immunohistochemistry

To collect cross sections, eyes were fixed in 4% wt/vol. paraformaldehyde for 1 h at room temperature and prepared as transverse sections. Eyes were cryoprotected in 30% wt/vol. sucrose and snap-frozen in an optimum cutting temperature compound (Tissue-Tek; Sakura Finetek, Thatcham, UK). Cryostat sections (10 μm) were then thaw-mounted onto glass slides. Sections were blocked for 1 h with 5% wt/vol. normal goat serum and permeabilised in 0.3% wt/vol. Triton X-100 in PBS (T-PBS). They were then incubated overnight with primary antibodies (Table 1) in blocking solution at room temperature. Secondary antibodies were applied for 1 h at room temperature, and the sections stained with 5 μmol/l DAPI. After the final washes, the tissue sections were mounted in Vectashield (Vector Laboratories, Peterborough, UK) and covered with a cover slip.
Table 1

List of antibodies for immunohistochemistry

Primary antibodies

Corresponding secondary antibodiesa

Antibody

Type and dilution

Manufacturer

Antibody

Type

Rabbit anti-collagen IV

Polyclonal 1:500

Bio-Rad, Hercules, CA, USA

Donkey anti-rabbit conjugated to Alexa Fluor 488 nm

Polyclonal

Rat anti-PECAM/CD31

Monoclonal 1:500

BD Biosciences, San Jose,

CA, USA

Donkey anti-rat conjugated to Alexa Fluor 647 nm

Polyclonal

Goat anti-IBA-1

Polyclonal 1:500

Abcam, Cambridge, UK

Donkey anti-goat conjugated to Alexa Fluor 488 nm

Polyclonal

Rabbit anti-GFAP

Polyclonal 1:500

Dako, Stockport, UK

Donkey anti-rabbit conjugated to Alexa Fluor 594 nm/488 nm

Polyclonal

Goat anti-BRN3A

Polyclonal 1:200

Millipore, Watford, UK

Donkey anti-goat conjugated to Alexa Fluor 488 nm

Polyclonal

aAll 1:1000; Thermo Fisher, Paisley, UK

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).

Leucostasis

Concanavalin A leucostasis was performed as described by Joussen et al [17]. 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 [18]. 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).

Electroretinography

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

Retinal thickness was measured from paraffin sections. The eyes were enucleated and fixed in 10% wt/vol. neutral buffered formalin and then processed as described in Table 2.
Table 2

Paraffin section processing

Station

Solution

Duration

1

Neutral buffered formalin

1 h

2

70% IDA

1 h

3

90% IDA

1 h

4–7

Absolute IDA

1.5 h each

8–11

Xylene

1 h each

12–14

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).

TUNEL assay

The protocol used for the TUNEL assay has previously been described in Foxton et al [18]. 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.

Fluorophotometry

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.

Statistical analysis

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.

Results

Obesity and hyperglycaemia

The lack of appetite control in the leptin-deficient BTBR ob/ob mice led to an increase in both body weight and degree of hyperglycaemia over time. The body weight and blood glucose concentrations of these mice were monitored to determine the rate at which they progressed into obesity and diabetes. The weight of the ob/ob mice began to significantly separate from those of the ob+/ob+ and ob+/ob mice at 2 weeks of age, so this age was considered to represent the onset of obesity (Fig. 1a). Whereas the ob+/ob+ and ob+/ob mice maintained their adult body weight at a mean of 23–39 g, the ob/ob mice continued to gain weight up to at least 20 weeks of age, reaching a maximum mean weight of 64–69 g.
Fig. 1

Body weight (a) and blood glucose levels (b) of all mouse phenotypes from 2 to 20 weeks of age. At 2 weeks of age, the body weights of both male and female ob/ob mice were significantly greater than those of ob+/ob+ and ob+/ob controls (unpaired t test ob+/ob+ and ob+/ob vs ob/ob, two-tailed, p < 0.05). Blood glucose measurements showed that 3-week-old male and female ob/ob mice exhibited an early onset of hyperglycaemia. Data are mean ± SEM. Both datasets in (a) and (b) were tested with one-way ANOVA, n = 6 animals; p < 0.001. The key next to (a) applies to both graphs. (c) Images showing the appearance of the ob+/ob+ and ob/ob mice at 6 and 20 weeks of age showed almost a doubling of girth in 6-week-old ob/ob mice in comparison to ob+/ob+ mice. The weight accumulated around the lower abdomen, as seen in the older (20-week-old) animals. With ageing, the fur around the eye and nose, in the mid-back and on the lower area of the front was gradually lost

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).

Inflammation

Immunocytochemical examination of the eyes in young mice (6 weeks of age) revealed signs of inflammation before the commonly observed features of diabetic retinopathy. Staining for glial fibrillary acid protein (GFAP) revealed glial activation during the development of diabetic retinopathy, with increased expression along the GCL and primary plexus (Fig. 2a) and the appearance of a more stellate phenotype with age (Fig. 2b). Although the expression and level of GFAP were not uniform across the retina, we nevertheless observed increased GFAP staining in the GCL, with more frequent and deeper Müller cell processes in the diabetic retina. The use of the microglia/macrophage marker ionised calcium-binding adapter molecule 1 (IBA-1) highlighted the deramification of the macrophages—from dendritic to amoeboid morphology with larger cell bodies—in the eyes of diabetic mice (Fig. 2c). The number of adherent leucocytes observed by perfusion of Rho-Con A into the retinal vessels (see the electronic supplementary material [ESM] Fig. 1a) increased more than twofold in 6-week-old diabetic mice compared with age-matched wild-type mice, and threefold at age 20 weeks (Fig. 2d).
Fig. 2

(a) GFAP is found along the GCL, and Müller cell processes are seen to project towards the plexiform layer. Representative images of GFAP expression (red) in transverse sections of eyes from 6-week-old ob/ob and ob+/ob+ mice reveal elevated GFAP expression in ob/ob mice along the GCL (white arrowheads) and OPL (yellow arrowheads), suggesting gliosis. Scale bar, 100 μm. (b) In the retinal whole-mounts, there were more GFAP-labelled Müller cells (green) in the primary plexus layer in ob/ob than ob+/ob+ mice. Gliosis by upregulated Müller cells reflects inflammation of the nervous system. Scale bar, 50 μm. (c) In the same layer of retinal whole-mounts, the IBA-1-labelled macrophages (green) were more abundant, amoeboid and activated in ob/ob mice. Scale bars, 150 μm. (d) Graph of the number of Rho-Con A-labelled leucocytes in the main retinal blood vessels in each eye, in 6- and 20-week-old ob+/ob+ and ob/ob mice. In both age groups, there were more leucocytes in the eyes of ob/ob than ob+/ob+ mice. This was significant at both 6 weeks (n = 6 eyes) and 20 weeks (n = 7 eyes); *p < 0.05 for comparisons shown, Mann–Whitney U test, one-tailed. Data are mean ± SEM

Retinal electrophysiology

Early loss of retinal function was shown using electroretinograms in 6-week-old mice. The eyes were subjected to increasing light stimuli under either scotopic (Fig. 3a–d) or photopic (Fig. 3e) conditions. The photoreceptor responses from the three mouse phenotypes are seen in the a-waves of the scotopic electroretinogram (Fig. 3a, c). The responses were weaker in retina from ob/ob mice compared with ob+/ob+ (−31%) or ob+/ob (−37%) animals, even in 6-week-old mice (Fig. 3a). The b-waves are responses from the inner retina, such as from bipolar cells, and were also weaker in the young ob/ob retina compared with age-matched ob+/ob+ (−37%) and ob+/ob (−20%) retina (Fig. 3b). Even in non-diabetic animals, these amplitudes generally decreased with age, suggesting that retinal function worsens. Under photopic conditions (Fig. 3e), b-wave responses were investigated using the maximum light level [2.87 log(cd × s/m2)]. The ob/ob animals consistently showed a reduced response between 6 and 20 weeks of age. These results were also seen in the oscillatory potential (ESM Fig. 2a).
Fig. 3

Electroretinography was performed to show the functioning of the retinal neuronal cells. There was a loss of retinal function in the ob/ob mice in comparison to the healthy ob+/ob+ and ob+/ob mice. Scotopic electroretinogram showing a-waves (a) and b-waves (b) in 6-week-old mice, and a-waves (c) and b-waves (d) in 20-week-old mice. The amplitudes of the a-waves, originating from the responses of the rod and cone photoreceptors, were reduced in the retinas from both 6- and 20-week-old ob/ob mice. The amplitude of the b-wave represents responses from cells in the inner retina, for example bipolar cells, and the same reductions in amplitude were seen in ob/ob mice. (e) Under photopic conditions, the b-wave responses were most different when the maximum light level [2.87 log(cd × s/m2)] was used. The ob/ob animals consistently showed a reduced response at 6, 10, 15 and 20 weeks of age. Overall, the electroretinogram recordings from ob/ob retina were weaker than in age-matched ob+/ob+ and ob+/ob mice between 6 and 20 weeks of age. Two-way ANOVA: age, p < 0.001; phenotype, p < 0.001 for all figure parts (ae). Data are mean ± SEM, n = 12 eyes. Black circles, ob+/ob+; blue squares ob+/ob; red triangles ob/ob

SD-OCT

Measurements of retinal layer thickness using SD-OCT in 6- and 20-week-old diabetic animals showed thinning of the retina with age (Fig. 4a, b). The differences in retinal thickness were more evident between the 20-week-old ob+/ob+ and ob/ob animals. In these older animals, nearly all measurements taken in the ob/ob animals showed a significant reduction in thickness of the retinal layer, inner and outer segments, ONL, INL and inner retina. Furthermore, counts of numbers of nuclei in the two nuclear layers in the retina suggested that the greatest reduction of cells was seen in the inner retina (Fig. 4c–e).
Fig. 4

(a) Representative image of 6-week-old ob+/ob+ retina captured using SD-OCT showing the different retinal layer thicknesses. Scale bar, 250 μm. IR, inner retina, IS, inner segment; OS outer segment. (b) Quantification of 12 images from each phenotype are represented in the bar graph. The thickness of the outer retinal layer was similar between the 6-week-old control and diabetic retina, but that of the INL and IR was reduced. This was exacerbated at 20 weeks of age, with the eyes of diabetic mice showing a reduction in thickness in the IS and OS layer, ONL, INL and IR. In particular, the ONL in diabetic mice was significantly reduced with age compared with non-diabetic mice. Two-way ANOVA: retinal layers, p < 0.001; phenotype, p < 0.001. *p < 0.05, **p < 0.01, ***p < 0.001 for comparisons shown, Mann–Whitney U test, one-tailed. Data are mean ± SEM, n = 12 eyes. Black circles, 6 week ob+/ob+; red squares, 6 week ob/ob; black triangles, 20 week ob+/ob+; red inverted triangles, 20 week ob/ob. (c) Representative H&E-stained, paraffin-embedded transverse retinal sections of 6- and 20-week-old ob+/ob+ and ob/ob mice. The histology was similar in the 6-week-old ob+/ob+ and ob/ob mice. However, in 20-week-old mice, the retinal layers were thinner in the ob/ob mice. Scale bar, 50 μm. (d) Number of INL nuclei stacked in 6- and 20-week-old ob+/ob+ and ob/ob mice. The data suggest that there were fewer cells in this layer in the young diabetic mice and that the number continued to decrease with age (ob+/ob+; ob/ob). (e) Number of ONL nuclei in the same animals, suggesting that there was a slight loss of photoreceptors in the aged diabetic mice. *p < 0.05, **p < 0.01 for comparisons shown, Mann–Whitney U test, one-tailed. Data are mean ± SEM, n = 6 eyes

Apoptosis assay

Retinal whole-mount staining using TUNEL as a marker for cell death (ESM Fig. 3a) revealed that the number of apoptotic cells in the GCL was significantly greater in 20-week-old ob/ob mice than age-matched ob+/ob+ mice, compared with younger 6 week ob/ob and ob+/ob+ mice (Fig. 5a). Specific staining of the retinal ganglion cell (RGC) marker using BRN3A revealed that the number of these cells declined significantly in the presence of diabetes and with age (Fig. 5b, ESM Fig. 3b). The axons of the RGCs that formed the optic nerve were examined. The axon density captured from semi-thin sections of the optic nerve revealed a significant reduction in number of these axons, which occurred as early as 6 weeks of age in diabetic mice compared with wild-type controls. By close examination of the IPL using TEM, the axons in aged diabetic retinas appeared either to be fused together or to have lost their cell membrane structures (ESM Fig. 4a).
Fig. 5

(a) Cell count of TUNEL-stained apoptotic cells in the retina of 6- and 20-week-old mice. There were over threefold more TUNEL-positive nuclei in the GCL in 20-week-old ob/ob than 6-week-old ob+/ob+, 6-week-old ob/ob or 20-week-old ob+/ob+ mice (*p < 0.05, **p < 0.01, ***p < 0.001 for comparisons shown, Mann–Whitney U test, one-tailed). One-way ANOVA, p = 0.0001; n = 4 eyes. (b) Cell counts of BRN3A-stained RGC cells in the GCL in each field of view with an area of 0.65 mm2. The number of ganglion cells decreased not only from 6 to 20 weeks, but also with hyperglycaemia between 20-week-old ob+/ob+ and ob/ob mice (*p < 0.05, **p < 0.01 for comparisons shown, Mann–Whitney U test, one-tailed); n = 4 eyes. (c) The brightfield images and quantification in the bar chart show that optic nerve axon density in RGC cells in the 6-week-old ob/ob mice is reduced by approximately one-third (*p < 0.05, Mann–Whitney U test, one tailed). The axons from the ob/ob eyes are more spread apart between supporting cells compared with those from ob+/ob+ eyes. Scale bar, 5 μm. Data are mean ± SEM, n = 4 nerves

Retinal vessel degeneration

Although inflammatory and neuronal factors are believed to drive the early manifestations of diabetic retinopathy, the earliest clinical signs and hallmarks of this disease are related to vascular damage. Microvascular damage such as loss of endothelial cells and increased vascular permeability was observed. We demonstrated a loss of endothelial cells by co-staining these cells (PECAM) with the basement membrane (collagen IV), to identify vascular degeneration in the primary plexus of the retina [19]. Considerably more vessels showed degeneration in the aged ob/ob mice than in aged ob+/ob+ mice, and also than in aged-matched controls (Fig. 6a, b). There were no significant differences between the groups of 6-week-old mice, or between retinas from young and older ob+/ob+ mice.
Fig. 6

With ageing, there is an increase in some physiological processes, such as blood vessel regression. (a) Representative images showing expression of collagen IV (red) and PECAM (green) in the retinal vascular primary plexus of 20-week-old ob/ob and ob+/ob+ mice. Collagen IV labels the structural scaffolding of the blood vessels, and PECAM labels endothelial cells. The white arrowheads point to microvascular degeneration where only the blood vessel scaffolding remained and the endothelial cells were absent. These regressed vessels were more frequently seen in ob/ob than ob+/ob+ mice. Scale bar, 50 μm. (b) Collagen IV-stained vessels not showing co-localisation of PECAM in the primary plexus were counted from six images captured from the intermediate region of each retina in 6- and 20-week-old ob+/ob+ and ob/ob mice. There were no significant differences between the two groups of 6-week-old mice, or between the ob+/ob+ retinas with age. However, there was significantly more microvascular degeneration in the 20-week-old ob/ob retina compared with the 6-week-old ob/ob retina and with the aged-matched wild-type retina; n = 7 eyes. (c) Retinal vessel leakage in the vitreous body was detected using fluorescein fluorophotometry in 6- and 20-week-old ob+/ob+ and ob/ob mice. There were no significant differences in the ob+/ob+ mice with age. In the diabetic mice, however, there were significant changes with age (n = 13 eyes). Retinal vascular leakage in older diabetic mice was significantly greater than in age-matched controls (n = 9 eyes). *p < 0.05, **p < 0.01, ***p < 0.001 for comparisons shown, Mann–Whitney U test, one-tailed. Data are mean ± SEM

Fluorescein fluorophotometry

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 [23]. 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.

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 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 [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.

Notes

Acknowledgements

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].

Contribution statement

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.

Funding

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.

Supplementary material

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References

  1. 1.
    Cheung N, Mitchell P, Wong TY (2010) Diabetic retinopathy. Lancet 376:124–136CrossRefPubMedGoogle Scholar
  2. 2.
    O'Brien PD, Hur J, Hayes JM, Backus C, Sakowski SA, Feldman EL (2015) BTBR ob/ob mice as a novel diabetic neuropathy model: neurological characterization and gene expression analyses. Neurobiol Dis 73:348–355CrossRefPubMedGoogle Scholar
  3. 3.
    Hudkins KL, Pichaiwong W, Wietecha T et al (2010) BTBR Ob/Ob mutant mice model progressive diabetic nephropathy. J Am Soc Nephrol 21:1533–1542CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zhi Z, Chao JR, Wietecha T, Hudkins KL, Alpers CE, Wang RK (2014) Noninvasive imaging of retinal morphology and microvasculature in obese mice using optical coherence tomography and optical microangiography. Invest Ophthalmol Vis Sci 55:1024–1030CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Berglund ED, Vianna CR, Donato J Jr et al (2012) Direct leptin action on POMC neurons regulates glucose homeostasis and hepatic insulin sensitivity in mice. J Clin Invest 122:1000–1009CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cummings BP, Bettaieb A, Graham JL et al (2011) Subcutaneous administration of leptin normalizes fasting plasma glucose in obese type 2 diabetic UCD-T2DM rats. Proc Natl Acad Sci U S A 108:14670–14675CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Coppari R, Ichinose M, Lee CE et al (2005) The hypothalamic arcuate nucleus: a key site for mediating leptin’s effects on glucose homeostasis and locomotor activity. Cell Metab 1:63–72CrossRefPubMedGoogle Scholar
  8. 8.
    Mittendorfer B, Horowitz JF, DePaoli AM, McCamish MA, Patterson BW, Klein S (2011) Recombinant human leptin treatment does not improve insulin action in obese subjects with type 2 diabetes. Diabetes 60:1474–1477CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Moon HS, Matarese G, Brennan AM et al (2011) Efficacy of metreleptin in obese patients with type 2 diabetes: cellular and molecular pathways underlying leptin tolerance. Diabetes 60:1647–1656CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Stoehr JP, Byers JE, Clee SM et al (2004) Identification of major quantitative trait loci controlling body weight variation in ob/ob mice. Diabetes 53:245–249CrossRefPubMedGoogle Scholar
  11. 11.
    Nadler ST, Stoehr JP, Schueler KL, Tanimoto G, Yandell BS, Attie AD (2000) The expression of adipogenic genes is decreased in obesity and diabetes mellitus. Proc Natl Acad Sci U S A 97:11371–11376CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Clee SM, Nadler ST, Attie AD (2005) Genetic and genomic studies of the BTBR ob/ob mouse model of type 2 diabetes. Am J Ther 12:491–498CrossRefPubMedGoogle Scholar
  13. 13.
    Ranheim T, Dumke C, Schueler KL, Cartee GD, Attie AD (1997) Interaction between BTBR and C57BL/6J genomes produces an insulin resistance syndrome in (BTBR x C57BL/6J) F1 mice. Arterioscler Thromb Vasc Biol 17:3286–3293CrossRefPubMedGoogle Scholar
  14. 14.
    Bogdanov P, Corraliza L, Villena JA et al (2014) The db/db mouse: a useful model for the study of diabetic retinal neurodegeneration. PLoS One 9:e97302CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Antonetti DA, Klein R, Gardner TW (2012) Diabetic retinopathy. N Engl J Med 366:1227–1239CrossRefPubMedGoogle Scholar
  16. 16.
    Simo R, Hernandez C, European Consortium for the Early Treatment of Diabetic Retinopathy (EUROCONDOR) (2014) Neurodegeneration in the diabetic eye: new insights and therapeutic perspectives. Trends Endocrinol Metab 25:23–33CrossRefPubMedGoogle Scholar
  17. 17.
    Joussen AM, Murata T, Tsujikawa A, Kirchhof B, Bursell SE, Adamis AP (2001) Leukocyte-mediated endothelial cell injury and death in the diabetic retina. Am J Pathol 158:147–152CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Foxton RH, Finkelstein A, Vijay S et al (2013) VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma. Am J Pathol 182:1379–1390CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Baluk P, Morikawa S, Haskell A, Mancuso M, McDonald DM (2003) Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors. Am J Pathol 163:1801–1815CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    McManus LM, Mitchell R (2014) Pathobiology of human disease: a dynamic encyclopedia of disease mechanisms. Academic Press, Elsevier, CambridgeGoogle Scholar
  21. 21.
    Sander B, Thornit DN, Colmorn L et al (2007) Progression of diabetic macular edema: correlation with blood retinal barrier permeability, retinal thickness, and retinal vessel diameter. Invest Ophthalmol Vis Sci 48:3983–3987CrossRefPubMedGoogle Scholar
  22. 22.
    Chen CL, Cheng WS, Chen JL, Chiang CH (2013) Potential of nonoral alpha-lipoic acid aqueous formulations to reduce ocular microvascular complications in a streptozotocin-induced diabetic rat model. J Ocul Pharmacol Ther 29:738–745CrossRefPubMedGoogle Scholar
  23. 23.
    McCullough PC, Koester CJ, Campbell CJ, Anderson EA (1983) An evaluation of the clinical role of vitreous fluorophotometry. Trans Am Ophthalmol Soc 81:130–148PubMedPubMedCentralGoogle Scholar
  24. 24.
    Olivares AM, Althoff K, Chen GF et al (2017) Animal models of diabetic retinopathy. Curr Diab Rep 17:93CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Li CR, Sun SG (2010) VEGF expression and cell apoptosis in NOD mouse retina. Int J Ophthalmol 3:224–227PubMedPubMedCentralGoogle Scholar
  26. 26.
    Barber AJ, Antonetti DA, Kern TS et al (2005) The Ins2Akita mouse as a model of early retinal complications in diabetes. Invest Ophthalmol Vis Sci 46:2210–2218CrossRefPubMedGoogle Scholar
  27. 27.
    Tzekov R, Arden GB (1999) The electroretinogram in diabetic retinopathy. Surv Ophthalmol 44:53–60CrossRefPubMedGoogle Scholar
  28. 28.
    Yu Y, Chen H, Su SB (2015) Neuroinflammatory responses in diabetic retinopathy. J Neuroinflammation 12:141CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Heo Y, Zhang Y, Gao D, Miller VM, Lawrence DA (2011) Aberrant immune responses in a mouse with behavioral disorders. PLoS One 6:e20912CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Careaga M, Schwartzer J, Ashwood P (2015) Inflammatory profiles in the BTBR mouse: how relevant are they to autism spectrum disorders? Brain Behav Immun 43:11–16CrossRefPubMedGoogle Scholar
  31. 31.
    Birnbaum Y, Bajaj M, Qian J, Ye Y (2016) Dipeptidyl peptidase-4 inhibition by saxagliptin prevents inflammation and renal injury by targeting the Nlrp3/ASC inflammasome. BMJ Open Diabetes Res Care 4:e000227CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    VanGuilder HD, Brucklacher RM, Patel K, Ellis RW, Freeman WM, Barber AJ (2008) Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retina. Eur J Neurosci 28:1–11CrossRefPubMedGoogle Scholar
  33. 33.
    Feit-Leichman RA, Kinouchi R, Takeda M et al (2005) Vascular damage in a mouse model of diabetic retinopathy: relation to neuronal and glial changes. Invest Ophthalmol Vis Sci 46:4281–4287CrossRefPubMedGoogle Scholar
  34. 34.
    Robinson R, Barathi VA, Chaurasia SS, Wong TY, Kern TS (2012) Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals. Dis Model Mech 5:444–456CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Hinder LM, O'Brien PD, Hayes JM et al (2017) Dietary reversal of neuropathy in a murine model of prediabetes and metabolic syndrome. Dis Model Mech 10:717–725CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Author(s) 2018

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Vivian K. Lee
    • 1
  • Brett M. Hosking
    • 1
  • Joanna Holeniewska
    • 1
  • Ewa C. Kubala
    • 1
  • Peter Lundh von Leithner
    • 1
  • Peter J. Gardner
    • 1
  • Richard H. Foxton
    • 1
  • David T. Shima
    • 1
  1. 1.Translational Vision Research, UCL Institute of OphthalmologyUniversity College LondonLondonUK

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