**3. Results**

### *3.1. HFD Mice Have Normal Glucose Levels but Are Insulin-Resistant*

We first sought to validate our model by confirming in our cohort that HFD feeding led to similar degrees of body weight gain as reported in the literature [10,35]. Mice on HFD showed increased body weights by 4 weeks of feeding (*p* = 0.0020). This increase was sustained throughout the 12-month observation period (Figure 1A). At 12 months, HFD mice had moderately higher lean mass (difference of 5.580 ± 1.003g, *p* < 0.0001) and water content (difference of 4.517 ± 0.876, *p* = 0.0001) but a markedly increased fat mass (difference of 21.15 ± 2.362, *p* < 0.0001) compared to LFD controls (Figure 1B). Unexpectedly, chronic high-fat feeding did not cause hyperglycemia. Despite feeding mice with a HFD for 12 months, the HbA1c levels were not different between HFD and LFD mice (Figure 1C). At 12 months, there was no difference in fasting blood glucose levels (Figure 1D, basal) and intraperitoneal glucose tolerance test (IP-GTT) did not show any significant differences between LFD and HFD mice (Figure 1D). However, due to the very high levels of insulin in HFD mice (0.6 ng/mL for LFD and 3.5 ng/mL for HFD; Figure 1E, basal), the insulin sensitivity index demonstrated that HFD mice had much lower insulin sensitivity compared to LFD mice (Figure 1E,F). Also, plasma total cholesterol levels were higher in HFD mice compared to LFD (174.4 vs. 114.9, *p* = 0.0008) (Figure 1G)

**Figure 1.** *Cont*.

**Figure 1.** Body weight, glucose levels, and insulin sensitivity of high-fat diet (HFD) mice vs. low-fat diet (LFD) mice. (**A**) Body weights as measured for mice on LFD (green) and HFD (red) for 12 months. (\* *p* < 0.000001; *n* = 6). (**B**) Lean mass, fat mass and water content of LFD mice vs. HFD mice. (**C**) Glycated hemoglobin (HbA1c) levels measured for the mice after 6 months and 12 months. (**D**–**G**) Glucose curves (*p* > 0.46 for all time points), insulin curves (*p* < 0.0018 for all time points), insulin sensitivity index, and total cholesterol levels for LFD mice vs. HFD mice, respectively, following intraperitoneal glucose tolerance test (IP-GTT) after 12 months of feeding.

### *3.2. HFD Mice Have Functional Deficits in Their Retinas*

Full-field ERG under both scotopic and photopic conditions was performed at 6 months and 12 months of HFD feeding (Figure 2A–D). HFD mice at 6 months showed significantly reduced aand b-wave amplitudes under scotopic conditions (*p* = 0.00125 and *p* = 0.000002 for 0.25 cd.s/m<sup>2</sup> and 2.5 cd.s/m<sup>2</sup> stimulus luminance, respectively) but not photopic conditions when compared to LFD mice. After 12 months of feeding of the respective diets, the difference was not significant (*p* = 0.183 and

*p* = 0.154 for 0.25 cd.s/m<sup>2</sup> and 2.5 cd.s/m<sup>2</sup> stimulus luminance respectively) (Figure 2C,D). Interestingly, when comparing 6 and 12 months of LFD feeding, the mice experienced marked reductions in both the a- and b- waves under both photopic and scotopic conditions at 12 months (Figure 2E,F), but no significant difference was noted in the HFD-fed mice (Figure 2G,H).

**Figure 2.** Assessment of retinal function of LFD mice versus HFD mice by electroretinogram (ERG). The amplitudes of a-waves and b-waves were assessed under both scotopic and photopic conditions for LFD mice and HFD mice after 6 months (**A**,**B**) and 12 months (**C**,**D**). LFD mice showed a significant reduction in retinal response between 6 months and 12 months of feeding (**E**,**F**), but HFD mice did not (**G**,**H**); (*n* = 4 for both groups).

### *3.3. Fundus Photography shows Neural Retinal Lesions in HFD Mice*

In humans, DR is associated with retinal lesions such as hemorrhages, microaneurysms, exudates, and "cotton wool spots" [36]. Fundus photography using Micron IV demonstrated retinal pathology in the HFD mice. Though not statistically significant, HFD mice showed a trend of increased numbers of "lipid-laden-like" lesions (Figure 3A) after 6 months (*p* = 0.057). However, with 12 months of feeding, HFD mice showed significantly higher number of lesions in the retina (Figure 3B).

**Figure 3.** Assessment of retinal lesions by fundus photography (**A**,**B**) and vascular leakage by fluorescein angiography (**C**,**D**). HFD mice developed more neural infarcts ((**A**,**B**), white arrows) than LFD mice. No infarct was observed for LFD after 6 months (**A**). However, vascular leakage was observed in HFD mice after 12 months of feeding ((**D**), white arrows).

### *3.4. Vascular Permeability Changes in HFD Mice*

A hallmark of DR in humans is increased vascular permeability, ultimately leading to diabetic macular edema in humans. To determine if HFD mice developed a breakdown in the blood–retinal barrier, we assessed vascular leakage by fluorescein angiography (FA). At 6 months of HFD feeding, FA did not show any evidence of retinal vascular leakage and were similar to FAs in LFD controls (Figure 3C). However, after 12 months of HFD feeding, increased leakage of fluorescein was observed in the retina compared to LFD control retinas (Figure 3D).

### *3.5. Acellular Capillary Formation in HFD Mice*

A well-established feature of diabetic microvascular dysfunction is an increase in the number of acellular capillaries in the retina, defined as basal membrane tubes lacking endothelial cells and pericyte nuclei. At 12 months of HFD feeding, there was no significant increase in acellular capillary numbers in the HFD mice (Figure 4B,C) compared to the LFD mice (Figure 4A,C). However, the HFD retinas showed lower vascular densities compared to LFD retinas (Figure 4D).

**Figure 4.** Enumeration of acellular capillaries in LFD and HFD mice after 12 months of feeding. Red arrows indicate acellular capillaries in the retinas of LFD ( **A**) and HFD (**B**) mice. There was no significant di fference in the number of acellular capillaries between both groups ( **C**) (*p* = 0.086). However, HFD retinas showed lesser vascular densities compared to LFD retinas ( **D**).

### *3.6. Retinal Damage, Hypoxia, and Lipid Transport in WD Mice*

While the HFD represents a diet with 60% fat content that is used as a model of obesity and T2D, the WD with 40% fat content has garnered popularity as it represents a regimen closer to that actually ingested by humans. Since the WD diet has lower fat content and is not associated with hyperglycemia, we hypothesized that if retinal changes were present they would be subtle compared to those we observed with HFD feeding. To test the validity of our hypothesis, we performed IHC studies and first examined whether there was evidence of glial activation by examining expression of the glial marker GFAP after 6 months of WD feeding. Although there was no statistically significant di fference (*p* = 0.88) in the total expression of GFAP between retinas of WD and LFD mice (Figure 5A–C), increased expression of GFAP was observed in selected Vimentin-positive Muller cells in the WD mice (Figure 5G–I) compared to LFD (Figure 5D–F). Increased expression of GFAP in Muller cells is supportive of increased oxidative stress and inflammation in these cells, and suggests that the impact of WD is not experienced uniformly across all Muller cells [37,38].

To assess whether WD feeding induced retinal hypoxia, changes in HIF-1 α expression were examined by IHC. After 6 months of WD feeding, a significant increase (*p* = 0.025) in expression of HIF-1 α was seen in WD mice (Figure 6D) compared to LFD mice (Figure 6C). This was not observed after 3 months of WD feeding (Figure 6A,B). Quantitation of HIF-1 α expression is shown in Figure 6E, demonstrating that WD-fed mice exhibit higher levels than LFD-fed mice. Co-localization with isolectin, a known vascular endothelial cell marker, showed increased expression of HIF- 1 α in some endothelial cells in WD mice (I–K) but not in LFD mice (F–H). Higher magnification images from two di fferent WD samples are shown in Figure 6L,M.

Retinal lipid content is regulated in part by liver X receptor beta (LXRβ) expression. We next examined changes in LXRβ expression in the two experimental cohorts. In control mice, LXRβ localized predominantly in the ganglion cell layer, as well as the inner nuclear layer (Figure 7A), which is the location of the bipolar cells, horizontal cells, and amacrine cells. There was a significant reduction in expression of LXRβ in WD only in the ganglion cell layer (*p* = 0.0079) after 3 months of feeding (Figure 7B). However, after 6 months of WD feeding, WD mice (Figure 7E) showed significantly reduced expression of LXRβ in the ganglion cell layer (*p* = 0.0374), inner nuclear layer (*p* < 0.0001), and outer nuclear layer, as well as in the photoreceptors of the outer nuclear layer (*p* = 0.0020). The expression of LXRβ was reduced after 6 months compared to 3 months of feeding in both LFD (*p* < 0.0001) and WD (*p* < 0.0001) in the nuclear and ganglion cell layers, suggesting an age-related loss in LXRβ.

**Figure 5.** *Cont*.

**Figure 5.** Retinal glial fibrillary acidic protein (GFAP) expression after 6 months of feeding. Some Muller cells in Western diet (WD) retinas express GFAP (**A**,**C**, white arrows), but not in LFD (**A**,**B**), indicating that the impact of WD is not uniform across all Muller cells. Co-localization with Vimentin, a known Mueller cell marker, showed increased expression of GFAP in some Mueller cells in WD mice (**G**–**I**) but not in LFD mice (**D**–**F**).

**Figure 6.** *Cont*.

**Figure 6.** Retinal hypoxia-inducible factor 1 alpha (HIF-1α) expression after 3 and 6 months of WD feeding. There was increased expression of HIF-1α in WD retinas (**D**, white arrows) compared to LFD retinas (**C**), as shown by quantification (**E**). Also, there was no significant difference in expression of HIF-1α after 3 months of feeding (**A**,**B**). Co-localization with isolectin, a known vascular endothelial cell marker, showed increased expression of HIF-1α in some endothelial cells in WD mice (**I**–**K**) but not in LFD mice (**F**–**H**). (**L**,**M**) Magnified merged images from two different WD samples.

**Figure 7.** Retinal liver X receptor beta (LXRβ) expression after 3 and 6 months of feeding. After 3 months of either WD or LFD feeding, there was significant reduction in the expression of LXRβ in only the ganglion cell layer of WD mice (**B**) compared to LFD mice (**A**). However, after 6 months of feeding, there was reduced expression of LXRβ in the ganglion cell layer as well as inner and outer nuclear layers of WD mice (**E**, white arrows) compared to LFD mice (**D**). Quantification of LXR in the inner nuclear layer (INL) and outer nuclear layer (ONL) at 3 months shows reductions in the ganglion cell (GC) layer (**C**). At 6 months, reductions are seen in the INL, ONL, and ganglion cell (GC) layer of the WD-fed mice when compared to LFD mice.
