Density of retinal circulation is unchanged in hyperglycemic mice in first year of life

Abstract

Purpose : A model for type 1 diabetes, the Ins2Akita mouse sustains an elevated blood glucose 4 weeks after birth. Here, we examine if vascular density changes in hyperglycemic mice compared to controls in the first year of life.

Methods : A mouse diabetic model (JAX#003548 C57BL/6-Ins2Akita/J) was crossed with mice having fluorescent smooth muscle cell and pericyte covered vessels (NG2DsRedBAC JAX#008241). Progeny included euglycemic mice (control group) and hyperglycemic mice (sustained blood glucose > 250 mg/dL, study group). 3 retinas of 3 male mice (controls, age 33-45 weeks) were dissected and flat mounted. Confocal microscopy (Nikon A1 Ti2 Eclipse, 60x mag) was used to capture sub-µm resolution Z-stacks containing all retinal vascular plexuses (eccentricity from optic nerve: 1012-2171 µm). Vessels were traced in the stacks by a masked grader using ImageJ’s SNT plugin. Imaging captured 9 locations in control mice and 7 locations in study group mice (5 retinas of 4 mice, age 7-54 weeks, eccentricity: 1029-2381 µm). Vessel path length in each stack as well as each plexus and connecting vessels were calculated using MATLAB.

Results : Average vascular path length was 84.5 mm/mm², SD 15.7 (study group), 75.9 mm/mm², SD 15.1 (control group). For study and control group, respectively, length of the superficial plexus was 21.3 mm/mm² (SD 9.3) vs. 16.6 mm/mm² (SD 3.4); intermediate 24.0 mm/mm² (SD 5.4) vs. 20.4 mm/mm² (SD 7.6); deep 25.9 mm/mm² (SD 9.3) vs. 27.3 mm/mm² (SD 4.5). Connecting vessels were rare, and comprised 13.3 mm/mm² (SD 3.8) vs. 11.6 mm/mm² (SD 5.4) in study and control groups, respectively. Neither age nor retinal location differed significantly between study and control mice. Unpaired t-testing (p = .05) showed no significant difference in vessel length between these two groups in any plexus, nor with respect to total path length.

Conclusions : While prior literature identifies anatomical changes within the first year of hyperglycemia in mice, this study finds the total vessel path length remains unchanged. Moreover, we do not see that any particular vascular stratification is preferentially impacted relative to others.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Fig. 1: Confocal en face slices (295 µm²) of hyperglycemic and euglycemic mouse retina expressing NG2DsRed vessels.

 

Fig. 2: 3D cubes of retinal circulation and manually traced vessels in superficial (green), intermediate (blue), and deep (red) plexuses. Connecting vessels in yellow.