Successful induction of diabetes in mice demonstrates no gender difference in development of early diabetic retinopathy

Abstract

Purpose: Female mice have been found to be resistant to streptozotocin (STZ)-induced diabetes, and pre-clinical research related to diabetic complications commonly omits females. The purpose of this study was to develop a method to induce diabetes in female mice, and to determine if retinas of diabetic female mice develop molecular changes and histopathological abnormalities comparable to those which develop in male diabetic mice.

Methods: To induce diabetes, animals of both sexes received daily intraperitoneal (i.p.) injection of STZ for 5 consecutive days at 55 mg/kg BW (a dose that is known to induce diabetes in male mice) or for females, 75 mg/kg BW of STZ. Retinal abnormalities that have been implicated in the development of the retinopathy (superoxide generation and expression of inflammatory proteins, iNOS and ICAM-1) were evaluated at 2 months of diabetes, and retinal capillary degeneration was evaluated at 8 months of diabetes.

Results: Daily i.p. injection of STZ for 5 consecutive days at a concentration of 55 mg/kg BW was sufficient to induce diabetes in 100% of male mice, but only 33% of female mice. However, females did become hyperglycemic when the dose of STZ administered was increased to 75 mg/kg BW. The resulting STZ-induced hyperglycemia in female and male mice was sustained for at least 8 months. After induction of the diabetes, both sexes responded similarly with respect to the oxidative stress, expression of iNOS, and degeneration of retinal capillaries, but differed in the limited population evaluated with respect to expression of ICAM-1.

Conclusions: The resistance of female mice to STZ-induced diabetes can be overcome by increasing the dose of STZ used. Female mice can, and should, be included in pre-clinical studies of diabetes and its complications.

Fig 1. Glycemia following administration of STZ to female mice at a dose of 75 mg/kg BW (grey lines) compared to males at 55 mg/kg BW (bold black lines).

In both sexes, the increase in (A) HbA1c or (B) blood glucose persisted for at least 8 months. Solid lines represent diabetic mice, and dashed lines represent nondiabetic control mice. Data are presented as mean ± SD (n = 8–13 per group). *** P ≤ 0.001.

Fig 2. Two month duration of diabetes increased superoxide levels in retinas of male and female mice compared to their respective nondiabetic controls.

Data are presented as mean ± SD (n = 5–6 per group). *** P ≤ 0.001. N; nondiabetic, D; diabetic.

Fig 3. Retinal explants from nondiabetic female and male mice incubated in 5 or 25 mM glucose for 24 hours reveal that retinal superoxide in high glucose is elevated similarly in both sexes.

Eyecups were obtained from wild type male and female mice at age 2–3 months. Data are presented as mean ± SD, n = 6–9 per group. * P ≤ 0.05.

Fig 4. Effects of STZ-induced diabetes on protein expression of iNOS and ICAM-1 in retinas of female and male mice diabetic for 8 months.

Summary graph for iNOS (A) and ICAM-1 (B) expression determined by image analysis. C representative immunoblots for iNOS, ICAM-1 and βactin. Data are expressed relative to the expression of βactin, a housekeeping protein in the same sample, and are expressed as a percent of the value of nondiabetic controls. Data are expressed as mean ± SD (n = 3–4). N, nondiabetic; D, diabetic. * P ≤ 0.05, ** P ≤ 0.001.

Fig 5. SD-OCT images (A) and quantification of data (B and C) show that 8 months duration of diabetes (dashed lines) resulted in essentially no loss of retinal photoreceptors (ONL thickness) in either male (bold black) or female (grey) mice compared to gender- and age-matched nondiabetic controls (solid lines).

Similarly, TRL, NFL, IPL, and INL were not significantly different between diabetic and nondiabetic. Data are presented as mean ± SD, n = 4 mice (8 retinas) per group. N, nondiabetic; D, diabetic; M, males; F, females; TR, total retina; NFL, nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer.

Fig 6. Physiologic testing on the effects of gender and diabetes on retinal function.

ERG response functions were recorded to evaluate the impact of gender and diabetes on retinal function under scotopic conditions; a-wave (A) and b-wave (B). Diabetes significantly reduced b-wave in both males and females, whereas a-wave was significantly different in female mice and tend to decrease in male mice (but did not reach significance). ERG recordings of both a- and b-wave were not different between genders in N mice. Data shown as Mean ± SEM (n = 16–20 eyes). * P ≤ 0.05; ** P ≤ 0.01.

Fig 7. Leukocyte-mediated cytotoxicity toward retinal endothelial cells is significantly increased in both diabetic males and females compared to nondiabetic controls.

Data are presented as mean ± SD, n = 6 per group. N, nondiabetic; D, diabetic. *P ≤ 0.05, **P ≤ 0.01.

Fig 8. Effects of STZ-induced diabetes (8 months) on capillary degeneration in retinas from male and female mice.

Representative photomicrographs depicting the retinal vasculature in nondiabetic and diabetic males and females (A). Data are graphed as degenerated capillaries per unit area of retina (B). Examples of degenerated capillaries are indicated by arrows. Data are presented as mean ± SD, n = 6 per group. N, nondiabetic; D, diabetic. **P ≤ 0.01, ***P ≤ 0.001.