Barber Alistair J, Antonetti David A, Kern Timothy S, Reiter Chad E N, Soans Rohit S, Krady J Kyle, Levison Steven W, Gardner Thomas W, Bronson Sarah K
Penn State Retina Research Group, The Ulerich Ophthalmology Research Laboratory, Penn State College of Medicine, Hershey, 17033, USA.
Invest Ophthalmol Vis Sci. 2005 Jun;46(6):2210-8. doi: 10.1167/iovs.04-1340.
This study tested the Ins2(Akita) mouse as an animal model of retinal complications in diabetes. The Ins2(Akita) mutation results in a single amino acid substitution in the insulin 2 gene that causes misfolding of the insulin protein. The mutation arose and is maintained on the C57BL/6J background. Male mice heterozygous for this mutation have progressive loss of beta-cell function, decreased pancreatic beta-cell density, and significant hyperglycemia, as early as 4 weeks of age.
Heterozygous Ins2(Akita) mice were bred to C57BL/6J mice, and male offspring were monitored for hyperglycemia, beginning at 4.5 weeks of age. After 4 to 36 weeks of hyperglycemia, the retinas were analyzed for vascular permeability, vascular lesions, leukostasis, morphologic changes of micro- and macroglia, apoptosis, retinal degeneration, and insulin receptor kinase activity.
The mean blood glucose of Ins2(Akita) mice was significantly elevated, whereas the body weight at death was reduced compared with that of control animals. Compared with sibling control mice, the Ins2(Akita) mice had increased retinal vascular permeability after 12 weeks of hyperglycemia (P < 0.005), a modest increase in acellular capillaries after 36 weeks of hyperglycemia (P < 0.0008), and alterations in the morphology of astrocytes and microglia, but no changes in expression of Muller cell glial fibrillary acidic protein. Increased apoptosis was identified by immunoreactivity for active caspase-3 after 4 weeks of hyperglycemia (P < 0.01). After 22 weeks of hyperglycemia, there was a 16.7% central and 27% peripheral reduction in the thickness of the inner plexiform layer, a 15.6% peripheral reduction in the thickness of the inner nuclear layer (P < 0.001), and a 23.4% reduction in the number of cell bodies in the retinal ganglion cell layer (P < 0.005). In vitro insulin receptor kinase activity was reduced (P < 0.05) after 12 weeks of hyperglycemia.
The retinas of heterozygous male Ins2(Akita) mice exhibit vascular, neural, and glial abnormalities generally consistent with clinical observations and other animal models of diabetes. In light of the relatively early, spontaneous onset of the disease and the popularity of the C57BL/6J inbred strain as a background for the generation and study of other genetic alterations, combining the Ins2(Akita) mutation with other engineered mutations will be of great use for studying the molecular basis of retinal complications of diabetes.
本研究将Ins2（Akita）小鼠作为糖尿病视网膜并发症的动物模型进行测试。Ins2（Akita）突变导致胰岛素2基因中的单个氨基酸替换，从而引起胰岛素蛋白错误折叠。该突变出现在C57BL/6J背景中并得以维持。携带此突变的杂合子雄性小鼠早在4周龄时就出现β细胞功能进行性丧失、胰腺β细胞密度降低以及显著的高血糖症。
将杂合子Ins2（Akita）小鼠与C57BL/6J小鼠杂交，并从4.5周龄开始监测雄性后代的高血糖情况。在高血糖4至36周后，分析视网膜的血管通透性、血管病变、白细胞淤滞、小胶质细胞和大胶质细胞的形态变化、细胞凋亡、视网膜变性以及胰岛素受体激酶活性。
Ins2（Akita）小鼠的平均血糖显著升高，而死亡时的体重与对照动物相比有所降低。与同窝对照小鼠相比，Ins2（Akita）小鼠在高血糖12周后视网膜血管通透性增加（P < 0.005），高血糖36周后无细胞毛细血管适度增加（P < 0.0008），星形胶质细胞和小胶质细胞形态发生改变，但穆勒细胞胶质纤维酸性蛋白的表达无变化。高血糖4周后，通过活性半胱天冬酶-3免疫反应性鉴定出细胞凋亡增加（P < 0.01）。高血糖22周后，内网状层厚度中央减少16.7%，周边减少27%，内核层厚度周边减少15.6%（P < 0.001），视网膜神经节细胞层细胞体数量减少23.4%（P < 0.005）。高血糖12周后，体外胰岛素受体激酶活性降低（P < 0.05）。
杂合子雄性Ins2（Akita）小鼠的视网膜表现出血管、神经和胶质细胞异常，总体上与糖尿病的临床观察结果和其他动物模型一致。鉴于该疾病相对较早自发发生，且C57BL/6J近交系作为其他基因改变产生和研究的背景很受欢迎，将Ins2（Akita）突变与其他工程突变相结合对于研究糖尿病视网膜并发症的分子基础将非常有用。
