The effect of conditional knocking out vascular endothelial growth factor gene on the mouse model of oxygen induced retinopathy_Chinese Journal of Ocular Fundus Diseases

Authors：

 Jin Ji , Lu Yi , Feng Jia , Ren Yanhong , Yang Lili , Gu Weizhong

Department of Ophthalmology, Children's Hospital, Zhejiang University School of Medicine, Hangzhou 310052, China (Lu Yi, standardized training doctor, Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiaotong University);

Corresponding author：

Jin Ji, Email: jinji2@sina.com.cn

Keywords：

Microglia; Mice, knockout; Vascular endothelial growth factors; Disease models, animal; Hypoxia-inducible factor 1, alpha subunit

DOI：

10.3760/cma.j.issn.1005-1015.2017.05.016

Video：

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ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x²=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.

Citation： Jin Ji, Lu Yi, Feng Jia, Ren Yanhong, Yang Lili, Gu Weizhong. The effect of conditional knocking out vascular endothelial growth factor gene on the mouse model of oxygen induced retinopathy. Chinese Journal of Ocular Fundus Diseases, 2017, 33(5): 508-512. doi: 10.3760/cma.j.issn.1005-1015.2017.05.016 Copy

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1. Broxterman EC, Hug DA. Retinopathy of prematurity: a review of current screening guidelines and treatment options[J]. Mo Med, 2016, 113(3): 187-190.
2. Rivera JC, Madaan A, Zhou TE, et al. Review of the mechanisms and therapeutic avenues for retinal and choroidal vascular dysfunctions in retinopathy of prematurity[J]. Acta Paediatr, 2016, 105(12): 1421-1433. DOI: 10.1111/apa.13586.
3. Vadlapatla RK, Vadlapudi AD, Mitra AK. Hypoxia-inducible factor-1 (HIF-1): a potential target for intervention in ocular neovascular diseases[J]. Curr Drug Targets, 2013, 14(8): 919-935.
4. Jiang Y, Wang H, Culp D, et al. Targeting Müller cell-derived VEGF164 to reduce intravitreal neovascularization in the rat model of retinopathy of prematurity[J]. Invest Ophthalmol Vis Sci, 2014, 55(2): 824-831. DOI: 10.1167/iovs.13-13755.
5. Sankar MJ, Sankar J, Mehta M, et al. Anti-vascular endothelial growth factor (VEGF) drugs for treatment of retinopathy of prematurity[J/OL]. Cochrane Database Syst Rev, 2016, 2: CD009734[2016-02-27]. http://dx.doi.org/10.1002/14651858.CD009734.pub2. DOI: 10.1002/14651858.CD009734.pub2.
6. Mintz-Hittner HA, Kennedy KA, Chuang AZ, et al. Efficacy of intravitreal bevacizumab for stage 3+ retinopathy of prematurity[J]. N Engl J Med, 2011, 364(7): 603-615. DOI: 10.1056/NEJMoa1007374.
7. Honda S, Hirabayashi H, Tsukahara Y, et al. Acute contraction of the proliferative membrane after an intravitreal injection of bevacizumab for advanced retinopathy of prematurity[J]. Graefe's Arch Clin Exp Ophthalmol, 2008, 246(7): 1061-1063. DOI: 10.1007/s00417-008-0786-7.
8. Jang SY, Choi KS, Lee SJ. Delayed-onset retinal detachment after an intravitreal injection of ranibizumab for zone 1 plus retinopathy of prematurity[J]. J AAPOS, 2010, 14(5): 457-459. DOI: 10.1016/j.jaapos.2010.05.011.
9. Ueki Y, Ash JD, Zhu M, et al. Expression of Cre recombinase in retinal Müller cells[J]. Vision Res, 2009, 49(6): 615-621. DOI: 10.1016/j.visres.2009.01.012.
10. Hansen TM, Moss AJ, Brindle NP. Vascular endothelial growth factor and angiopoietins in neurovascular regeneration and protection following stroke[J]. Curr Neurovasc Res, 2008, 5(4): 236-245.
11. Sato T, Wada K, Arahori H, et al. Serum concentrations of bevacizumab (Avastin) and vascular endothelial growth factor in infants with retinopathy of prematurity[J]. Am J Ophthalmol, 2012, 153(2): 327-333. DOI: 10.1016/j.ajo.2011.07.005.
12. Syed MA, Choo-Wing R, Homer RJ, et al. Role of nitric oxide isoforms in vascular and alveolar development and lung injury in vascular endothelial growth factor overexpressing neonatal mice lungs[J/OL]. PLoS One, 2016, 11(1): 0147588[2016-01-22]. http://dx.plos.org/10.1371/journal.pone.0147588. DOI: 10.1371/journal.pone.0147588. eCollection 2016.

Chinese Journal of Ocular Fundus Diseases

The effect of conditional knocking out vascular endothelial growth factor gene on the mouse model of oxygen induced retinopathy

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