Please wait a minute...
 Home  About the Journal Editorial Board Aims & Scope Peer Review Policy Subscription Contact us
Early Edition  //  Current Issue  //  Open Special Issues  //  Archives  //  Most Read  //  Most Downloaded  //  Most Cited
Aging and disease    2018, Vol. 9 Issue (6) : 1122-1133     DOI: 10.14336/AD.2018.0711
Orginal Article |
Remote Ischemic Conditioning Protects Diabetic Retinopathy in Streptozotocin-induced Diabetic Rats via Anti-Inflammation and Antioxidation
Ren Changhong1,5, Wu Hang1,2, Li Dongjie1,2, Yang Yong3, Gao Yuan1,2, Jizhang Yunneng4, Liu Dachuan2, Ji Xunming1,5, Zhang Xuxiang1,2,*
1Beijing Key Laboratory of Hypoxia Conditioning Translational Medicine, Beijing, China.
2Department of Ophthalmology, Xuanwu Hospital, Capital Medical University, Beijing, China.
3Department of Herbal Formula Science Medicine, Chinese Medicine College, Beijing University of Chinese Medicine, Beijing, China.
4Center of Cerebrovascular Disease Research, University of Pittsburgh School of Medicine, Pittsburgh, USA.
5Center of Stroke, Beijing Institute for Brain Disorder, Beijing 100069, China
Download: PDF(1126 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks    

Ischemic conditioning inhibits oxidative stress and inflammatory response in diabetes. However, whether limb remote ischemic conditioning (LRIC) has beneficial effects on diabetic retinopathy (DR) remains unknown. This study aims to investigate the protective effects of LRIC in retinal ganglion cell in streptozotocin (STZ) induced Type 1 diabetic rats. A total of 48 healthy male Sprague-Dawley (200-220g) rats were randomly assigned to the normal group, normal+LRIC group, diabetes mellitus (DM) group and DM+LRIC group. Streptozotocin (STZ, 60 mg/kg) was intraperitoneally injected into the rats to establish the diabetic model. LRIC was conducted by tightening a tourniquet around the upper thigh and releasing for three cycles daily (10 mins x 3 cycles). Retinas were harvested after 12 weeks of LRIC treatment for histopathologic, Western blot and ELISA analysis. Plasma were collected at the same time for ELISA analysis. LRIC alleviated diabetic retinopathy symptoms as evidenced by the increased number of retinal ganglion cells (P<0.01) and decreased glial fibrillary acidic protein (GFAP) expression level (P<0.01) in the rat retina. LRIC in DM rats exhibited anti-inflammatory and antioxidative effects as confirmed by the down-regulation of pro-inflammatory cytokine: interleukin-6 (IL-6), and the up-regulation of antioxidants: superoxide dismutase (SOD), and glutathione (GSH)/oxidized glutathione (GSSG). Furthermore, LRIC significantly downregulated VEGF protein expression in the retina (P<0.01). These results suggest that the antioxidative and anti-inflammatory activities of LRIC may be important mechanisms involved in the protective effect of LRIC in STZ-induced diabetic rats.

Keywords diabetes mellitus      diabetic retinopathy      limb remote ischemic conditioning      oxidative stress     
Corresponding Authors: Zhang Xuxiang   
About author:

Current address: Department of Orthopaedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, China.

Issue Date: 07 December 2018
E-mail this article
E-mail Alert
Articles by authors
Ren Changhong
Wu Hang
Li Dongjie
Yang Yong
Gao Yuan
Jizhang Yunneng
Liu Dachuan
Ji Xunming
Zhang Xuxiang
Cite this article:   
Ren Changhong,Wu Hang,Li Dongjie, et al. Remote Ischemic Conditioning Protects Diabetic Retinopathy in Streptozotocin-induced Diabetic Rats via Anti-Inflammation and Antioxidation[J]. Aging and disease, 2018, 9(6): 1122-1133.
URL:     OR
Figure 1.  Effect of LRIC on blood glucose levels and body weight at 12 weeks after onset of diabetes. (A) Representative sketches of the experiment. (B) Quantification of blood glucose levels. (C) Quantification of body weight. Data are expressed as mean±SD, * P<0.05 (DM vs. Normal group). N=10 each group.
Figure 2.  LRIC treatment ameliorated Brn3a+ retinal ganglion cell loss in diabetic rats. Immunohistochemical analysis of retinas of normal control group (A), normal+LRIC group (B), DM group (C) and DM+LRIC group (D). Arrows indicate Brn3a+ RGCs, scale bar=50 μm. (E) Bar graphs depicting the average number of Brn3a+ RGCs in each group. Data are expressed as mean±SD, * P<0.05. N=5 each group.
Figure 3.  LRIC treatment ameliorated retinal Müller cell activation in diabetic rats. There was a significant increase in the level of GFAP expression in the diabetic retina (C) compared with the control retina (A). After 12 weeks of LRIC treatment, GFAP immunostaining decreased significantly in the diabetic retina (D). However, GFAP expression in the control retina was not affected by LRIC (B). Scale bar=50 μm. (E) Bar graphs depicting the the density of GFAP in each group. Data are expressed as mean±SD, ** P<0.01, *** P<0.001. N=5 each group.
Figure 4.  LRIC treatment attenuated oxidative stress induced by hyperglycemia. ROS production in the retina was evaluated at 12 weeks after LRIC treatment. The graph showed the relative reactive oxygen species levels in each group. *P<0.05, **P<0.01, N=7 each group.
Figure 5.  LRIC altered antioxidant enzyme levels in diabetic rats. Antioxidant enzyme levels in the retina were analyzed at 12 weeks after LRIC treatment. (A) Analysis of SOD activity. Data represent mean±SD of six independent experiments. (B) The analysis of total glutathione level. Data represent mean±SD of three independent experiments. (C) Analysis of SOD/CAT ratio. (D) Analysis of GSH/GSSG ratio. Data represent mean±SD of six independent experiments. *P< 0.05, ** P<0.01, N=7 each group.
Figure 6.  LRIC treatment attenuated retinal inflammation in diabetic rats. The inflammatory markers were analyzed in the diabetic rat retina and plasma at 12 weeks after LRIC treatment by using ELISA. (A) Inflammatory cytokines TNF-α, IL-1β and IL-6 levels in the rat retina. (B) Inflammatory cytokines TNF-α, IL-1β, IL-6 levels in the rat plasma. Data represent mean±SD of six independent experiments. *P<0.05, **P<0.01, N=7 each group.
Figure 7.  LRIC treatment decreased the level of VEGF in diabetic rats. The expression of VEGF was analyzed in the diabetic rat retina and plasma at 12 weeks after LRIC treatment. (A) The expression of VEGF in retinas was detected by ELISA for the di?erent groups. (B) The expression of VEGF in retinas was detected by Western blot for the di?erent groups. Data represent mean±SD of six independent experiments. *P<0.05, **P<0.01, N=7 each group.
Figure 8.  Hypothesis regarding the mechanism of LRIC’s protective effects against diabetic retinopathy.
[1] Fong DS, Aiello L, Gardner TW, King GL, Blankenship G, Cavallerano JD, et al. (2004). Retinopathy in diabetes. Diabetes Care, 27 Suppl 1: S84–87
[2] Jindal V (2015). Neurodegeneration as a primary change and role of neuroprotection in diabetic retinopathy. Mol Neurobiol, 51: 878–884
[3] Hendrick AM, Gibson MV, Kulshreshtha A (2015). Diabetic Retinopathy. Prim Care, 42: 451–464
[4] SooHoo JR, Seibold LK, Kahook MY (2014). The link between intravitreal antivascular endothelial growth factor injections and glaucoma. Curr Opin Ophthalmol, 25: 127–133
[5] Yang Y, Ren C, Zhang Y, Wu X (2017). Ginseng: An Nonnegligible Natural Remedy for Healthy Aging. Aging Dis, 8: 708–720
[6] Luo X, Wu J, Jing S, Yan LJ (2016). Hyperglycemic Stress and Carbon Stress in Diabetic Glucotoxicity. Aging Dis, 7: 90–110
[7] Murugeswari P, Shukla D, Rajendran A, Kim R, Namperumalsamy P, Muthukkaruppan V (2008). Proinflammatory cytokines and angiogenic and anti-angiogenic factors in vitreous of patients with proliferative diabetic retinopathy and eales' disease. Retina, 28: 817–824
[8] Masuda T, Shimazawa M, Hara H (2017). Retinal Diseases Associated with Oxidative Stress and the Effects of a Free Radical Scavenger (Edaravone). Oxid Med Cell Longev, 2017: 9208489
[9] Augustin AJ, Keller A, Koch F, Jurklies B, Dick B (2001). [Effect of retinal coagulation status on oxidative metabolite and VEGF in 208 patients with proliferative diabetic retinopathy]. Klin Monbl Augenheilkd, 218: 89–94
[10] Heusch G (2013). Cardioprotection: chances and challenges of its translation to the clinic. Lancet, 381: 166–175
[11] Li X, Ren C, Li S, Han R, Gao J, Huang Q, et al. (2017). Limb Remote Ischemic Conditioning Promotes Myelination by Upregulating PTEN/Akt/mTOR Signaling Activities after Chronic Cerebral Hypoperfusion. Aging Dis, 8: 392–401
[12] Fernandez DC, Sande PH, Chianelli MS, Aldana Marcos HJ, Rosenstein RE (2011). Induction of ischemic tolerance protects the retina from diabetic retinopathy. Am J Pathol, 178: 2264–2274
[13] Salido EM, Dorfman D, Bordone M, Chianelli MS, Sarmiento MI, Aranda M, et al. (2013). Ischemic conditioning protects the rat retina in an experimental model of early type 2 diabetes. Exp Neurol, 240: 1–8
[14] Meng R, Asmaro K, Meng L, Liu Y, Ma C, Xi C, et al. (2012). Upper limb ischemic preconditioning prevents recurrent stroke in intracranial arterial stenosis. Neurology, 79: 1853–1861
[15] Brandli A (2015). Remote Limb Ischemic Preconditioning: A Neuroprotective Technique in Rodents. J Vis Exp
[16] Meng R, Ding Y, Asmaro K, Brogan D, Meng L, Sui M, et al. (2015). Ischemic Conditioning Is Safe and Effective for Octo- and Nonagenarians in Stroke Prevention and Treatment. Neurotherapeutics, 12: 667–677
[17] Zhang X, Jizhang Y, Xu X, Kwiecien TD, Li N, Zhang Y, et al. (2014). Protective effects of remote ischemic conditioning against ischemia/reperfusion-induced retinal injury in rats. Vis Neurosci, 31: 245–252
[18] Ren C, Gao M, Dornbos D3rd, Ding Y, Zeng X, Luo Y, et al. (2011). Remote ischemic post-conditioning reduced brain damage in experimental ischemia/reperfusion injury. Neurol Res, 33: 514–519
[19] Liu ZJ, Chen C, Li XR, Ran YY, Xu T, Zhang Y, et al. (2016). Remote Ischemic Preconditioning-Mediated Neuroprotection against Stroke is Associated with Significant Alterations in Peripheral Immune Responses. CNS Neurosci Ther, 22: 43–52
[20] Rolova T, Dhungana H, Korhonen P, Valonen P, Kolosowska N, Konttinen H, et al. (2016). Deletion of Nuclear Factor kappa B p50 Subunit Decreases Inflammatory Response and Mildly Protects Neurons from Transient Forebrain Ischemia-induced Damage. Aging Dis, 7: 450–465
[21] Geng X, Fu P, Ji X, Peng C, Fredrickson V, Sy C, et al. (2013). Synergetic neuroprotection of normobaric oxygenation and ethanol in ischemic stroke through improved oxidative mechanism. Stroke, 44: 1418–1425
[22] Urzua U, Chacon C, Lizama L, Sarmiento S, Villalobos P, Kroxato B, et al. (2017). Parity History Determines a Systemic Inflammatory Response to Spread of Ovarian Cancer in Naturally Aged Mice. Aging Dis, 8: 546–557
[23] Zheng H, Wu J, Jin Z, Yan LJ (2017). Potential Biochemical Mechanisms of Lung Injury in Diabetes. Aging Dis, 8: 7–16
[24] Nadal-Nicolas FM, Jimenez-Lopez M, Salinas-Navarro M, Sobrado-Calvo P, Alburquerque-Bejar JJ, Vidal-Sanz M, et al. (2012). Whole number, distribution and co-expression of brn3 transcription factors in retinal ganglion cells of adult albino and pigmented rats. PLoS One, 7: e49830
[25] Nadal-Nicolas FM, Jimenez-Lopez M, Sobrado-Calvo P, Nieto-Lopez L, Canovas-Martinez I, Salinas-Navarro M, et al. (2009). Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci, 50: 3860–3868
[26] Scott TM, Foote J, Peat B, Galway G (1986). Vascular and neural changes in the rat optic nerve following induction of diabetes with streptozotocin. J Anat, 144: 145–152
[27] Lopes de Faria JM, Russ H, Costa VP (2002). Retinal nerve fibre layer loss in patients with type 1 diabetes mellitus without retinopathy. Br J Ophthalmol, 86: 725–728
[28] Rungger-Brandle E, Dosso AA, Leuenberger PM (2000). Glial reactivity, an early feature of diabetic retinopathy. Invest Ophthalmol Vis Sci, 41: 1971–1980
[29] Dehdashtian E, Mehrzadi S, Yousefi B, Hosseinzadeh A, Reiter RJ, Safa M, et al. (2018). Diabetic retinopathy pathogenesis and the ameliorating effects of melatonin; involvement of autophagy, inflammation and oxidative stress. Life Sci, 193: 20–33
[30] Ren C, Li S, Liu K, Rajah G, Han R, Liu Y, et al. (2017). Enhanced oxidative stress response and neuroprotection of combined limb remote ischemic conditioning and atorvastatin after transient ischemic stroke in rats. Brain Circulation, 3: 204–212
[31] Joussen AM, Poulaki V, Le ML, Koizumi K, Esser C, Janicki H, et al. (2004). A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J, 18: 1450–1452
[32] Mocan MC, Kadayifcilar S, Eldem B (2006). Elevated intravitreal interleukin-6 levels in patients with proliferative diabetic retinopathy. Can J Ophthalmol, 41: 747–752
[33] Gustavsson C, Agardh CD, Hagert P, Agardh E (2008). Inflammatory markers in nondiabetic and diabetic rat retinas exposed to ischemia followed by reperfusion. Retina, 28: 645–652
[34] Costagliola C, Daniele A, dell'Omo R, Romano MR, Aceto F, Agnifili L, et al. (2013). Aqueous humor levels of vascular endothelial growth factor and adiponectin in patients with type 2 diabetes before and after intravitreal bevacizumab injection. Exp Eye Res, 110: 50–54
[35] Dongare S, Gupta SK, Mathur R, Saxena R, Mathur S, Agarwal R, et al. (2016). Zingiber officinale attenuates retinal microvascular changes in diabetic rats via anti-inflammatory and antiangiogenic mechanisms. Mol Vis, 22: 599–609
[36] Zhao G, Cheng XW, Piao L, Hu L, Lei Y, Yang G, et al. (2017). The Soluble VEGF Receptor sFlt-1 Contributes to Impaired Neovascularization in Aged Mice. Aging Dis, 8: 287–300
[37] Kastelan S, Tomic M, Gverovic Antunica A, Salopek Rabatic J, Ljubic S (2013). Inflammation and pharmacological treatment in diabetic retinopathy. Mediators Inflamm, 2013: 213130s
[38] Joussen AM, Poulaki V, Qin W, Kirchhof B, Mitsiades N, Wiegand SJ, et al. (2002). Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo. Am J Pathol, 160: 501–509
[39] Ueno K, Samura M, Nakamura T, Tanaka Y, Takeuchi Y, Kawamura D, et al. (2016). Increased plasma VEGF levels following ischemic preconditioning are associated with downregulation of miRNA-762 and miR-3072-5p. Sci Rep, 6: 36758
[40] Ara J, Fekete S, Frank M, Golden JA, Pleasure D, Valencia I (2011). Hypoxic-preconditioning induces neuroprotection against hypoxia-ischemia in newborn piglet brain. Neurobiol Dis, 43: 473–485
[41] Hausenloy DJ, Yellon DM (2011). The therapeutic potential of ischemic conditioning: an update. Nat Rev Cardiol, 8: 619–629
[42] Fernandez DC, Pasquini LA, Dorfman D, Aldana Marcos HJ, Rosenstein RE (2012). Early distal axonopathy of the visual pathway in experimental diabetes. Am J Pathol, 180: 303–313
[43] Hess DC, Blauenfeldt RA, Andersen G, Hougaard KD, Hoda MN, Ding Y, et al. (2015). Remote ischaemic conditioning-a new paradigm of self-protection in the brain. Nat Rev Neurol, 11: 698–710
[1] Luca Antonina, Calandra Carmela, Luca Maria. Molecular Bases of Alzheimer’s Disease and Neurodegeneration: The Role of Neuroglia[J]. Aging and disease, 2018, 9(6): 1134-1152.
[2] Yong-Fei Zhao, Qiong Zhang, Jian-Feng Zhang, Zhi-Yin Lou, Hen-Bing Zu, Zi-Gao Wang, Wei-Cheng Zeng, Kai Yao, Bao-Guo Xiao. The Synergy of Aging and LPS Exposure in a Mouse Model of Parkinson’s Disease[J]. Aging and disease, 2018, 9(5): 785-797.
[3] Fabiana Morroni,Giulia Sita,Agnese Graziosi,Eleonora Turrini,Carmela Fimognari,Andrea Tarozzi,Patrizia Hrelia. Neuroprotective Effect of Caffeic Acid Phenethyl Ester in A Mouse Model of Alzheimer’s Disease Involves Nrf2/HO-1 Pathway[J]. A&D, 2018, 9(4): 605-622.
[4] Yao-Chih Yang,Cheng-Yen Tsai,Chien-Lin Chen,Chia-Hua Kuo,Chien-Wen Hou,Shi-Yann Cheng,Ritu Aneja,Chih-Yang Huang,Wei-Wen Kuo. Pkcδ Activation is Involved in ROS-Mediated Mitochondrial Dysfunction and Apoptosis in Cardiomyocytes Exposed to Advanced Glycation End Products (Ages)[J]. A&D, 2018, 9(4): 647-663.
[5] Tao Yan,Poornima Venkat,Michael Chopp,Alex Zacharek,Peng Yu,Ruizhuo Ning,Xiaoxi Qiao,Mark R. Kelley,Jieli Chen. APX3330 Promotes Neurorestorative Effects after Stroke in Type One Diabetic Rats[J]. A&D, 2018, 9(3): 453-466.
[6] Jie Zhen,Tong Lin,Xiaochen Huang,Huiqiang Zhang,Shengqi Dong,Yifan Wu,Linlin Song,Rong Xiao,Linhong Yuan. Association of ApoE Genetic Polymorphism and Type 2 Diabetes with Cognition in Non-Demented Aging Chinese Adults: A Community Based Cross-Sectional Study[J]. A&D, 2018, 9(3): 346-357.
[7] Meng Zhang,Yong-Ning Deng,Jing-Yi Zhang,Jie Liu,Yan-Bo Li,Hua Su,Qiu-Min Qu. SIRT3 Protects Rotenone-induced Injury in SH-SY5Y Cells by Promoting Autophagy through the LKB1-AMPK-mTOR Pathway[J]. A&D, 2018, 9(2): 273-286.
[8] Mari L. Sbardelotto,Giulia S. Pedroso,Fernanda T. Pereira,Helen R. Soratto,Stella MS. Brescianini,Pauline S. Effting,Anand Thirupathi,Renata T. Nesi,Paulo CL. Silveira,Ricardo A. Pinho. The Effects of Physical Training are Varied and Occur in an Exercise Type-Dependent Manner in Elderly Men[J]. A&D, 2017, 8(6): 887-898.
[9] Guofen Gao, Nan Zhang, Yue-Qi Wang, Qiong Wu, Peng Yu, Zhen-Hua Shi, Xiang-Lin Duan, Bao-Lu Zhao, Wen-Shuang Wu, Yan-Zhong Chang. Mitochondrial Ferritin Protects Hydrogen Peroxide-Induced Neuronal Cell Damage[J]. A&D, 2017, 8(4): 458-470.
[10] Hong Zheng,Jinzi Wu,Zhen Jin,Liang-Jun Yan. Potential Biochemical Mechanisms of Lung Injury in Diabetes[J]. A&D, 2017, 8(1): 7-16.
[11] Kyung Soo Kim,Jin Wook Kwak,Su Jin Lim,Yong Kyun Park,Hoon Shik Yang,Hyun Jik Kim. Oxidative Stress-induced Telomere Length Shortening of Circulating Leukocyte in Patients with Obstructive Sleep Apnea[J]. A&D, 2016, 7(5): 604-613.
[12] Amelia Maria Gaman,Adriana Uzoni,Aurel Popa-Wagner,Anghel Andrei,Eugen-Bogdan Petcu. The Role of Oxidative Stress in Etiopathogenesis of Chemotherapy Induced Cognitive Impairment (CICI)-“Chemobrain”[J]. A&D, 2016, 7(3): 307-317.
[13] Haiping Zhao,Ziping Han,Xunming Ji,Yumin Luo. Epigenetic Regulation of Oxidative Stress in Ischemic Stroke[J]. A&D, 2016, 7(3): 295-306.
[14] Liwei Ma,Hongjun Wang,Chunyan Wang,Jing Su,Qi Xie,Lu Xu,Yang Yu,Shibing Liu,Songyan Li,Ye Xu,Zhixin Li. Failure of Elevating Calcium Induces Oxidative Stress Tolerance and Imparts Cisplatin Resistance in Ovarian Cancer Cells[J]. A&D, 2016, 7(3): 254-266.
[15] Sasanka Chakrabarti,Kochupurackal P. Mohanakumar. Aging and Neurodegeneration: A Tangle of Models and Mechanisms[J]. A&D, 2016, 7(2): 111-113.
Full text



Copyright © 2014 Aging and Disease, All Rights Reserved.
Address: Aging and Disease Editorial Office 3400 Camp Bowie Boulevard Fort Worth, TX76106 USA
Fax: (817) 735-0408 E-mail:
Powered by Beijing Magtech Co. Ltd