Establishment of a novel alloxan-induced rabbit model exhibiting unique diabetic retinal neuropathy features assessed via ERG + VEP

Abstract

Background: Diabetic retinal neuropathy (DRN) leads to significant visual impairment; however, no existing animal model fully replicates its neural alterations, and inconsistent induction protocols with high mortality rates hinder long-term investigations.

Methods: Adult male rabbits were randomly assigned to four experimental groups, each receiving a single intravenous injection of varying doses of alloxan and one control group. The safety and efficacy of alloxan in inducing diabetes were evaluated to determine the optimal dose. At 9 weeks following injection with alloxan, retinal function was assessed using full-field electroretinography (ERG) and visual evoked potentials (VEPs). Retinal structure was examined in rabbits using spectral-domain optical coherence tomography (SD-OCT), Optos ultra-widefield (Optos UWF) false-color imaging, and widefield fundus fluorescein angiography (WF-FFA).

Results: Rabbits in the 80 mg/kg alloxan group exhibited fewer complications, lower mortality, and a higher model success rate compared to other groups. At 9 weeks post-injection, these rabbits demonstrated significantly elevated hemoglobin A1c and total cholesterol (p < 0.05) relative to controls. ERG revealed statistically significant reductions in oscillatory potential and b-wave amplitudes (p < 0.05), while VEP indicated decreased P2 amplitude (p < 0.001) and prolonged P2 latency (p < 0.05). SD-OCT, Optos UWF imaging, and WF-FFA demonstrated no significant changes in vascular abnormalities. Additionally, Hematoxylin and Eosin staining revealed retinal swelling (p < 0.05), and immunofluorescence confirmed glial activation and neuronal loss.

Conclusions: A single intravenous injection of 80 mg/kg alloxan effectively and safely induced DRN in rabbits, resulting in neural retina damage, thereby establishing this model as an ideal model for DRN research.

Keywords

alloxan / diabetic retinal neuropathy / ERG+ops / rabbit / VEP

References

[1] Tan T-E, Wong TY. Diabetic retinopathy: looking forward to 2030. Front Endocrinol. 2022; 13: 1077669.

[2] Xie Q, Liu Y, Huang H, et al. An innovative method for screening and evaluating the degree of diabetic retinopathy and drug treatment based on artificial intelligence algorithms. Pharmacol Res. 2020; 159: 104986.

[3] Simó R, Stitt AW, Gardner TW. Neurodegeneration in diabetic retinopathy: does it really matter? Diabetologia. 2018; 61(9): 1902-1912.

[4] Oshitari T. The pathogenesis and therapeutic approaches of diabetic neuropathy in the retina. Int J Mol Sci. 2021; 22(16): 9050.

[5] Jonsson KB, Frydkjaer-Olsen U, Grauslund J. Vascular changes and neurodegeneration in the early stages of diabetic retinopathy: which comes first? Ophthalmic Res. 2016; 56(1): 1-9.

[6] Ahmed TS, Shah J, Zhen YNB, et al. Ocular microvascular complications in diabetic retinopathy: insights from machine learning. BMJ Open Diabetes Res Care. 2024; 12(1): e003758.

[7] Baba T. Detecting diabetic retinal neuropathy using fundus perimetry. Int J Mol Sci. 2021; 22(19): 10726-10737.

[8] Bikbova G, Oshitari T, Bikbov M. Diabetic neuropathy of the retina and inflammation: perspectives. Int J Mol Sci. 2023; 24(11): 2927-2940.

[9] Niaz K, Maqbool F, Khan F, Hassan FI, Momtaz S, Abdollahi M. Comparative occurrence of diabetes in canine, feline, and few wild animals and their association with pancreatic diseases and ketoacidosis with therapeutic approach. Vet World. 2018; 11(4): 410-422.

[10] Mansour SZ, Hatchell DL, Chandler D, Saloupis P, Hatchell MC. Reduction of basement membrane thickening in diabetic cat retina by sulindac. Invest Ophthalmol Vis Sci. 1990; 31(3): 457-463.

[11] Kador PF, Takahashi Y, Akagi Y, et al. Effect of galactose diet removal on the progression of retinal vessel changes in galactose-fed dogs. Invest Ophthalmol Vis Sci. 2002; 43(6): 1916-1921.

[12] Kleinwort KJH, Amann B, Hauck SM, et al. Retinopathy with central oedema in an INS C94Y transgenic pig model of long-term diabetes. Diabetologia. 2017; 60(8): 1541-1549.

[13] Degroote RL, Schmalen A, Renner S, Wolf E, Hauck SM, Deeg CA. Diabetic retinopathy from the vitreous proteome perspective: the INSC94Y transgenic pig model study. Proteomics. 2024; 24(20): e2300591.

[14] Yao F, Jiang X, Qiu L, et al. Long-term oral administration of salidroside alleviates diabetic retinopathy in db/db mice. Front Endocrinol. 2022; 13: 861452.

[15] Zhang L, Wang X, Chang L, et al. Quercetin improves diabetic kidney disease by inhibiting ferroptosis and regulating the Nrf2 in streptozotocin-induced diabetic rats. Ren Fail. 2024; 46(1): 2327495.

[16] Helfenstein T, Fonseca FA, Ihara SS, et al. Impaired glucose tolerance plus hyperlipidaemia induced by diet promotes retina microaneurysms in New Zealand rabbits. Int J Exp Pathol. 2011; 92(1): 40-49.

[17] Drago F, La Manna C, Emmi I, Marino A. Effects of sulfinpyrazone on retinal damage induced by experimental diabetes mellitus in rabbits. Pharmacol Res. 1998; 38(2): 97-100.

[18] Ozaki H, Hayashi H, Vinores SA, Moromizato Y, Campochiaro PA, Oshima K. Intravitreal sustained release of VEGF causes retinal neovascularization in rabbits and breakdown of the blood-retinal barrier in rabbits and primates. Exp Eye Res. 1997; 64(4): 505-517.

[19] Gardner TW, Abcouwer SF, Barber AJ, Jackson GR. An integrated approach to diabetic retinopathy research. Arch Ophthalmol. 2011; 129(2): 230-235.

[20] Sachdeva MM. Retinal neurodegeneration in diabetes: an emerging concept in diabetic retinopathy. Curr Diab Rep. 2021; 21(12): 65.

[21] Millán I, Desco MDC, Torres-Cuevas I, et al. Pterostilbene prevents early diabetic retinopathy alterations in a rabbit experimental model. Nutrients. 2019; 12(1): 82.

[22] Zhang Y, Zhao X, Liu Y, Yang X. Sulforaphane and ophthalmic diseases. Food Sci Nutr. 2024; 12(8): 5296-5311.

[23] Lee GH, Jeon C, Mok JW, et al. Smart wireless near-infrared light emitting contact lens for the treatment of diabetic retinopathy. Adv Sci (Weinh). 2022; 9(9): e2103254.

[24] Datlibagi A, Zein-El-Din A, Frohly M, Willermain F, Delporte C, Motulsky E. Experimental models to study epithelial-mesenchymal transition in proliferative vitreoretinopathy. Int J Mol Sci. 2023; 24(5): 4509.

[25] Sun W-T, Lei C-L, Bi C-C, Chen Z-L, Zhang L. Effect of alloxan time administerDrug on establishing diabetic rabbit model. Int J Ophthalmol. 2010; 3(3): 200-202.

[26] Bacevic M, Rompen E, Radermecker R, Drion P, Lambert F. Practical considerations for reducing mortality rates in alloxan-induced diabetic rabbits. Heliyon. 2020; 6(6): e04103.

[27] Chiang ST, Yeh SM, Chen YC, Lin SL, Tseng JK. Investigation of the protective effects of taurine against alloxan-induced diabetic retinal changes via electroretinogram and retinal histology with New Zealand white rabbits. Int J Endocrinol. 2014; 2014: 631549.

[28] Deng C-L, Hu C-B, Ling S-T, et al. Photoreceptor protection by mesenchymal stem cell transplantation identifies exosomal MiR-21 as a therapeutic for retinal degeneration. Cell Death Differ. 2021; 28(3): 1041-1061.

[29] Rösch S, Werner C, Müller F, Walter P. Photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea (MNU) in rabbits: a pilot study. Graefes Arch Clin Exp Ophthalmol. 2017; 255(2): 317-331.

[30] Rai V, Moellmer R, Agrawal DK. Clinically relevant experimental rodent models of diabetic foot ulcer. Mol Cell Biochem. 2022; 477(4): 1239-1247.

[31] Lin F, Chen Y, Liang H, Tan S. Echistatin prevents posterior capsule opacification in diabetic rabbit model via integrin linked kinase signaling pathway. Int J Clin Exp Pathol. 2015; 8(11): 14294-14304.

[32] Marzec E, Nowakowska De Rossi M, Paolucci S, Olszewski J. Dielectric properties of healthy and diabetic alloxan-induced lenses in rabbits. Bioelectrochemistry. 2020; 135: 107583.

[33] Maurissen TL, Spielmann AJ, Schellenberg G, et al. Modeling early pathophysiological phenotypes of diabetic retinopathy in a human inner blood-retinal barrier-on-a-chip. Nat Commun. 2024; 15(1): 1372.

[34] Safwat A, Sabry D, Ragiae A, Amer E, Mahmoud RH, Shamardan RM. Adipose mesenchymal stem cells-derived exosomes attenuate retina degeneration of streptozotocin-induced diabetes in rabbits. J Circ Biomark. 2018; 7: 1849454418807827.

[35] Li Y, Mitchell W, Elze T, Zebardast N. Association between diabetes, diabetic retinopathy, and glaucoma. Curr Diab Rep. 2021; 21(10): 38.

[36] Eid S, Sas KM, Abcouwer SF, et al. New insights into the mechanisms of diabetic complications: role of lipids and lipid metabolism. Diabetologia. 2019; 62(9): 1539-1549.

[37] Chen P, Zha Y, Wang L, et al. Evaluation of bone marrow texture and trabecular changes with quantitative DCE-MRI and QCT in Alloxan-induced diabetic rabbit models. Front Endocrinol. 2021; 12: 785604.

[38] Liu T, Zhao H, Li J, Korantzopoulos P, Li G. Rosiglitazone attenuates atrial structural remodeling and atrial fibrillation promotion in alloxan-induced diabetic rabbits. Cardiovasc Ther. 2014; 32(4): 178-183.

[39] Hu L, Zha YF, Wang L, et al. Quantitative evaluation of vertebral microvascular permeability and fat fraction in alloxan-induced diabetic rabbits. Radiology. 2018; 287(1): 128-136.

[40] Damek-Poprawa M, Both S, Wright AC, Maity A, Akintoye SO. Onset of mandible and tibia osteoradionecrosis: a comparative pilot study in the rat. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013; 115(2): 201-211.

[41] Mao X, Gu H, Kim D, et al. Aplp1 interacts with Lag3 to facilitate transmission of pathologic α-synuclein. Nat Commun. 2024; 15(1): 4663.

[42] Keum DH, Kim S-K, Koo J, et al. Wireless smart contact lens for diabetic diagnosis and therapy. Sci Adv. 2020; 6(17): eaba3252.

[43] Wang Z, Zhang N, Lin P, Xing Y, Yang N. Recent advances in the treatment and delivery system of diabetic retinopathy. Front Endocrinol. 2024; 15: 1347864.

[44] Chalke SD, Kale PP. Combinational approaches targeting neurodegeneration, oxidative stress, and inflammation in the treatment of diabetic retinopathy. Curr Drug Targets. 2021; 22(16): 1810-1824.

[45] Gopal K, Karwi QG, Tabatabaei Dakhili SA, et al. Aldose reductase inhibition alleviates diabetic cardiomyopathy and is associated with a decrease in myocardial fatty acid oxidation. Cardiovasc Diabetol. 2023; 22(1): 73.

[46] Yuan Y, Shi M, Li L, et al. Mesenchymal stem cell-conditioned media ameliorate diabetic endothelial dysfunction by improving mitochondrial bioenergetics via the Sirt1/AMPK/PGC-1α pathway. Clin Sci. 2016; 130(23): 2181-2198.

[47] Zhou P, Xie W, Meng X, et al. Notoginsenoside R1 ameliorates diabetic retinopathy through PINK1-dependent activation of mitophagy. Cells. 2019; 8(3): 213.

[48] Song S, Bao S, Zhang C, et al. Stimulation of AMPK prevents diabetes-induced photoreceptor cell degeneration. Oxid Med Cell Longev. 2021; 2021: 5587340.

[49] Yune TY, Lee JY, Jung GY, et al. Minocycline alleviates death of oligodendrocytes by inhibiting pro-nerve growth factor production in microglia after spinal cord injury. J Neurosci. 2007; 27(29): 7751-7761.

[50] Midena E, Torresin T, Longhin E, Midena G, Pilotto E, Frizziero L. Early microvascular and oscillatory potentials changes in human diabetic retina: amacrine cells and the intraretinal neurovascular crosstalk. J Clin Med. 2021; 10(18): 4035.

[51] McAnany JJ, Liu K, Park JC. Electrophysiological measures of dysfunction in early-stage diabetic retinopathy: no correlation between cone phototransduction and oscillatory potential abnormalities. Doc Ophthalmol. 2020; 140(1): 31-42.

[52] Kim T-H, Wang B, Lu Y, Son T, Yao X. Functional optical coherence tomography enables in vivo optoretinography of photoreceptor dysfunction due to retinal degeneration. Biomed Opt Express. 2020; 11(9): 5306-5320.

[53] Pescosolido N, Barbato A, Stefanucci A, Buomprisco G. Role of electrophysiology in the early diagnosis and follow-up of diabetic retinopathy. J Diabetes Res. 2015; 2015: 319692.

[54] Miura G. Visual evoked potentials for the detection of diabetic retinal neuropathy. Int J Mol Sci. 2023; 24(8): 7361.

[55] Kapila Sharma A, Mohan L, Mittal S, Bahadur A, Mirza AA, Thapiyal M. Assessment of visual evoked potential to detect visual pathway dysfunction in gestational diabetes mellitus: a longitudinal case-control study with postpartum follow-up. Cureus. 2023; 15(11): e49619.

[56] Silva PS, Liu D, Glassman AR, et al. Assessment of fluorescein angiography nonperfusion in eyes with diabetic retinopathy using ultrawide field retinal imaging. Retina. 2022; 42(7): 1302-1310.

[57] Li S, Zheng L, Zhang J, Liu X, Wu Z. Inhibition of ferroptosis by up-regulating Nrf2 delayed the progression of diabetic nephropathy. Free Radic Biol Med. 2021; 162: 435-449.

[58] de Bem AF, Portella RdL RL, Colpo E, et al. Diphenyl diselenide decreases serum levels of total cholesterol and tissue oxidative stress in cholesterol-fed rabbits. Basic Clin Pharmacol Toxicol. 2009; 105(1): 17-23.

[59] Sriwimol W, Choosongsang P, Choosongsang P, et al. Strong correlation and high comparability of capillary electrophoresis and three different methods for HbA1c measurement in a population without hemoglobinopathy. Scand J Clin Lab Invest. 2020; 80(2): 139-150.

[60] Kumar B, Gupta SK, Nag TC, et al. Retinal neuroprotective effects of quercetin in streptozotocin-induced diabetic rats. Exp Eye Res. 2014; 125: 193-202.

[61] Liu Y, Huang H, Sun Y, et al. Monosodium glutamate-induced mouse model with unique diabetic retinal neuropathy features and artificial intelligence techniques for quantitative evaluation. Front Immunol. 2022; 13: 862702.

[62] Mahmoudi N, Kiasalari Z, Rahmani T, et al. Diosgenin attenuates cognitive impairment in Streptozotocin-induced diabetic rats: underlying mechanisms. Neuropsychobiology. 2021; 80(1): 25-35.

[63] Sohn EH, van Dijk HW, Jiao C, et al. Retinal neurodegeneration may precede microvascular changes characteristic of diabetic retinopathy in diabetes mellitus. Proc Natl Acad Sci. 2016; 113(19): E2655-E2664.

[64] Lechner J, O'Leary OE, Stitt AW. The pathology associated with diabetic retinopathy. Vision Res. 2017; 139: 7-14.

[65] Li Y, Zhang W, Nguyen VP, et al. Retinal safety evaluation of photoacoustic microscopy. Exp Eye Res. 2021; 202: 108368.

[66] Llorian-Salvador M, Cabeza-Fernandez S, Gomez-Sanchez JA, de la Fuente AG. Glial cell alterations in diabetes-induced neurodegeneration. Cell Mol Life Sci. 2024; 81(1): 47.

[67] Zhang W, Wang Y, Kong J, Dong M, Duan H, Chen S. Therapeutic efficacy of neural stem cells originating from umbilical cord-derived mesenchymal stem cells in diabetic retinopathy. Sci Rep. 2017; 7(1): 408.

[68] Zhao X, Lin Z, Yu S, et al. An artificial intelligence system for the whole process from diagnosis to treatment suggestion of ischemic retinal diseases. Cell Rep Med. 2023; 4(10): 101197.

[69] Ljubimov AV, Burgeson RE, Butkowski RJ, et al. Basement membrane abnormalities in human eyes with diabetic retinopathy. J Histochem Cytochem. 1996; 44(12): 1469-1479.

[70] Paulus YM, Wang X, Nguyen VP, Zhang W, Tian C. Novel photoacoustic microscopy and optical coherence tomography dual-modality Chorioretinal imaging in living rabbit eyes. J Vis Exp. 2018; (132).

[71] Liu L, Li X, Cai W, et al. Coadministration of compound Danshen dripping pills and bezafibrate has a protective effect against diabetic retinopathy. Front Pharmacol. 2022; 13: 1014991.

[72] de Moraes G, Layton CJ. Therapeutic targeting of diabetic retinal neuropathy as a strategy in preventing diabetic retinopathy. Clin Exp Ophthalmol. 2016; 44(9): 838-852.

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