Gene expression profiling in a mouse model of retinal vein occlusion induced by laser treatment reveals a predominant inflammatory and tissue damage response

Purpose
Retinal vein occlusion (RVO) has been investigated in several laser-induced animal models using pigs, rabbits and rats. However, laser-induced RVO has been rarely reported in mice, despite the impressive number of available mutants, ease of handling and cost effectiveness. The aim of this study was to further assess the feasibility of a RVO mouse model for gene expression analysis and its possible use to investigate effects of hypoxia.
Methods
C57Bl/6J mice were injected with eosin Y for photo-sensitization. Subsequently, large retinal veins were laser-treated in one eye to induce vascular occlusion. Contralateral control eyes received non-occlusive retinal laser treatment sparing large vessels. The animals were followed for up to eight days and assessed by funduscopy, angiography, hypoxyprobe staining, histopathology and gene expression analysis by qPCR and RNA sequencing (RNAseq). Another group of mice was left untreated and studied at a single time point to determine baseline characteristics.
Results
Laser-induced RVO persisted in half of the treated veins for three days, and in a third of the veins for the whole observation period of 8 days. Funduscopy revealed large areas of retinal swelling in all laser-treated eyes, irrespective of vascular targeting or occlusion status. Damage of the outer retina, retinal pigment epithelium (RPE), and even choroid and sclera at the laser site was observed in histological sections. Genes associated with inflammation or cell damage were highly up-regulated in all laser-treated eyes as detected by RNAseq and qPCR. Retinal hypoxia was observed by hypoxyprobe staining in all RVO eyes for up to 5 days with a maximal extension at days 2 and 3, but no significant RVO-dependent changes in gene expression were detected for angiogenesis- or hypoxia-related genes.
Conclusion
The laser-induced RVO mouse model is characterized by a predominant general inflammatory and tissue damage response, which may obscure distinct hypoxia- and angiogenesis-related effects. A non-occlusive laser treatment control is essential to allow for proper data interpretation and should be mandatory in animal studies of laser-induced RVO to dissect laser-induced tissue damage from vascular occlusion effects.

研究目的
视网膜静脉阻塞（Retinal vein occlusion, RVO）已在多种激光诱导的动物模型中得到研究，所用动物包括猪、兔与大鼠。尽管小鼠拥有大量可用的突变品系、操作便捷且成本效益优异，但激光诱导RVO在小鼠中的相关研究仍鲜有报道。本研究旨在进一步评估RVO小鼠模型用于基因表达分析的可行性，以及其用于探究缺氧效应的潜在应用价值。

研究方法
向C57Bl/6J小鼠注射伊红Y（eosin Y）以进行光致敏。随后对单侧眼的视网膜大静脉实施激光照射，以诱导血管阻塞；对侧对照眼则接受非阻塞性视网膜激光照射，避开大静脉。对实验动物进行最长8天的随访，通过眼底检查、血管造影、缺氧探针染色、组织病理学检测，以及qPCR与RNA测序（RNA sequencing, RNAseq）开展基因表达分析。另有一组小鼠未接受任何处理，仅在单个时间点进行检测以确定基线特征。

研究结果
半数接受激光处理的静脉在3天内维持激光诱导的RVO，另有三分之一的静脉在整个8天观察期内持续阻塞。眼底检查显示，所有接受激光照射的眼睛均出现大范围视网膜肿胀，与是否靶向血管或发生阻塞无关。组织切片观察发现，激光照射位点的视网膜外层、视网膜色素上皮（retinal pigment epithelium, RPE），甚至脉络膜与巩膜均出现损伤。通过RNAseq与qPCR检测发现，所有接受激光照射的眼睛中，与炎症或细胞损伤相关的基因均显著上调。缺氧探针染色显示，所有RVO模型眼在最长5天内均出现视网膜缺氧，缺氧范围在第2、3天达到峰值，但未检测到与血管生成或缺氧相关的基因出现显著的RVO依赖性表达变化。

研究结论
激光诱导的RVO小鼠模型以显著的全身性炎症与组织损伤反应为主要特征，这可能掩盖缺氧与血管生成相关的特异性效应。为确保数据能够得到合理解读，非阻塞性激光照射对照是必不可少的；在激光诱导RVO的动物研究中，应强制设置此类对照，以区分激光诱导的组织损伤与血管阻塞所产生的效应。