Supplementary Material for: Effects of light exposure duration on severity and long-term neurodevelopment following photothrombotic stroke in a neonate

Introduction
Perinatal stroke causes lasting neurological deficits and there are currently no effective treatment options. Established animal models of perinatal stroke do not always mimic the clinical presentation of neonatal injury or are technically challenging to perform. The photothrombotic (PT) stroke model is a minimally invasive method that replicates focal ischemic injury. Few studies have applied the PT model in neonatal contexts, and none have examined both short- and long-term effects across varying injury severities. This study aimed to optimise a protocol to create a mild model of perinatal stroke and subsequently characterize injury progression, neuropathological impact, and motor deficits over time.
Methods
On post-natal day (PND) 10 we used the PT method to induce perinatal stroke in rat pups. Pups were exposed to various light exposure times (10, 20 or 30 minutes) to determine the optimal time needed to produce a mild and reproducible cortical stroke injury. Behavioural assessments were conducted on days 4, 10, 20, and 30 post-injury. Brains were collected for analysis on days 3 and 40 post-injury.
Results
3 days post-injury, the 20 and 30-minute group had significant focal lesions and microbleeds were present in each of the PT groups. All PT groups showed significant neuron loss in the penumbra and the thalamus, and microglia activation in multiple brain regions. As 30 minutes of light exposure showed extensive cortical tissue loss (>70%), we excluded the 30-minute group from long-term assessment. 40 days post-injury, the 10 and 20-minute groups demonstrated significant tissue loss and neuronal loss in the penumbra and thalamus, but only the 20-minute group showed neuron loss in the hippocampus. The 10- and 20-minute groups both demonstrated ongoing motor deficits.
Conclusion
Our results demonstrate that increasing light exposure time in PT stroke results in a more severe stroke phenotype. 30 minutes of light exposure resulted in a severe injury at only 3 days post insult, therefore, was not further investigated. 10 and 20 minutes of light exposure had a similar effect at 3 days, however after 40 days the 20-minute exposure time created a moderate injury phenotype. From this study we propose that 10 minutes of light exposure is optimal to create a mild stroke phenotype and is associated with motor deficits and altered neuropathology. This injury phenotype provides a focal and reproducible injury, while still being mild enough to feasibly test therapeutics.

引言
围产期卒中会导致永久性神经功能缺损，目前尚无有效的治疗方案。现有围产期卒中动物模型要么无法准确模拟新生儿损伤的临床表型，要么操作技术难度较大。光血栓卒中（photothrombotic, PT）模型是一种微创方法，可重现局灶性缺血性损伤。目前鲜有研究将PT模型应用于新生儿场景，且尚无研究针对不同损伤严重程度，同时探究其短期与长期效应。本研究旨在优化实验方案，构建围产期卒中轻度模型，并依次表征损伤进展、神经病理学影响以及随时间变化的运动功能缺损情况。

方法
本研究于生后第10天（PND 10），采用PT方法诱导新生大鼠幼崽发生围产期卒中。为确定构建轻度且可重复的皮层卒中损伤所需的最优光照时长，我们为幼崽设置了10、20或30分钟三种不同的光照时长。分别于损伤后第4、10、20和30天进行行为学评估；分别于损伤后第3天和第40天采集脑组织用于后续分析。

结果
损伤后第3天，20分钟和30分钟光照组出现显著局灶性病变，所有PT组均存在微出血。所有PT组均在半暗带与丘脑出现显著神经元丢失，并在多个脑区观察到小胶质细胞激活。由于30分钟光照组出现了超过70%的大面积皮层组织丢失，因此我们将30分钟光照组排除在长期评估之外。损伤后第40天，10分钟与20分钟光照组均在半暗带和丘脑出现显著组织丢失与神经元丢失，但仅20分钟光照组在海马体出现神经元丢失。10分钟与20分钟光照组均表现出持续的运动功能缺损。

结论
本研究结果表明，PT卒中模型中光照时长越长，卒中表型越严重。30分钟光照仅在损伤后第3天就造成了严重损伤，因此未进行后续研究。10分钟与20分钟光照组在损伤后第3天的损伤效果相近，但在损伤后第40天，20分钟光照组构建出中度损伤表型。基于本研究结果，我们认为10分钟光照时长是构建轻度卒中表型的最优参数，该模型会伴随运动功能缺损与神经病理学改变。该损伤表型可形成局灶性且可重复的损伤，同时损伤程度足够轻微，可用于治疗手段的可行性测试。