The study design, including the treatment and care of the animals, was approved and supervised by the Ethics Committee of Capital Medical University. All research protocols and procedures followed the Association for Research in Ophthalmology statement for the use of animals in ophthalmic and vision research. The study included male pigmented three-week-old guinea pigs that were reared in cycles of 12-hour light (450–500 lux) and 12-hour dark (∼0 lux) with room temperature maintained at 25°C. All animals had free access to food and water. 
研究设计，包括动物的治疗和护理，得到了首都医科大学伦理委员会的批准和监督。所有研究方案和程序都遵循眼科研究协会关于在眼科和视力研究中使用动物的声明。该研究包括雄性色素沉着的三周龄豚鼠，这些豚鼠以 12 小时光照（450-500 勒克斯）和 12 小时黑暗（∼0 勒克斯）的周期饲养，室温保持在 25°C。 所有动物都可以免费获得食物和水。

We performed five experimental protocols (the details for the doses applied are described in the Supplementary Materials and Methods). 
我们执行了五种实验方案（应用剂量的详细信息在补充材料和方法中描述）。

Everolimus and erlotinib, and MHY1485 (GlpBio Technology, Montclear, CA, USA) are insoluble in water. To keep a constant drug concentration during injection, suspensions were prepared by adding 5% polysorbate 80 (Sigma-Aldrich Co., St. Louis, MO, USA) to phosphate-buffered saline solution (PBS; Sigma-Aldrich Co.). All suspensions were immediately aliquoted and stored at −20°C. Intravitreal injections were performed under topical anesthesia using 0.5% proxymetacaine hydrochloride eye drops. The suspensions were thawed and shaken well before use. A 26-gauge needle (0.26 mm inner diameter) was used as a trocar needle to first make a vitreous entry port 2 mm posterior to the limbus. A 32-gauge Hamilton microsyringe (outer diameter: 0.235 mm, Hamilton Microliter syringe; Sigma-Aldrich Co.) delivered 5 µL of the solution into the eyes through the 26-gauge trocar needle. 
依维莫司和厄洛替尼以及MHY1485（GlpBio Technology，Montclear，CA，USA）不溶于水。为了在注射过程中保持恒定的药物浓度，通过向磷酸盐缓冲盐水溶液（PBS;Sigma-Aldrich公司）。立即将所有悬浮液等分并储存在-20°C下。 玻璃体内注射在局部麻醉下使用0.5%盐酸甲氧基美卡因滴眼液进行。悬浮液在使用前解冻并摇晃。使用 26 号针（内径 0.26 mm）作为套管针，首先在角膜缘后方 2 mm 处形成玻璃体入口。32 号汉密尔顿微量注射器（外径：0.235 mm，汉密尔顿微升注射器;Sigma-Aldrich公司）通过 26 号套管针将 5 μL 溶液输送到眼睛中。

All guinea pigs that received intravitreal injections of drugs into their right eyes received intravitreal injections of 5 µL of PBS with 5% polysorbate 80 into their left eyes. The guinea pigs in the negative control group and the NLIAE-only group received 5 µL of PBS with 5% polysorbate 80 once a week in both eyes. 
所有接受玻璃体内注射药物的豚鼠右眼都接受了玻璃体内注射 5 μL PBS 和 5% 聚山梨醇酯 80 的左眼。阴性对照组和仅NLIAE组的豚鼠每周一次双眼接受5μLPBS和5%聚山梨醇酯80。

To induce axial elongation, goggles were fitted with −10.0 diopter lenses (polymethyl methacrylate; diameter: 12.7 mm; Supplementary Fig. S1a) and taped onto the orbital rims of both eyes of the guinea pigs. Care was taken to ensure that the guinea pigs could open their eyes and blink freely while wearing the goggles. The refractive power of the lens and its centration were measured and verified before application. The goggles were examined daily to ensure that the lenses were clean and in place; otherwise, the goggles were detached and replaced with new goggles. The goggles were removed weekly for biometric examinations of the eyes. 
为了诱导轴向伸长，护目镜配备了-10.0屈光度镜片（聚甲基丙烯酸甲酯;直径：12.7毫米;补充图。S1a）并贴在豚鼠双眼的眼眶边缘。注意确保豚鼠在戴上护目镜时可以睁开眼睛并自由眨眼。在应用前测量和验证晶状体的屈光度及其中心。每天检查护目镜，以确保镜片清洁到位;否则，护目镜被拆下并更换为新的护目镜。每周取下护目镜，对眼睛进行生物特征检查。

All guinea pigs underwent optical coherence tomography (OCT) imaging of the ocular fundus (SS-OCT, VG200D, SVision Imaging, Ltd., Guangdong, China) without anesthesia. The OCT scans were performed using a star scan pattern centered on the optic disc center. The horizontal and vertical scan images were exported. For each guinea pig, the choroidal thickness was measured in the horizontal (3 o'clock and 9 o'clock positions) and vertical meridians (12 o'clock and 6 o'clock positions) at distances of one and three horizontal disc diameters from the optic disc center. The mean choroidal thickness was calculated as the average of the four measurement points at each distance. Under topical anesthesia, we measured the axial length by ocular ultrasonography (A-scan mode scan; oscillator frequency: 11 MHz; Quantel Co., Les Ulis, France). The ultrasound velocities used were 1557.5 m/s for the cornea and aqueous humor, 1723.3 m/s for the lens, and 1540 m/s for the vitreous cavity.^19,20 For each guinea pig, five measurements were performed, and the mean values were recorded. Fundus photography was performed using a wide-field fundus camera (ZEISS CLARUS 500; Carl Zeiss Meditec AG, Jena, Germany). 
所有豚鼠均在无麻醉的情况下接受眼底光学相干断层扫描（OCT）成像（SS-OCT，VG200D，SVision Imaging，Ltd.，Guangdong，China）。OCT扫描是使用以光盘中心为中心的星形扫描模式进行的。水平和垂直扫描图像已导出。对于每只豚鼠，在水平（3 点钟和 9 点钟位置）和垂直经络（12 点钟和 6 点钟位置）测量脉络膜厚度，距离视盘中心为 1 个和 3 个水平圆盘直径。平均脉络膜厚度计算为每个距离处四个测量点的平均值。在局部麻醉下，我们通过眼部超声检查（A扫描模式扫描;振荡器频率：11 MHz;Quantel Co.， Les Ulis， 法国）。角膜和房水使用的超声速度为 1557.5 m/s，晶状体为 1723.3 m/s，玻璃体腔为 1540 m/s。 ^{19 20} 对于每只豚鼠，进行了五次测量，并记录了平均值。眼底摄影使用宽视场眼底相机（蔡司 CLARUS 500;Carl Zeiss Meditec AG，德国耶拿）。

Guinea pigs were anesthetized by intraperitoneal injection of urethane (1000 mg/kg). After the animals were killed, the eyes were enucleated. For Western blot examination, the cornea, lens, and vitreous were first removed, and then the retina-choroid tissue and the sclera were harvested separately under a microscope. All samples were immediately stored in liquid nitrogen and transferred to a −80°C freezer. The frozen tissues were analyzed within one week. For histopathological examination, the enucleated eyeballs were immediately fixed in 10 mL of FAS Eyeball Fixative Solution (Wuhan Servicebio Technology Co. Ltd. Wuhan, China) for 24 hours and then embedded in paraffin. 
豚鼠通过腹膜内注射氨基甲酸乙酯（1000mg / kg）麻醉。动物被杀死后，眼睛被摘除。对于蛋白质印迹检查，首先去除角膜、晶状体和玻璃体，然后在显微镜下分别采集视网膜脉络膜组织和巩膜。所有样品立即储存在液氮中，并转移到-80°C冰箱中。在一周内对冷冻组织进行分析。为了进行组织病理学检查，将摘除的眼球立即固定在 10 mL FAS 眼球固定液（Wuhan Servicebio Technology Co.， Ltd. Wuhan， China）中 24 小时，然后包埋在石蜡中。

The details of the TUNEL staining have been described previously.⁷ After fixation and embedding in paraffin, histological slides (thickness: 8 µm) of three eyes were prepared following a routine protocol. We performed TUNEL staining (Cell Death Detection kit; Kaiji Biotechnology Co. Ltd., Jiangsu, China) to detect apoptotic cells in the retina. The histological sections were deparaffinized, and 200 µL of Proteinase K (10 µg/mL) were added to completely cover each section before incubation for 10 minutes at room temperature followed by rinsing in PBS three times. The sections were incubated with terminal deoxyribonucleotide transferase enzyme mixture solution (45 µL of equilibration buffer, 1.0 µL of biotin-11-dUTP, 4.0 µL of TdT enzyme) at 37°C for one hour and washed with PBS solution three times. Afterward, the sections were labeled with streptavidin-fluorescein (50 µL) for 30 minutes and counterstained with 4,6-diamidino-2-phenylindole (300 nM, 50–100 µL) for five minutes. Three sections from each eye were photographed, and the TUNEL-positive cells in the retina were counted. The mean of the counts obtained from three images representing the eyes of three different animals was recorded. 
TUNEL染色的细节在前面已经描述过。 ⁷ 固定并包埋在石蜡中后，按照常规方案制备三只眼睛的组织学载玻片（厚度：8μm）。我们进行了TUNEL染色（细胞死亡检测试剂盒;Kaiji Biotechnology Co.， Ltd.， Jiangsu， China）来检测视网膜中的凋亡细胞。对组织学切片进行脱蜡，加入200μL蛋白酶K（10μg/mL）以完全覆盖每个切片，然后在室温下孵育10分钟，然后在PBS中冲洗3次。将切片与末端脱氧核糖核苷酸转移酶混合物溶液（45 μL平衡缓冲液、1.0 μL生物素-11-dUTP、4.0 μL TdT酶）在37°C下孵育1小时，并用PBS溶液洗涤3次。之后，用链霉亲和素-荧光素（50μL）标记切片30分钟，并用4,6-二脒基-2-苯基吲哚（300nM，50-100μL）复染5分钟。拍摄每只眼睛的三个切片，并计数视网膜中的TUNEL阳性细胞。记录从代表三种不同动物眼睛的三张图像中获得的计数平均值。

After deparaffinization and antigen retrieval of histological slides, the sections were treated with PBS containing 0.25% Triton X-100 for 10 minutes and washed with PBS solution three times. Nonspecific bindings were blocked by incubation in 50 to 100 µL goat serum for 20 minutes at room temperature before incubation overnight with anti-phosphorated P70S6 kinase (Phospho T389, ab2571; Abcam, Cambridge, MA, USA; at 1:200 dilution) as the primary antibody. The sections were then incubated with a FITC-conjugated goat anti-rabbit secondary antibody (1:1000 dilution, 50-100 µL; AiFang Biology, China) for one hour in a dark chamber at 37°C. Finally, the sections were repeatedly immersed in PBS (three minutes) three times. The cell nuclei were counterstained with 4,6-diamidino-2-phenylindole for five minutes in dim light at room temperature. The sections were examined using a biological fluorescence inverted microscope (Olympus-CKX53; Olympus Co., Tokyo, Japan). 
在组织学载玻片脱蜡和抗原修复后，用含有0.25%Triton X-100的PBS处理切片10分钟，并用PBS溶液洗涤3次。在室温下在 50 至 100 μL 山羊血清中孵育 20 分钟，然后用抗磷酸化 P70S6 激酶孵育过夜，从而阻断非特异性结合（Phospho T389，ab2571;Abcam，美国马萨诸塞州剑桥市;以 1：200 稀释度）作为一抗。然后将切片与FITC偶联的山羊抗兔二抗（1：1000稀释度，50-100μL;AiFang Biology， China）在37°C的暗室中放置一小时。 最后，这些部分被反复浸泡在PBS（三分钟）中三次。在室温下，在昏暗的光线下用4,6-二脒基-2-苯基吲哚复染细胞核5分钟。使用生物荧光倒置显微镜（Olympus-CKX53;奥林巴斯公司，日本东京）。

Frozen retina-choroid and scleral tissues were homogenized and lysed in cold lysis buffer (RIPA; Amresco, Solon City, OH, USA) supplemented with protease inhibitors (Roche 11697498001; Roche, Basel, Switzerland) and phosphatase inhibitors (Roche 04906837001; Roche). The tissue extracts were separated on 8% SDS‒PAGE gels and transferred to nitrocellulose membranes according to a standard protocol. The membranes were blocked with 5% skimmed milk in TBST (Tris-HCl, NaCl, and Tween 20) for two hours and sequentially incubated with primary antibodies overnight and with secondary antibodies for two hours on the following day. Signals were assessed with an enhanced chemiluminescence kit (Millipore, Burlington, MA, USA), and images were taken with the Total Lab Quant V11.5 (TotalLab Ltd., Gosforth, UK). The target bands were quantified and analyzed using ImageJ (NIH, Bethesda, MD, USA) with β-tubulin as an internal control. The antibody details are listed in the Table. 
将冷冻的视网膜脉络膜和巩膜组织均质化并在冷裂解缓冲液（RIPA;Amresco， Solon City， OH， USA）补充蛋白酶抑制剂（Roche 11697498001;罗氏，巴塞尔，瑞士）和磷酸酶抑制剂（罗氏04906837001;罗氏）。在8%SDS\u2012PAGE凝胶上分离组织提取物，并根据标准方案转移到硝酸纤维素膜上。用 TBST（Tris-HCl、NaCl 和 Tween 20）中的 5% 脱脂牛奶封闭膜 2 小时，并与一抗连续孵育过夜，第二天与二抗孵育 2 小时。使用增强型化学发光试剂盒（Millipore，Burlington，MA，USA）评估信号，并使用Total Lab Quant V11.5（TotalLab Ltd.，Gosforth，UK）拍摄图像。使用 ImageJ（NIH，Bethesda，MD，USA）以 β-微管蛋白作为内部对照对目标条带进行定量和分析。抗体详细信息列于表中。

Using a website-based tool (http://powerandsamplesize.com/), we calculated the sample size before the start of the study. The standard deviation of axial length measurements was <0.05 mm. We assumed an axial length difference of 0.2 mm between eyes with NLIAE and eyes of the negative control group at the end of the study period of three weeks.⁷ Fewer than ten guinea pigs were needed to detect a 0.08 mm intergroup difference with 90% power. Thus each group contained 10 guinea pigs. The other statistical analyses were conducted using the Stata 17.0 software program (StataCorp, College Station, TX, USA) and GraphPad Prism 9.3.1 (GraphPad Software, San Diego, CA, USA). Unless stated otherwise, continuous variables are presented as the mean ± standard error. Comparisons of two samples were performed using the two-tailed Student's t-test. Paired t-tests were used to compare data from the two eyes of individual animals, whereas unpaired t-tests were used for data from different groups. Comparisons of multiple measurements from the same set of animals at different time points were performed by applying repeated-measures ANOVA and then using a Tukey honestly significant difference post hoc analysis to identify which differences between pairs of means were significant. For Western blot analysis, protein abundance is expressed as the ratio of each protein to β-tubulin, and the first control values (relative intensities of each protein/β-tubulin signal ratios) are set to 1. P values <0.05 were considered to indicate statistical significance. 
使用基于网站的工具（http://powerandsamplesize.com/），我们在研究开始前计算了样本量。轴向长度测量的标准偏差为 <0.05 mm。 我们假设在三周的研究期结束时，NLIAE的眼睛和阴性对照组的眼睛之间的轴向长度差异为0.2毫米。 ⁷ 需要少于10只豚鼠才能以90%的功效检测0.08 mm的组间差异。因此，每组包含10只豚鼠。其他统计分析使用Stata 17.0软件程序（StataCorp，College Station，TX，USA）和GraphPad Prism 9.3.1（GraphPad Software，San Diego，CA，USA）进行。除非另有说明，否则连续变量表示为平均±标准误差。使用双尾学生 t 检验对两个样本进行比较。配对 t 检验用于比较来自个体动物的两只眼睛的数据，而未配对的 t 检验用于来自不同组的数据。通过应用重复测量方差分析，然后使用Tukey诚实的显著差异事后分析来比较同一组动物在不同时间点的多个测量值，以确定平均值对之间的哪些差异是显着的。对于蛋白质印迹分析，蛋白质丰度表示为每种蛋白质与β-微管蛋白的比率，第一个对照值（每种蛋白质的相对强度/β-微管蛋白信号比值）设置为 1。P值<0.05被认为具有统计学意义。

Three weeks of negative lens induction resulted in an increase in axial length by 0.23 ± 0.04 mm (P < 0.001; Supplementary Fig. S1a). The NLIAE did not affect the body weight of the guinea pigs (P > 0.20 for all time points; Supplementary Fig. S1b). Compared to the negative control, NLIAE was associated with activation of the downstream signaling pathway of EGFR, including ERK1/2 (0.775 ± 0.128 in negative control vs. 1.256 ± 0.197 in NLIAE, relative abundance to β-tubulin, P = 0.045) and PI3K (2.707 ± 1.046 in negative control vs. 4.833 ± 1.010 in NLIAE, relative abundance to β-tubulin, P = 0.006) in the retina-choroid tissue of the guinea pigs (Figs. 1a, 1b). The activated ERK1/2 and PI3K pathways were associated with increased phosphorylation of ribosomal protein S6 kinase beta-1 kinase (p70S6K), a key substrate of mTORC1 (1.737 ± 0.234 in negative control vs. 2.848 ± 0.172 in NLIAE, P < 0.001, relative abundance to β-tubulin, Fig. 1c).¹¹ Compared to the contralateral eye, the unilateral intravitreal injection of the EGFR inhibitor erlotinib (dose: 20 µg) significantly reduced axial elongation by 0.09 ± 0.02 mm (P < 0.001; Fig. 1d). The injection of erlotinib was associated with a reduction in the phosphorylation of p70S6K in the eyes with NLIAE compared with the contralateral eyes (0.882 ± 0.073 in NLIAE vs. 0.569 ± 0.072 in NLIAE + erlotinib, relative abundance to β-tubulin, P = 0.004; Fig. 1e). NLIAE was not significantly associated with AKT (protein kinase B) phosphorylation (0.606 ± 0.205 in negative control vs. 0.661 ± 0.153 in NLIAE, relative abundance to β-tubulin, P = 0.43; Supplementary Fig. S1c). Because mTORC2 can activate AKT, the unchanged AKT phosphorylation levels suggested that only mTORC1, not mTORC2, was involved in NLIAE.¹¹
三周的负晶状体感应导致轴向长度增加0.23±0.04 mm（P < 0.001; 补充图。 S1a）。 NLIAE对豚鼠的体重没有影响（所有时间点的P>0.20;补充图S1b）。与阴性对照相比，NLIAE与EGFR下游信号通路的激活相关，包括ERK1/2（阴性对照为0.775±0.128，NLIAE为1.256±0.197，与β-微管蛋白的相对丰度，P=0.045）和PI3K（阴性对照为2.707±1.046，NLIAE为4.833±1.010，与β-微管蛋白的相对丰度，P=0.006）（图1a， 激活的ERK1/2和PI3K通路与核糖体蛋白S6激酶β-1激酶（p70S6K）的磷酸化增加有关，mTORC1的关键底物（阴性对照为1.737±0.234，NLIAE为2.848±0.172，P<0.001，与β-微管蛋白的相对丰度，图1c）。 ¹¹ 与对侧眼相比，单侧玻璃体内注射EGFR抑制剂厄洛替尼（剂量：20μg）显着降低了0.09±0.02mm（P<0.001;图1d）。与对侧眼相比，注射厄洛替尼与NLIAE眼中p70S6K磷酸化降低有关（NLIAE为0.882±0.073，NLIAE+厄洛替尼为0.569±0.072，与β-微管蛋白的相对丰度，P=0.004;图1e）。NLIAE与AKT（蛋白激酶B）磷酸化无显著相关性（阴性对照为0.606±0.205，NLIAE为0.661±0.153，与β-微管蛋白相对丰度，P=0.43;补充图S1c）。由于 mTORC2 可以激活 AKT，因此 AKT 磷酸化水平不变表明只有 mTORC1 而不是 mTORC2 参与 NLIAE。 ¹¹  

To explore the involvement of mTORC1 signaling during NLIAE, we assessed the effect of an intravitreally applied mTORC1 inhibitor (everolimus) on axial elongation. Compared to the contralateral eyes undergoing NLIAE alone, weekly monocular injections of everolimus in the eyes with NLIAE was associated with a significant and dose-dependent reduction in mTORC1 activation and a reduction in axial elongation (1.737 ± 0.234 in negative control, 2.848 ± 0.172 in NLIAE, 1.946 ± 0.157 in NLIAE + 2 µg everolimus, 2.061 ± 0.215 in NLIAE + 10 µg everolimus, 1.630 ± 0.230 in NLIAE + 20 µg everolimus, relative abundance to β-tubulin; Figs. 2a, 2b, Supplementary Fig. S2a). After three weekly unilateral injections of everolimus in doses of 2 µg, 10 µg, and 20 µg, axial elongation was decreased by 0.05 ± 0.01 mm (P = 0.04), 0.09 ± 0.01 mm (P < 0.001), and 0.14 ± 0.01 mm (P < 0.001), respectively, when compared to the contralateral eyes undergoing NLIAE alone. 
为了探索 mTORC1 信号转导在 NLIAE 过程中的参与，我们评估了玻璃体内应用的 mTORC1 抑制剂（依维莫司）对轴向伸长率的影响。与单独接受NLIAE的对侧眼相比，每周单眼注射依维莫司与NLIAE的眼睛中与mTORC1激活的显着和剂量依赖性减少和轴向伸长率的减少有关（阴性对照组为1.737±0.234，NLIAE为2.848±0.172，NLIAE为1.946±0.157 + 2μg依维莫司，NLIAE为2.061±0.215 + 10μg依维莫司， 1.630 ± 0.230 的 NLIAE + 20 μg 依维莫司，相对丰度为 β-微管蛋白;图2a、2b，补充图S2a）。在每周三次以2μg，10μg和20μg的剂量单侧注射依维莫司后，与单独接受NLIAE的对侧眼睛相比，轴向伸长率分别降低了0.05±0.01mm（P = 0.04），0.09±0.01mm（P < 0.001）和0.14±0.01 mm（P < 0.001）。

The changes in axial length were due mainly to changes in the depth of the vitreous compartment, with only minor changes in the anterior chamber depth and lens thickness (Fig. 3). After three weekly unilateral injections of everolimus in doses of 2 µg, 10 µg, and 20 µg, the increase in vitreous depth was reduced by 0.05 ± 0.01 mm (P = 0.18), 0.08 ± 0.02 mm (P < 0.001), and 0.10 ± 0.02 mm (P < 0.001), respectively, when compared to the contralateral eyes undergoing NLIAE alone. In contrast, the injections were not significantly associated with a significant difference in either anterior chamber depth (everolimus 2 µg: 0.02 ± 0.01 mm; P = 0.23; everolimus 10 µg −0.00 ± 0.01 mm P = 0.99; and everolimus 20 µg: 0.00 ± 0.01 mm; P = 0.86) or in lens thickness (everolimus 2 µg: −0.01 ± 0.02 mm, P = 0.93; everolimus 10 µg: −0.01 ± 0.01 mm, P = 0.92; and everolimus 20 µg: 0.03 ± 0.01 mm, P = 0.44), when compared to contralateral eyes undergoing NLIAE alone. Well-defined choroidal vessels at the posterior pole were observed in guinea pigs with NLIAE. In contrast, such well-defined choroidal vessels were almost undetectable in guinea pigs with both NLIAE and intravitreal mTORC1 inhibition (Supplementary Figs. S2b–d). 
轴向长度的变化主要是由于玻璃体室深度的变化，前房深度和晶状体厚度只有微小的变化（图3）。在每周三次以2μg，10μg和20μg的剂量单侧注射依维莫司后，玻璃体深度的增加分别减少了0.05±0.01mm（P = 0.18），0.08±0.02mm（P < 0.001）和0.10±0.02 mm（P < 0.001），与单独接受NLIAE的对侧眼睛相比。相比之下，注射与前房深度的显着差异没有显着相关性（依维莫司 2 μg：0.02 ± 0.01 mm;P = 0.23;依维莫司 10 μg −0.00 ± 0.01 mm P = 0.99;依维莫司20μg：0.00±0.01mm;P = 0.86）或晶状体厚度（依维莫司 2 μg：-0.01 ± 0.02 mm，P = 0.93;依维莫司 10 μg：-0.01 ± 0.01 mm，P = 0.92;依维莫司 20 μg：0.03 ± 0.01 mm，P = 0.44），与单独接受 NLIAE 的对侧眼睛相比。在患有NLIAE的豚鼠中观察到后极的脉络膜血管边界清晰。相比之下，在同时抑制 NLIAE 和玻璃体内 mTORC1 的豚鼠中几乎无法检测到这种定义明确的脉络膜血管（补充图 S2b-d）。

The contralateral eyes intravitreally injected with vehicle solution and the eyes intravitreally injected with 10 µg everolimus did not differ significantly in axial length or in the fundus appearance (Fig. 2b, Supplementary Figs. S2a, S3). TUNEL staining also did not show significant differences in the count of TUNEL-positive cells in the inner nuclear and outer nuclear layer of the retina (0.33 ± 0.33 cells vs. 0.33 ± 0.33 cells; P > 0.99) between the eyes receiving intravitreal everolimus injections and the contralateral eyes receiving vehicle injections (Fig. 2c). 
玻璃体内注射载体溶液的对侧眼和玻璃体内注射10μg依维莫司的眼睛在轴向长度或眼底外观上没有显着差异（图2b，补充图S2a，S3）。TUNEL染色也未显示视网膜内核和外核层中TUNEL阳性细胞计数的显着差异（0.33±0.33个细胞对0.33个±0.33个细胞;P > 0.99） 在接受玻璃体内注射依维莫司的眼睛和接受载体注射的对侧眼睛之间（图 2c）。

To explore the location of mTORC1 activation during NLIAE, we examined the pattern of immunofluorescence staining of eyes undergoing NLIAE. The eyes undergoing NLIAE exhibited phosphorylated P70S6K primarily in the RPE layer (Fig. 4). Eyes receiving NLIAE and everolimus injections (10 µg) showed less intense immunofluorescence for phosphorylated P70S6K than eyes undergoing NLIAE alone. 
为了探索 NLIAE 期间 mTORC1 激活的位置，我们检查了接受 NLIAE 的眼睛的免疫荧光染色模式。接受NLIAE的眼睛主要在RPE层中表现出磷酸化的P70S6K（图4）。与单独接受NLIAE的眼睛相比，接受NLIAE和依维莫司注射（10μg）的眼睛对磷酸化P70S6K的免疫荧光较弱。

Sclera hypoxia and local inflammation have been found to be associated with the development and progression of myopia.^21,22 To better understand the mechanisms of mTORC1 signaling, we investigated the association of the mTORC1 signaling pathway with scleral hypoxia and local inflammation. As evaluated by in vivo OCT imaging (Figs. 5a, 5b), the intravitreal application of 10 µg everolimus partially attenuated NLIAE-induced choroidal thinning by 2.95 ± 0.94 µm (P = 0.002) and 9.20 ± 2.08 µm (P = 0.003), measured at a distance of one and three horizontal disc diameters from the optic disc center (Figs. 5c, 5d, Supplementary Figs. S4a, S4b). Compared to NLIAE alone, intravitreally injected MHY1485 combined with NLIAE promoted NLIAE-induced choroidal thinning by 3.28 ± 0.94 µm (P = 0.040) and 9.42 ± 2.10 µm (P = 0.008), measured at a distance of one and three horizontal disc diameters from the optic disc center (Figs. 5c 5d, Supplementary Fig. S4a, S4b). As the choroid contributes to the oxygen supply of the sclera, choroidal thinning may cause scleral hypoxia. Therefore, we examined the expression of hypoxia-inducible factor-1α (HIF-1α) in the scleral tissue. Compared to the negative control group, the NLIAE group showed an upregulation of HIF-1α expression in the sclera (1.603 ± 0.395 in negative control group vs. 3.288 ± 0.295 in the NLIAE group, relative abundance to β-tubulin, P = 0.022) (Figs. 5e, 5f). Intravitreal mTORC1 inhibition significantly downregulated scleral HIF-1α in a dose-dependent manner (3.288 ± 0.295 in the NLIAE group vs. 2.086 ± 0.198 in the NLIAE + 10 µg everolimus group, P = 0.021; 3.288 ± 0.295 in the NLIAE group vs. 0.546 ± 0.346 in the NLIAE + 20 µg everolimus group, relative abundance to β-tubulin, P = 0.038) (Figs. 5e, 5f). In addition, after three weeks of NLIAE treatment, NF-κB was significantly upregulated, but this effect was partially reversed by mTORC1 inhibition (1.246 ± 0.277 in the negative control group vs. 2.166 ± 0.192 in the NLIAE group, P = 0.045; 2.166 ± 0.192 in the NLIAE group vs. 1.552 ± 0.283 in the NLIAE + 10 µg everolimus group, P = 0.024; 2.166 ± 0.192 in the NLIAE group vs. 1.308 ± 0.110 in the NLIAE + 20 µg everolimus group, relative abundance to β-tubulin, P = 0.014) (Supplementary Fig. S5a). Monocyte chemoattractant protein–1 expression in the retina-choroid tissue was independent of mTORC1 activity and intravitreal application of everolimus (1.303 ± 0.234 in the negative control group, 1.316 ± 0.207 in the NLIAE group, 1.025 ± 0.228 in the NLIAE + 2 µg everolimus group, 1.194 ± 0.149 in the NLIAE + 10 µg everolimus group, 1.047 ± 0.224 in the NLIAE + 20 µg everolimus group, relative abundance to β-tubulin, P > 0.20 for all comparisons) (Supplementary Fig. S5b). 
巩膜缺氧和局部炎症与近视的发展和进展有关。 ^{21 22} 为了更好地了解 mTORC1 信号传导的机制，我们研究了 mTORC1 信号通路与巩膜缺氧和局部炎症的关联。通过体内OCT成像（图5a，5b）进行评估，玻璃体内施用10μg依维莫司可部分减弱NLIAE诱导的脉络膜变薄2.95±0.94μm（P = 0.002）和9.20±2.08μm（P = 0.003），在距视盘中心1和3个水平圆盘直径的距离处测量（图5c，5d，补充图S4a， S4b）。与单独使用NLIAE相比，玻璃体内注射MHY1485与NLIAE联合使用，在距视盘中心1个和3个水平圆盘直径的距离处测量，NLIAE诱导的脉络膜变薄±0.94μm（P=0.040）和9.42±2.10μm（P = 0.008）促进了3.28 （0.040）。由于脉络膜有助于巩膜的氧气供应，脉络膜变薄可能导致巩膜缺氧。因此，我们检查了缺氧诱导因子-1α （HIF-1α） 在巩膜组织中的表达。与阴性对照组相比，NLIAE组在巩膜中HIF-1α表达上调（阴性对照组为1.603±0.395，NLIAE组为3.288±0.295，与β-微管蛋白的相对丰度，P = 0.022）（图5e，5f）。玻璃体内 mTORC1 抑制以剂量依赖性方式显着下调巩膜 HIF-1α（NLIAE 组为 3.288 ± 0.295，NLIAE 组为 2.086 ± 0.198，P = 0.021;NLIAE 组为 3.288 ± 0.295，NLIAE 组为 0.546 ± 0.346，与 β-微管蛋白组相对丰度，P = 0.038）（图 5e、5f）。 此外，在NLIAE治疗3周后，NF-κB显著上调，但mTORC1抑制部分逆转了这种作用（阴性对照组为1.246±0.277，NLIAE组为2.166±0.192，P=0.045;NLIAE组为2.166±0.192，NLIAE组为1.552±0.283，P=0.024;NLIAE组为2.166±0.192，NLIAE组为1.308±0.110，nliae组为1.3080.110， β-微管蛋白的相对丰度，P = 0.014）（补充图S5a）。视网膜脉络膜组织中单核细胞趋化蛋白-1的表达与mTORC1活性和依维莫司的玻璃体内应用无关（阴性对照组为1.303±0.234，NLIAE组为1.316±0.207，NLIAE组为1.025±0.228 + 2μg依维莫司组，NLIAE组为1.194±0.149 + 10μg依维莫司组，NLIAE组为1.047±0.224 + 20μg依维莫司组， 与β-微管蛋白的相对丰度，所有比较的P>0.20）（补充图S5b）。

Compared to NLIAE alone, weekly intravitreal injections of MHY1485 and NLIAE significantly increased axial elongation. After three months of NLIAE and MHY1485 injections, the guinea pigs showed an additional axial elongation of 0.43 ± 0.03 mm (P < 0.001; Supplementary Fig. S6). The fundus images and OCT scan of negative controls and eye received NLIAE only were exhibited in Figures 6a through 6d. In parallel to the increased axial elongation, fundus photography showed large well-defined choroidal vessels at the posterior pole in all guinea pigs. After three months of NLIAE and MHY1485 injections, diffuse choroidal atrophy was found in the peripapillary region of 2 out of 8 (25%) guinea pigs (Fig. 6e). The diffuse choroidal atrophy areas corresponded to marked choroidal thinning on the OCT scans (Fig. 6f). 
与单独使用NLIAE相比，每周玻璃体内注射MHY1485和NLIAE可显著增加轴向伸长率。经过三个月的NLIAE和MHY1485注射后，豚鼠显示出0.43±0.03 mm的额外轴向伸长率（P < 0.001;补充图S6）。阴性对照的眼底图像和OCT扫描和仅接受眼睛NLIAE显示在图6a至6d中。在增加轴向伸长率的同时，眼底照相显示所有豚鼠的后极都有轮廓分明的大脉络膜血管。经过三个月的NLIAE和MHY1485注射后，在8只豚鼠中有2只（25%）的状周围区域发现弥漫性脉络膜萎缩（图6e）。弥漫性脉络膜萎缩区域对应于 OCT 扫描上明显的脉络膜变薄（图 6f）。

In this study, NLIAE was found to be associated with an activation of mTORC1, which could be suppressed by an intravitreally applied mTORC1 inhibitor (everolimus). The mTORC1 inhibition attenuated choroidal thinning during NLIAE. Immunofluorescence revealed that the RPE cell layer was the primary location of mTORC1 activation after NLIAE treatment. OCT examination showed that mTORC1 inhibition can partially attenuated NLIAE-induced choroidal thinning and upregulation of sclera HIF-1α expression. Compared with NLIAE alone, combining NLIAE and intravitreal injections of an mTORC1 agonist (MHY1485) further enhanced axial elongation, choroidal thinning, and parapapillary choroidal atrophy. 
在这项研究中，发现 NLIAE 与 mTORC1 的激活有关，mTORC1 可以被玻璃体内应用的 mTORC1 抑制剂（依维莫司）抑制。mTORC1 抑制减弱了 NLIAE 期间的脉络膜变薄。免疫荧光显示，NLIAE处理后，RPE细胞层是mTORC1激活的主要位置。OCT 检查显示，mTORC1 抑制可以部分减弱 NLIAE 诱导的脉络膜变薄和巩膜 HIF-1α 表达的上调。与单独使用 NLIAE 相比，结合 NLIAE 和玻璃体内注射 mTORC1 激动剂 （MHY1485） 可进一步增强轴向伸长、脉络膜变薄和旁脉络膜萎缩。

The results of our study agree with observations made in previous investigations suggesting that the EGFR signaling pathway may play a role in the process of axial elongation. Previous studies have linked EGFR activation with axial elongation of eyes with NLIAE in a guinea pig model of myopia.^7–9 Correspondingly, a single-gene polymorphism of amphiregulin (rs12511037), an EGFR ligand, is significantly associated with myopic refractive error, and this association interacts with high educational attainment in Asians.²³ In agreement with previous studies showing that the RPE has receptors for EGF and EGF family members such as amphiregulin,²⁴ we found that RPE cells were the primary site of mTORC1 activation. These findings indicate that the RPE and mTORC1 signaling may play a role in axial elongation. 
我们的研究结果与先前研究中的观察结果一致，表明EGFR信号通路可能在轴向伸长过程中发挥作用。先前的研究已将 EGFR 激活与豚鼠近视模型中 NLIAE 的眼睛轴向伸长联系起来。 ^{7 – 9} 相应地，EGFR配体两栖调节蛋白（rs12511037）的单基因多态性与近视屈光不正显著相关，并且这种关联与亚洲人的高教育程度相互作用。 ²³ 与先前的研究一致，表明 RPE 具有 EGF 和 EGF 家族成员（如两栖调节蛋白）的受体， ²⁴ 我们发现 RPE 细胞是 mTORC1 激活的主要位点。这些发现表明，RPE 和 mTORC1 信号转导可能在轴向伸长中发挥作用。

The upstream regulation of mTORC1 in axial elongation may involve several signaling pathways. In this study, we applied a dose of 20 µg (0.051 µmol) of erlotinib in a vitreous volume of guinea pig eyes of approximately 90 µL, resulting in an erlotinib concentration of 5.65*10² µM.¹⁴ Considering that the IC50 (half maximal inhibitory concentration) of a cell-based assay for EGFR inhibition is 0.42 µM, 20 µg of erlotinib results in an intraocular concentration 1000-fold higher than the IC50 in cell-based assays for EGFR inhibition.²⁵ However, the magnitude of attenuation of axial elongation was considerably lower than that achieved with the mTORC1 inhibitor (NLIAE + 20 µg everolimus vs. NLIAE: −0.14 ± 0.01 mm, NLIAE + 20 µg erlotinib vs. NLIAE −0.09 ± 0.02 mm for erlotinib). One of the reasons for the difference could be the lower molecular weight of erlotinib (393.4 g/mol) compared to everolimus (958.2 g/mol), which might result in faster clearance of erlotinib from the vitreous.¹⁵ Other mechanisms may also have contributed to mTORC1 activation. Local inflammation has been reported to be associated with myopia.^26,27 In guinea pigs eyes undergoing form deprivation-induced myopia, the retinal TNFα- and IL-17–related proinflammatory pathways are activated.²⁶ After treating hamster eyes that underwent form deprivation with the anti-inflammatory agent diacerein, retinal proinflammatory cytokine levels and axial elongation decreased.²⁸ In contrast, administrating proinflammatory cytokines, such as TNF-α and IL-6, to the conjunctival sacs of hamsters induced myopia ranging from −2.1D to −3.1D after 21 days.²² In our study, we also found that in the retina-choroid tissue of guinea pigs, the expression of NF-κB was significantly upregulated after NLIAE treatment. Proinflammatory cytokines can activate mTORC1 through inhibition of the tuberous sclerosis complex.¹¹ Because of its immunosuppressive effect, everolimus may suppress local proinflammatory cytokines in the retina and choroid. This effect may contribute to the inhibitory effect of everolimus on axial elongation.²⁹
mTORC1 在轴向伸长过程中的上游调控可能涉及多种信号通路。在这项研究中，我们在大约 90 μL 的豚鼠眼睛玻璃体中施用 20 μg（0.051 μmol）剂量的厄洛替尼，导致厄洛替尼浓度为 5.65*10 ² μM。 ¹⁴ 考虑到基于细胞的 EGFR 抑制测定的 IC50（半最大抑制浓度）为 0.42 μM，20 μg 厄洛替尼导致眼内浓度比基于细胞的 EGFR 抑制测定中的 IC50 高 1000 倍。 ²⁵ 然而，轴向伸长率的衰减幅度明显低于mTORC1抑制剂（NLIAE + 20μg依维莫司vs.NLIAE：-0.14±0.01mm，NLIAE + 20μg厄洛替尼vs.厄洛替尼-0.09±0.02mm）。造成这种差异的原因之一可能是厄洛替尼（393.4 g/mol）的分子量低于依维莫司（958.2 g/mol），这可能导致厄洛替尼从玻璃体中更快地清除。 ¹⁵ 其他机制也可能促成了 mTORC1 的激活。据报道，局部炎症与近视有关。 ^{26 27} 在经历形式剥夺诱发的近视的豚鼠眼睛中，视网膜 TNFα 和 IL-17 相关的促炎通路被激活。 ²⁶ 在用抗炎剂双醋瑞因治疗经历形式剥夺的仓鼠眼睛后，视网膜促炎细胞因子水平和轴向伸长率降低。 ²⁸ 相反，将促炎细胞因子（如 TNF-α 和 IL-6）施用于仓鼠的结膜囊，可在 21 天后诱导 -2.1D 至 -3.1D 的近视。 ²² 在我们的研究中，我们还发现，在豚鼠的视网膜脉络膜组织中，NLIAE治疗后NF-κB的表达显着上调。促炎细胞因子可以通过抑制结节性硬化症复合物来激活 mTORC1。 ¹¹ 由于其免疫抑制作用，依维莫司可抑制视网膜和脉络膜中的局部促炎细胞因子。这种作用可能有助于依维莫司对轴向伸长率的抑制作用。 ²⁹  

The downstream mechanism of mTORC1 activation in axial elongation involved upregulation of scleral HIF-1α. In a previous study, myopization-associated hypoxia promoted myofibroblast transdifferentiation.²¹ A cell-based assay demonstrated that hypoxia downregulated collagen-1 expression in human scleral fibroblasts, suggesting that scleral hypoxia may reduce scleral collagen synthesis, thereby promoting axial elongation.²¹ Furthermore, the anti-hypoxia drugs salidroside and formononetin ameliorated form deprivation-induced axial elongation in mice, and scleral HIF-1α knock-down led to hyperopia in mice.^21,30 The choroid supplies nutrients and oxygen to the scleral stroma. The axial elongation–associated thinning of the choroid may lead to an insufficient oxygen supply to the sclera. In agreement with previous studies, we found that scleral HIF-1α expression was upregulated during NLIAE. Furthermore, we found that the upregulated scleral HIF-1α expression could be suppressed by intravitreal mTORC1 inhibition. These results suggests that the downstream mechanism of mTORC1 activation in axial elongation may involve sclera hypoxia. When mTORC1 agonists were administered in combination with NLIAE, guinea pigs showed enhanced axial elongation, choroidal thinning, and peripapillary choroidal atrophy compared to those seen with NLIAE alone. 
轴向伸长中 mTORC1 激活的下游机制涉及巩膜 HIF-1α 的上调。在之前的一项研究中，近视相关的缺氧促进了肌成纤维细胞的转分化。 ²¹ 一项基于细胞的检测表明，缺氧下调了人巩膜成纤维细胞中胶原蛋白-1的表达，表明巩膜缺氧可能会减少巩膜胶原蛋白的合成，从而促进轴向伸长。 ²¹ 此外，抗缺氧药物水景苷和毛松素改善了小鼠形式剥夺诱导的轴向伸长，巩膜 HIF-1α 敲低导致小鼠远视。 ^{21 30} 脉络膜为巩膜基质提供营养和氧气。脉络膜的轴向伸长相关变薄可能导致巩膜供氧不足。与之前的研究一致，我们发现巩膜 HIF-1α 表达在 NLIAE 期间上调。此外，我们发现上调的巩膜 HIF-1α 表达可以通过玻璃体内 mTORC1 抑制来抑制。这些结果表明，轴向伸长中mTORC1激活的下游机制可能涉及巩膜缺氧。当 mTORC1 激动剂与 NLIAE 联合使用时，与单独使用 NLIAE 的患者相比，豚鼠表现出增强的轴向伸长、脉络膜变薄和状周围脉络膜萎缩。

The limitations of our study should be considered. First, although mTORC1 signaling was associated with axial elongation in our experiments, the downstream pathway of mTORC1 activation in RPE cells has remained unclear. Considering that the complex regulatory network underlying myopia development involves the retina, choroid, and sclera, further studies are needed to explore the details of the downstream mechanism of mTORC1. Second, we did not assess either refractive error or corneal curvature in the current study. Although enhanced axial elongation is the most important risk factor for myopia-related complications, such measurements need to be included in future studies to comprehensively evaluate the effects of mTORC1 inhibition and overactivation on NLIAE.³¹ Third, we used −10D lenses for exploring the effect of long-term mTORC1 overactivation on axial elongation. However, axial elongation during three months of NLIAE would likely compensate for the imposed −10D defocus, thereby potentially confounding the interpretation of our results. For future studies, one may consider using defocusing lenses with incremental refractive power. 
应考虑我们研究的局限性。首先，尽管在我们的实验中，mTORC1信号传导与轴向伸长有关，但RPE细胞中mTORC1激活的下游途径仍不清楚。考虑到近视发展背后的复杂调控网络涉及视网膜、脉络膜和巩膜，需要进一步研究来探索 mTORC1 下游机制的细节。其次，在目前的研究中，我们没有评估屈光不正或角膜曲率。尽管增强的轴向伸长是近视相关并发症的最重要危险因素，但此类测量需要纳入未来的研究中，以全面评估 mTORC1 抑制和过度激活对 NLIAE 的影响。 ³¹ 第三，我们使用−10D透镜来探索长期mTORC1过度激活对轴向伸长率的影响。然而，在NLIAE的三个月内，轴向伸长可能会补偿施加的-10D散焦，从而可能混淆我们结果的解释。对于未来的研究，可以考虑使用具有增量屈光力的散焦透镜。

In conclusion, NLIAE was associated with activation of mTORC1. Suppression of mTORC1 activation by intravitreal injection of the mTORC1 inhibitor, everolimus, was dose-dependently associated with a reduction in axial elongation and choroidal thinning during NLIAE. In contrast, compared to NLIAE alone, intravitreal injection of the mTORC1 activator MHY1485 was associated with greater axial elongation, advanced choroidal thinning, and parapapillary choroidal atrophy, compared to NLIAE alone. The findings suggest that mTORC1 may play a role in axial elongation, and that its blockade by everolimus could be helpful in reducing axial elongation and slowing myopia progression. 
总之，NLIAE与mTORC1的激活有关。玻璃体内注射 mTORC1 抑制剂依维莫司抑制 mTORC1 活化与 NLIAE 期间轴向伸长率和脉络膜变薄的减少呈剂量依赖性相关。相比之下，与单独使用 NLIAE 相比，玻璃体内注射 mTORC1 激活剂 MHY1485 与单独的 NLIAE 相比，与更大的轴向伸长率、晚期脉络膜变薄和旁脉络膜萎缩相关。研究结果表明，mTORC1可能在轴向伸长中发挥作用，依维莫司阻断其可能有助于减少轴向伸长和减缓近视进展。

Supported by the National Natural Science Foundation of China (82220108017, 82141128), the Capital Health Research and Development of Special (2020-1-2052) and the Science & Technology Project of Beijing Municipal Science & Technology Commission (Z201100005520045, Z181100001818003). 
由国家自然科学基金（82220108017,82141128），首都健康研究与发展专项（2020-1-2052）和北京市科委科学技术计划（Z201100005520045，Z181100001818003）资助。

Disclosure: R. Zhang, None; L. Dong, None; H. Wu, None; X. Shi, None; W. Zhou, None; H. Li, None; Y. Li, None; C. Yu, None; Y. Li, None; Y. Nie, None; L. Shao, None; C. Zhang, None; Y. Liu, None; J.B. Jonas, EP 3 271 392 (P), JP 2021-119187 (P), US 2021 0340237 A1 (P); W. Wei, None; Q. Yang, None 
披露：R. Zhang，无;L. Dong，无;H. Wu，无;X. Shi，无;W. 周，无;H. Li，无;Y. Li，无;C. Yu，无;Y. Li，无;Y. Nie，无;L. Shao，无;C. Zhang，无;Y. Liu，无;J.B. Jonas， EP 3 271 392 （P）， JP 2021-119187 （P）， US 2021 0340237 A1 （P）;W. Wei，无;Q. 杨，无