Characterization and validation of a chronic retinal neovascularization rabbit model by evaluating the efficacy of anti-angiogenic and anti-inflammatory drugs

INTRODUCTION

Animals with no ocular anomalies, as examined and confirmed by slit-lamp biomicroscopy and indirect ophthalmoscopy, were enrolled onto the study. Ocular discharge and hyperemia was observed after IVT administration of DL-AAA, anti-VEGF and TAA on dosing day but resolved in most animals by the next day,confirming that they were related to ⅠVT injection procedure.Occasional recurrences of ocular discharge were observed in a subset of animals, along with, more rarely, instances of ocular swelling. These symptoms of ocular irritation were likely due to underlying ocular inflammation. Post 12wk of DL-AAA administration, all the animals were presented with moderate to severe retinal/choroidal inflammation and were accompanied by faint to intense vitreous flare and sparse to numerous cells in the vitreous humor (VH). These findings likely reflect infiltration of inflammatory cells into the VH subsequent to persistent posterior inflammation and retinal hemorrhage. A subset of eyes also exhibited posterior lens opacities, likely due to cells from the vitreous space precipitating onto the posterior lens capsule. Sluggish pupillary response was noted in a subset of eyes, was likely also a result of persistent ocular inflammation.Eyes treated with 1 µg/eye of bevacizumab, ranibizumab,aflibercept and TAA had mild inflammation accompanied by very mild vitreous flare and low number of inflammatory cells into the VH, however, controls eyes treated with BSS had moderate to severe retinal/choroidal inflammation with intense vitreous flare and presence of significant number of cells in the VH, suggesting suppression of inflammation and vascular leakage post bevacizumab, ranibizumab, aflibercept and TAA administration. Ocular anomalies were significantly low by day 30 in the eyes treated with bevacizumab, ranibizumab, and aflibercept. However, TAA treated eyes had lowest level on day 7.

Endeavors in ocular medicine has encouraged the ocular research community to not only focus on the development of novel anti-VEGF agents, but to also develop novel sustained drug delivery systems that increase the duration of action for currently available therapies. This is applicable to IVT administration as well as other routes like topical,trans-conjunctival, trans-scleral or suprachoroidal. This approach helps to reduce the frequency, and consequently the risks related to multiple IVT injections and post injection complications like endophthalmitis. The bigger obstacle however to the discovery and development of improved therapies for retinal vascular diseases is the lack of animal models with larger eyes that can mimic the chronic phenotype of human ocular vascular diseases.

Currently available animal models that can be used to test the duration of action of newly developed therapies have some limitations such as a short efficacy window or retinal ocular vascular pathologies which heal rapidly over time. It has been shown that post laser treatment in the laser induced choroidal neovascularization (CNV) model, that VEGF levels reach a peak on day 5 but decline quickly thereafter causing the CNV lesion to completely heal by day 14. The VEGF induced retinopathy model has retinal vascular leakage which peaks on day 3 but returns to baseline levels on day 7 post VEGF IVT injection, resulting in the requirement for repeat IVT injections of active VEGF which in turn may cause post IVT injection related complications. In the current study,we have developed and characterized a DL-2-aminoadipic acid(DL-AAA) (retinal glial cell toxin)induced retinopathy model in pigmented Dutch Belted rabbits and evaluated the duration of action of currently available anti-VEGF and antiinflammatory drugs over a period of 2mo. The DL-AAA rabbits were tested for sustained vascular leakage over 36mo with fluorescein angiography (FA). Additionally, a novel FA grading system was developed to enable accurate and consistent comparison between the drug’s efficacy and it’s duration of action. In summary, we demonstrated that the DLAAA rabbit model represents the most ideal chronic model of retinal vascular leakage that can be utilized to test and develop novel therapies for ocular vascular diseases over an extended period of time.

MATERIALS AND METHODS

All animal experiments adhered to the Association for Research in Vision and Ophthalmology(ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The project was supervised by the Institutional Animal Care and Use Committee (IACUC) at Absorption Systems, a Pharmaron company, San Diego (CA, USA).

关于公司，郑斌董事长又一次强调“精益求精”四个字，恰如精密达一直专注于印后。智能化是公司发展的方向，郑斌董事长对此也有自己的理解：于精密达而言，智能化是一个“在路上”的过程，企业要一直进步和创新。精密达成立的24年里，正是在不断调整中顺应趋势，经历必然的发展过程。印刷设备要实现智能化，前提是机械化和自动化。如今，机械化已经实现，自动化正在实现，具有选择性能的智能化正在努力实现，而在智能化的基础上还要再实现“互联网+”，远程控制等。

Thirty-eight naïve (19 male and 19 female) Dutch Belted () rabbits, approximately 1.5 to 2.5 kg, were purchased from Western Oregon Rabbit Company(OR, USA). Animals were acclimated for 10d before the enrollment on the study. All animals were maintained with a room temperature between 18℃ and 26℃, a relative humidity between 30% and 70%, and a 12-hour light-dark cycle inhouse under pathogen-free conditions.

Statistical analysis was performed by using the GraphPad Prism 5.0 (GraphPad Software, Inc.,San Diego, CA, USA). Percent of FA scores was compared and plotted among treatments groups. Data was presented as mean±standard error of the mean (SEM).

Animals were anesthetized with an intramuscular injection of ketamine hydrochloride (20 mg/kg) and xylazine (5 mg/kg) prior to surgical procedures. Prior to test/control articles IVT administration, pupils were dilated with topical application of one drop each of 10% phenylephrine and 1% tropicamide on each cornea. A 5% Betadine solution was used to clean the eye and surrounding area. Betadine was applied for 5min,after which the eye was rinsed with balanced salt solution(BSS). After the area was surgically prepared, one to two drops of topical 0.5% proparacaine hydrochloride anesthetic were applied to the animal’s eyes. After surgical procedure,atipamezole hydrochloride (1 mg/kg via intramuscular injection) was used as a reversal agent and after full anesthesia recovery animal received one injection of buprenorphine(0.02 mg/kg via subcutaneous injection). Allimaging procedures were performed without anesthesia.

桩身完整性是检验基础桩是否满足承载要求的重要指标。通常采用低应变反射法进行测试，这种测试方法的依据是：在激振锤敲击基础桩桩顶时，其质点的震动会产生应力波，在应力波从桩顶传至桩底并反弹回时，桩身的阻抗变化会对应力波形成反射，使得反射信号传感器接收到的信号在进行计算机分析时，造成曲线的波形、相位、振幅等因素的改变。工作人员会根据这些变化，分析出桩内是否存在缩径、扩径、离析、夹泥等情况，进而了解基础桩的桩长、质量是否达标，并采取必要的措施进行改进。

Once stable retinal leakage (12wk post DL-AAA induction) was established in rabbits, following the IVT injection procedure described in the previous section,the right eye was injected with either 40 µL of bevacizumab(solution 25 mg/mL, Genentech), 100 µL of ranibizumab(10 mg/mL, Genentech ), 25 µL of aflibercept (solution 40 mg/mL, Regeneron) at a dose of 1 mg/eye or triamcinolone(TAA; 40 mg/mL, Bristol-Meyers Squibb) at a dose of 2 mg/eye (Table 1). The contralateral eye of each animal received equal volume of BSS. All drugs were delivered into the mid vitreous cavity.

Prior to placement on study,each animal underwent clinical ophthalmic examinations (slitlamp biomicroscopy and indirect ophthalmoscopy) and ocular findings were scored according to a modified McDonald-Shadduck Scoring System. The acceptance criteria for placement on study was scores of “0” for all ocular variables.All animals were assigned to one experimental groups based on body weight for DL-AAA induction for retinal leakage.Post 12wk of DL-AAA induction, 20 animals were assigned a numeric rank from 1 to 20 according their FA scores in a decreasing order (animal with highest FA score was assigned rank =1) into four groups (Table 1).

A 31G beveled needle attached to 0.3 mL insulin syringe was inserted (right eye approximately 11 o’clock position and left eye approximately 1 o’clock)3-4 mm away from the limbus into the vitreous body and 80 µL of the 80 mmol/L DL-AAA solution was administered into the mid vitreous. The needle was removed slowly to reduce risk of back flow from the injection track, and the eye was rinsed with BSS. Triple antibiotic ophthalmic ointment was administered to all eyes after dosing.

The 80 mmol/L DL-AAA solution(Sigma-Aldrich Corp.) was freshly prepared based on published procedures. IVT injections were performed in 2 staggers (Stagger 1:10 males and 9 females; stagger 2:9 males and 10 females). In brief, 120 mg of DL-AAA was dissolved in 1 mL 1 mol/L hydrochloric acid (HCl) to create a stock solution. The components were gently swirled for 5min until a clear solution formed. For IVT administration, DL-AAA stock solution was diluted by adding 4 mL of 0.9% sterile normal saline solution, followed by sufficient 1 mol/L sodium hydroxide (NaOH) to adjust the pH of the solution to 7.4.Sufficient volume of saline was then added to bring the DL‐AAA concentration to 80 mmol/L, the pH was re-tested, and a minimal volume of NaOH (4-5 µL) was added to bring the pH back to 7.4. The solution was then sterile filtered through a disposable 0.2 µm syringe filter to remove any potential particulates. DL-AAA solution was administrated within 15min of formulation preparation. Solutions were kept at room temperature until injection.

FA was performed using Heidelberg Retina Angiograph (HRA) device from Heidelberg Engineering (Heidelberg, Germany), the Spectralis ophthalmic imaging system on both eyes of all animals at baseline (prior to DL-AAA administration) and on weeks 2, 4, 6, 8, and 12 post DL-AAA administration. Additional FA was performed on days 7, 30, and 60 post IVT administration of bevacizumab,ranibizumab, aflibercept and TAA. Ⅰn brief, sodium fluorescein was injected intravenously (IV) and FA time-course images were captured on both eyes between 30s to at least 10min post fluorescein injection. Laser intensity was kept at constant(55%) to avoid any overexposure of FA images. FA images were captured in three areas for retinal vascular leakage: optic nerve head, nasal optic streak, and temporal optic streak. In addition to leakage, any other associated pathology that was secondary to the retinal leakage (such as hemorrhage and retinal detachment) was also imaged. A novel grid based FA grading system was developed based on the published FA scoring system on monkey and rabbits and used for retinal vascular leakage grading.

Infrared (IR) and color fundus imaging were performed at baseline and 2wk post DL-AAA administration.Spectral-domain optical coherence tomography (SD-OCT)imaging was performed using HRA-OCT device from Heidelberg Engineering (Heidelberg, Germany), the Spectralis ophthalmic imaging system on both eyes of all animals at baseline (prior to DL-AAA administration) and on weeks 2, 4, 6,8, and 12 post DL-AAA administration. In brief, OCT sessions were taken on superior/inferior/nasal/temporal and center retinal at 55° field of view using the high-resolution mode(signal quality ≥24 dB) with scan speed of 40 000 A‐scans per second. The image scalingandwere 1.10 μm per pixel and 3.87 μm per pixel, respectively. The optimal focus depth was approximately 3 diopters. Axial resolution was 7 μm optical and 3.5 μm digital. SD‐OCT data were exported as 8‐bit grayscale image.

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参照《中国2型糖尿病防治指南》中提出的周围神经病变诊断标准进行诊断，对比有无周围神经病变对象的心电图检查自主神经功能指标。同时将以上指标，与正中神经、尺神经运动神经传导速度（MCV）、感觉神经传导速度(SCV）进行相关性分析。

RESULTS

Angiogenesis is a complex physiological process,important for both vascular development and pathological condition like neovascularization. Dysregulated cascade of angiogenesis in the eye causes pathologies in retinal/choroidal vascular bed that may lead to partial or complete vision loss, such as retinopathy of prematurity(ROP), diabetic retinopathy (DR), neovascular age-related macular degeneration (nAMD), neovascular glaucoma, and corneal neovascularization. Ocular vascular pathological conditions are characterized by increased vascular permeability and growth of new vessels which may bleed or leak exudates and caused retinal edema followed by fibrous scar that destroys the photoreceptor cells in the retina. Human and animals studied revealed that vascular endothelial growth factor(VEGF) is a key pathogenic factor for aforementioned ocular vascular diseases, therefore, intravitreal (IVT) administration of anti-VEGF therapies have become the most common treatment modality for many retinal diseases. In past two decades, anti-VEGF therapies have had tremendous success and revolutionized the treatment for ocular vascular diseases.Nonetheless, current anti-VEGF (bevacizumab, ranibizumab and aflibercept) therapies have several limitations within clinical use, namely 1) short duration effect, 2) repeated IVT injections, 3) post IVT injections complications like endophthalmitis (a sight-threatening infection), subconjunctival hemorrhage, raised intraocular pressure and rhegmatogenous retinal detachmentIn addition, repeat IVT treatment posed a significant burden to patients and the healthcare system; for example overall annual cost of AMD care in the United States was estimated at $575 million in 2004 prior to the widespread use of anti-VEGF IVT injections and is projected to increase to$845 million over the next 15y.

A novel FA grading system was developed for grading of DL-AAA rabbit vascular leakage.FA images were exported as .jpeg files. FA images comprised of central, nasal and temporal retinal vasculature were aligned and 4 grids were placed on temporal (R1, R2, R3, and R4)and nasal (R5, R6, R7, and R8) retinal vasculature areas. At each time point vascular leakage was graded using following criteria, Grade 0: Major vessels straight some tortuosity of smaller vessels, no vessel dilation; Grade 1: Increased tortuosity of major vessels and/or some vessel dilation; Grade 2: Leakage between major vessels, significant vessel dilation;Grade 3: Leakage between major and minor vessels, minor vessels still visible; Grade 4: Leakage between major and minor vessels, minor vessels poorly/not visible (Figure 1). A total score consisting of the sum of all individual region scores was calculated for each eye at each time point.

Maximum retinal vascular leakage was observed at 2wk after DL-AAA injection. In stagger 1 animals, a reduction in mean FA scores was observed in week 4; mean FA scores then increased again slightly, reaching a level that was also reduced from maximum values in week 2, but still showed robust leakage. This level of leakage then persisted as evidenced by stable mean FA scores in weeks 8 and 12. Stagger 2 animals were not assessedFA at weeks 4 and 6, but showed comparable FA mean scores in weeks 8 and 12,, week 8 mean FA values that were reduced from the high values in week 2, but still reflective of robust leakage, and that remained stable at this level in week 12 (Figure 2B, 2C). A cohort of DL-AAA animals (=10, received either 1 or 2 DL-AAA injections) was screened with FA at 36mo post DL-AAA induction. All the animals presented with same level of retinal vascular leakage suggesting that DL-AAA rabbit model consistently has stable vascular leakage up to 36mo (Figure 2E).

FA scores reflected substantial vascular leakage in all the eyes of 38 animals after DL-AAA injection. The retinal vascular leakage observed in week 2 was accompanied by retinal hemorrhage and the growth of retinal neovessels (RNV). Neovessels continued to extend in weeks 4 and 6 in all eyes assessed at these time points. By week 8, retinal neovessels in all eyes remained tortuous and dilated and continued to extend. Vessels were still tortuous and dilated in week 12; the robust vascular leakage observed at this time point was now restricted to the tips of the telangiectatic neovessels (Figure 2A, red arrows).Temporal regions [regions 5-8 in right eyes (OD), regions 1-4 in left eyes (OS)] were more severely affected, while nasal regions (regions 1-4 OD, regions 5-8 OS) were affected less(Figure 2A). Notably, the injection site was located temporally(11 o’clock position OD, 1 o’clock position OS) in all eyes.Interestingly, in an additional cohort of rabbit (=10), we demonstrated that homogeneous (both temporal and nasal retinal vessels) retinal leakage can be achieved by delivering DL-AAAtwo 40 µL of 80 mmol/L DL-AAA injections per eye, 15min apart, one in each superior or inferior hemisphere(temporal at 11 or 8 o’clock position and nasal at 1 or 4 o’clock position; Figure 2D).

1.2.2 CKK-8法检测SiHa细胞增殖 将稳定表达miR-145SiHa细胞接种至96孔板（1.0×105个/孔），培养0、12、24、36、48、60、72 h后，加入CKK-8试剂，按照说明书操作，酶标仪检测450 nm下各孔细胞光密度（optic density，OD），绘制细胞生长曲线。

IR and color fundus imaging showed that all animals had tortuous vessels, retinal hemorrhage (Figure 3B, yellow star) and growth of new epiretinal vessels towards retinal periphery and area centralis within 2wk post DL-AAA IVT injection (Figure 3A, green arrows). Baseline before DL-AAA dose, OCT scans confirmed normal retina in all study animals.Week 2, OCT scans showed moderate to severe intraretinal separation with retinal degeneration (Figure 3B, red arrow)evidenced in the detached areas. Progressive degeneration of the retinal ganglion cell layer was observed in weeks 4 and 6 on OCT scans. By week 8, retinal degeneration and thinning in the detachment areas had progressed further, and by week 12, the retinal neuronal layer in these areas had completely disappeared (Figure 3B, green arrows).

幻想是人类的天性，在史前的蒙昧时期神话传说已经诞生，思维的最初形式就是将认知修辞化，这也正是儿童文学最本源的能量。然而儿童文学始于18世纪，显示出一个漫长的历史缺位，原因在于文学话语主体的缺位。当神话探索逐渐转向事件记载，再到故事的虚构，成人因素成为文学发展的主宰，儿童的声音淹没在话语时空中，儿童接受层面的经验和需求不被纳入文学话语的建构过程。在这个历史空档中，儿童的阅读接受是成人话语的附庸，或是被说教准则束缚的被动接受。这种表达的缺陷导致了话语信息传输的断层，除了教条式的话语复制外，难以得到应有的行为回报。当人们能够主动全面地考察文学活动的接受主体时，儿童文学才真正产生了能量。

On day 7, mean FA score showed that 1 µg/eye of bevacizumab,ranibizumab, aflibercept suppress 65%, 90%, and 100%retinal vascular leakage, respectively, however, TAA 2 µg/eye showed only 50% suppression of vascular leakage.Day 30, bevacizumab and TAA continued to show 66% and 44% suppression of vascular leakage while ranibizumab effect was becoming less effective (68%) and reached a similar level as bevacizumab. Ⅰn contrast, 1 µg/eye aflibercept was still able to fully (100%) suppress vascular leakage on Day 30. On day 60, bevacizumab, ranibizumab and TAA showed suppression of 7%, 12%, and 9% retinal vascular leakage, respectively,however, aflibercept continued to be more effective showing 50% suppression of vascular leakage on day 60. The day 60 angiogram showed that vascular leakage levels returned close to the baseline for bevacizumab, ranibizumab and TAA animals which is consistent with drug elimination, while also indicating the reversible nature of vascular leakage of the DL-AAA model. Control (OS) eyes treated with BSS in all groups didn’t show any change in vascular leakage though out the study, suggesting that there was no crossover effect with either treatment (Figures 4 and 5).

DISCUSSION

We have established and validated a rabbit model with stable and chronic retinal vascular leakage, inducedIVT administration of DL-AAA, a glutamine synthetase inhibitor and retinal glial (Müller) cell toxin. Retinal Müller cells play an essential role in regulating neuronal activity and maintaining the integrity of the blood-retinal barrier. The mechanism by which DL-AAA causes retinal disruption and neovascularization development are not well understood.After IVT delivery, DL-AAA mainly target and damage the connecting bridges between retinal nerves cells and blood vessels (blood-retinal barrier), which causes metabolic dysfunction of retina, ischemia and retinal inflammationand resulted an increase in VEGF levels in the retina.Higher levels of VEGF in the retina increase retinal vascular permeability and induced chronic condition of RNV. IVT delivery of DL-AAA in primates, rodents and rabbits has been shown to induce RNV. In a recent report, pigmented rabbit are shown to develop persistent and measurable RNV at a greater success rate, compared to non-pigmented rabbit.In this study, IVT injection of 1.03 mg/eye DL-AAA in pigmented Dutch Belted rabbits was associated with substantial retinal vascular leakage, moderate to severe intraretinal separation with retinal degeneration in the areas of detachment,moderate to severe retinal/choroidal inflammation, retinal hemorrhage, tortuous and dilated retinal vessels, and RNV with epiretinal vessels growing towards the retinal periphery and area centralis. RNV was progressive, with neovessels continuing to extend for at least 8wk after DL-AAA injection.Retinal degeneration in the areas of detachment was likewise progressive, with continually more severe retinal thinning and degeneration of the retinal ganglion cell layer, culminating in degeneration of the optic streak and a complete disappearance of the retinal neuronal layer in the detachment areas by 12wk post-dose. However, these features do not exactly mimic all the pathological and genetic features of ocular vascular diseases, for example, DL-AAA rabbits are not presented with CNV like AMD or retinal edema like diabetic macular edema (DME). Nonetheless, the key pathognomonic feature of DL-AAA rabbit is RNV and retinal vessels leakage which is the most striking feature of most ocular vascular diseases.DL-AAA model had vascular leakage at maximal 2wk after DL-AAA injection, decreased to lower but still robust levels by 4wk post-dose, and then increased again slightly to a level that was below the initial maximum, but still substantial by 6wk post-dose and finally becomes stable at 12wk post DL-AAA administration. Liand Caoobserved vascular leakage up to 18 and 12mo, respectively, in a DL-AAA rabbit model. In this study, we observed levels of vascular leakage remained stable and restricted to the tips of the telangiectatic neovessels up to 36mo. Furthermore, consistency in developed retinal phenotypes and stable FA scores up to 36mo among all the animals, in two different staggers, showed that DL-AAA rabbit is an excellent preclinical model to test the long term drug efficacy for vascular inflammation and leakage.

Presence of vitreous flare, inflammatory cells in the VH, retinal inflammation including retinal hemorrhage and posterior lens capsule opacities did not interfere withimaging, and show thatimaging in combination with clinical exams can provide long term efficacy evaluation of drugs that are designed to target vascular inflammation and leakage. No effect of the test article on body weights was observed during the study suggesting no systemic burden or toxicity in this experimental model.

Despite the fact that DL-AAA model does not mimic all the features of existing human retinal diseases, but, it still presents features like stable and persistent chronic retinal neovascular pathology and leakage similar to human vascular disease like nAMD, DME, and retinal vein occlusion. Furthermore,higher vitreous VEGF levels in DL-AAA eyes compared to naïve animalsand longer duration of stable vascular leakage compared to other available animals models like laser induced CNV rodent model, are valuable features of DL-AAA rabbit model. The rabbit’s large eye size with similar anatomical and physiological characteristics to human, is also well suited to test the efficacies of novel formulations and delivery of ocular devices. Therefore, collectively the described features of this experimental DL-AAA rabbit model, provide wide applicability making this model a valuable tool for the investigation of efficacy and duration of novel treatment strategies for retinal angiogenic diseases.

选取2015年2月～2017年11月本院接诊的长期住院老年精神分裂症患者20例作为研究对象，其中，男9例、女11例，年龄61～70岁，平均年龄（65.3±0.2）岁，病程1～8年，平均病程（5.0±0.3）年，均满足《中国精神疾病分类方案与诊断标准》[1] 里精神分裂症诊断标准。

We thank Avani Mody, Matthew Lyulkin and Manindra Singh for comments on the manuscript.

Kumar S designed and performed experiments, analyzed data, supervised the study, and wrote the manuscript; Quach J, Cook N performed fluorescein angiography; Gum G helped with the clinical ophthalmic examinations; Naageshwaran V, Gum G supported the overall operations of the project, review and comments on the manuscript.

None;None;None;None;None.