鼠青光眼模型的眼压测量方法

青光眼是主要的致盲眼病之一,对于该病病因、病理、预防、诊断和治疗的研究都需要青光眼动物模型。近年青光眼鼠模型应用报道越来越多。但由于鼠眼较小,对其眼压测量有一定特殊性,我们对鼠青光眼模型的眼压测量方法,尤其是各种方法的原理、特点进行综述。     

Characterization of intraocular pressure pattern and changes of retinal ganglion cells in DBA2J glaucoma mice

AIM: To characterize the pattern of intraocular pressure (IOP) change and the deficit of retinal ganglion cells (RGCs) in DBA2J, which is most wellcharacterized chronic glaucoma mouse model and wild type (WT) C57bl/6 mice, and to study the relationship between IOP change and RGCs deficit. METHODS: IOP was monitored with a rebound tonometer in WT C57bl/6 and DBA2J mice from 3 to 15-monthold. Retinal function was evaluated by dark-adapted electroretinogram (ERG) in DBA2J and WT mice of 15monthold. A dye (Neurobiotin) was applied to optic nerve stump to retrograde label RGCs. TO-PRO-3 visualized all nuclei of cells in the RGC layer. RESULTS: The IOP in WT mice was 9.03±0.6 mm Hg on average and did not increase significantly as aging. The IOP in DBA2J mice, arranging from 7.2 to 28 mm Hg, was increasing significantly as aging, and it was normal at 3monthold compared with WT mice, slightly increased from 7-monthold and increased in 50% animals at 11monthold and in 38% animals at 15-monthold. The RGCs density in DBA2J mice started reducing by 7month-old, continuously decreased until reached about 20% of RGC in WT retina by 15monthold. RGC density was not linearly correlated with IOP in 15-monthold DBA2J mice. The amplitude of positive scotopic threshold response, and negative scotopic threshold response of ERG were significantly reduced in DBA2J mice of 15-monthold than that in agepaired WT mice. CONCLUSION: The present study found that DBA2J mice display pathological and functional deficits of the retina that was not linearly correlated with IOP.     

补精益视片对大鼠慢性高眼压模型视网膜损害的影响

目的　观察补精益视片对大鼠慢性高眼压（ｅｌｅｖａｔｅｄｉｎｔｒａｏｃｕｌａｒｐｒｅｓｓｕｒｅ,ＥＩＯＰ）模型视网膜损害的干预作用,探讨其作用机理。方法　将３０只ＳＤ大鼠随机分为３组:对照组、给药组、模型组,每组１０只。采用烙闭上巩膜静脉法对给药组、模型组ＳＤ大鼠建立慢性ＥＩＯＰ模型,观察补精益视片对慢性ＥＩＯＰ大鼠眼压和视网膜病理形态学变化的影响。结果　给药组、模型组大鼠在造模后即刻直至造模后８周与造模前相比眼压均升高,差异均有统计学意义（均为Ｐ＜０．０１）,说明ＥＩＯＰ模型造模成功。造模后８周眼压与造模后即刻相比,给药组有降低趋势,差异有统计学意义（Ｐ＜０．０１）,模型组差异无统计学意义（Ｐ＞０．０５）。造模后８周,视网膜神经节细胞（ｒｅｔｉｎａｌｇａｎｇｌｉｏｎｃｅｌｌｓ,ＲＧＣｓ）数量给药组（１４．５７±１．９７）与模型组（１０．７６±２．１９）均明显低于对照组（１７．４７±１．９７）,差异均有统计学意义（Ｐ＜０．０５,Ｐ＜０．０１）;视网膜厚度模型组为（１５０．８３±１７．９１）μｍ低于对照组的（２１９．７２±３２．２４）μｍ,差异有统计学意义（Ｐ＜０．０１）,给药组为（２１５．５１±５１．２３）μｍ,与对照组相比差异无统计学意义（Ｐ＞０．０５）;给药组ＲＧＣｓ数量及视网膜厚度均优于模型组,差异均有统计学意义（均为Ｐ＜０．０１）。ＲＧＣｓ超微结构显示给药组较模型组明显改善。结论　补精益视片能保护ＳＤ大鼠慢性ＥＩＯＰ模型视功能,表现为降低眼压、提高ＲＧＣｓ数量、增加视网膜厚度、改善ＲＧＣｓ超微结构。     

补精益视片对大鼠慢性高眼压模型视网膜p-Akt表达的影响

目的　观察补精益视片对大鼠慢性高眼压（ｅｌｅｖａｔｅｄｉｎｔｒａｏｃｕｌａｒｐｒｅｓｓｕｒｅ,ＥＩＯＰ）模型ｐ-Ａｋｔ表达的干预作用,探讨其作用机理。方法　将３０只ＳＤ大鼠随机分为３组:对照组、模型组、给药组,模型组和给药组采用烙闭上巩膜静脉法建立ＳＤ大鼠慢性ＥＩＯＰ模型,给药组每天予１．８ｇ·ｋｇ－１体质量补精益视片混悬液灌胃,对照组、模型组每天予３ｍＬ生理盐水灌胃,连续给药８周,并于８周末处死大鼠,观察各组大鼠眼压、视网膜ｐ-Ａｋｔ表达的情况。结果　模型组、给药组大鼠造模后即刻直至造模后８周与造模前眼压相比均升高,差异均有统计学意义（均为Ｐ＜０．０１）;造模后８周眼压与造模后即刻相比,给药组有降低趋势,差异有统计学意义（Ｐ＜０．０１）,模型组无降低趋势,差异无统计学意义（Ｐ＞０．０５）。造模后８周视网膜ｐ-Ａｋｔ表达免疫组织化学分析示:给药组总面积、平均光密度和积分光密度分别为（７４６３１．８１±１２８４１．３０）μｍ２,９３９．２６±１７５．１７和３７８１４．９６±５８２２．７１,与模型组的（３７９４７．２６±８０５０．５１）μｍ２、４８１．４５±１６１．０２和１８９０７．６４±４３６７．２９相比,差异均有显著统计学意义（均为Ｐ＜０．０１）;给药组平均黑度为１３８．５５±８．５８,虽高于模型组的１３６．５２±３．８１,但差异无统计学意义（Ｐ＞０．０５）。结论　补精益视片通过降低眼压、上调视网膜ＰＩ３Ｋ／Ａｋｔ信号转导通路中ｐ-Ａｋｔ的表达而保护慢性ＥＩＯＰ模型ＳＤ大鼠的视功能。     

青光眼小鼠基因模型的研究进展

青光眼作为目前全球首位不可逆性致盲眼病,是具有遗传倾向的多因素复杂疾病,病理性眼压升高是其危险因素。关于青光眼的发病机制尚不完全清楚,现有研究多建立在动物模型的基础上,以小鼠为主要研究对象,通过实验诱导手段或转基因操作复建青光眼病理损伤过程,进一步研究相关的发病机制和病理变化。实验诱导构建青光眼小鼠模型技术经过诸多学者的研究,逐渐趋于成熟。而随着分子生物学和遗传学的研究深入,越来越多的研究集中在青光眼的相关疾病基因上,转基因小鼠模型成为近年的热点,与实验操作控制单一因素相比,基因编辑更能模拟出疾病发病的复杂过程。本文主要通过阐述相关青光眼小鼠转基因模型的研究进展,为今后研究中模型的选择提供更完整的方向与策略。     

Endothelin and its potential role in glaucoma

Endothelin is a potent vasoactive peptide occurring in 3 isotypes, ET-1, ET-2, and ET-3. Through its two main receptors, endothelin A and endothelin B, it is responsible for a variety of physiological functions, primarily blood flow control. Recent evidence from both human and experimental optic neuropathies shows involvement of endothelin and upregulation of its receptors (principally endothelin B). Experimental studies have shown that chronic ET-1 administration to the optic nerve immediately behind the globe causes neuronal damage, activation of astrocytes, the major glial cell in the anterior optic nerve, and upregulation of endothelin B receptors. This review outlines the ubiquitous role of endothelin and its potential involvement in glaucoma.     

Pathophysiology of human glaucomatous optic nerve damage: Insights from rodent models of glaucoma

Understanding mechanisms of glaucomatous optic nerve damage is essential for developing effective therapies to augment conventional pressure-lowering treatments. This requires that we understand not only the physical forces in play, but the cellular responses that translate these forces into axonal injury. The former are best understood by using primate models, in which a well-developed lamina cribrosa, peripapillary sclera and blood supply are most like that of the human optic nerve head. However, determining cellular responses to elevated intraocular pressure (IOP) and relating their contribution to axonal injury require cell biology techniques, using animals in numbers sufficient to perform reliable statistical analyses and draw meaningful conclusions. Over the years, models of chronically elevated IOP in laboratory rats and mice have proven increasingly useful for these purposes. While lacking a distinct collagenous lamina cribrosa, the rodent optic nerve head (ONH) possesses a cellular arrangement of astrocytes, or glial lamina, that ultrastructurally closely resembles that of the primate. Using these tools, major insights have been gained into ONH and the retinal cellular responses to elevated IOP that, in time, can be applied to the primate model and, ultimately, human glaucoma.     

Factors associated with topographic changes of the optic nerve head induced by acute intraocular pressure reduction in glaucoma patients

Purpose

To investigate factors associated with changes in optic nerve head (ONH) topography after acute intraocular pressure (IOP) reduction in patients with primary open-angle glaucoma (POAG).

Methods

Untreated POAG patients (IOP >21 mm Hg) were prospectively enrolled. Systemic and ocular information were collected, including central corneal thickness (CCT) and corneal hysteresis (CH). All patients underwent confocal scanning laser ophthalmoscopy and tonometry (Goldmann) before and 1 h after pharmacological IOP reduction. The mean of three measurements was considered for analysis. Changes in each ONH topographic parameter were assessed (one eye was randomly selected), and those that changed significantly were correlated with patient''s systemic and ocular characteristics.

Results

A total of 42 patients were included (mean age, 66.7±11.8 years). After a mean IOP reduction of 47.3±11.9%, significant changes were observed in cup area and volume, and in rim area and volume (P<0.01), but not in mean cup depth (P=0.80). Multiple regression analysis (controlling for baseline IOP and magnitude of IOP reduction) showed that CH (r²=0.17, P<0.01) and diabetes diagnosis (r²⩾0.21, P<0.01) were negatively correlated with the magnitude of changes in ONH parameters, whereas the cup-to-disc ratio was positively correlated (r²=0.30, P<0.01). Age, race, disc area, and CCT were not significant (P⩾0.12). Including all significant factors in a multivariable model, only the presence of diabetes remained significantly associated with all ONH parameters evaluated (P<0.01).

Conclusions

Different systemic and ocular factors, such as diabetes, CH, and the relative size of the cup, seem to be associated with the magnitude of changes in ONH topography after acute IOP reduction in POAG patients. These associations partially explain the ONH changes observed in these patients and suggest that other factors are possibly implicated in an individual susceptibility to IOP.     

大鼠实验性慢性高眼压对视网膜神经节细胞的损伤

目的:制作高眼压大鼠模型,观察高眼压对视神经的损害。方法:成年Wistar雄性大鼠65只,烧灼右眼上方2支和外侧1支巩膜上静脉,建立慢性高眼压模型。左眼作为对照眼。对造模成功的,分别于造模后1d,1,2,3,4,6,8,10wk各摘除6只大鼠双眼。在取出眼球前24h,用Fluoro-gold进行视网膜神经节细胞(retinal ganglion cells,RGC)逆行性染色,做视网膜铺片计数RGC,观察不同时段高眼压对RGC的影响。结果:右眼巩膜上静脉烧灼后各时间点造模眼平均眼压分别为42.2±1.8mmHg,37.9±2.3mmHg,36.1±2.0mmHg,33.6±2.2mmHg,32.2±2.4mmHg,30.1±2.0mmHg,30.5±2.1mmHg和27.6±1.3mmHg。术后各时间点的成模率分别为80.0%,76.9%,74.5%,71.7%,63.8%,56.1%,42.9%,41.4%。成模率和成模眼的眼压随时间延长呈下降趋势。实验组和对照组的RGC密度在早期(巩膜上静脉烧灼术后3wk内)没有显著差别。造模后4wk高眼压组RGC密度明显低于对照组(P<0.05),随着时间的推移差别越来越显著。结论:巩膜上静脉烧灼法能诱导出持续的肯定的大鼠慢性高眼压模型,成模眼的眼压和随时间而下降。高眼压持续的时间越长,RGC的损失越多。     

Mouse models of retinal ganglion cell death and glaucoma

Once considered too difficult to use for glaucoma studies, mice are now becoming a powerful tool in the research of the molecular and pathological events associated with this disease. Often adapting technologies first developed in rats, ganglion cell death in mice can be induced using acute models and chronic models of experimental glaucoma. Similarly, elevated IOP has been reported in transgenic animals carrying defects in targeted genes. Also, one group of mice, from the DBA/2 line of inbred animals, develops a spontaneous optic neuropathy with many features of human glaucoma that is associated with IOP elevation caused by an anterior chamber pigmentary disease. The advent of mice for glaucoma research is already having a significant impact on our understanding of this disease, principally because of the access to genetic manipulation technology and genetics already well established for these animals.     

Pathogenesis of block of rapid orthograde axonal transport by elevated intraocular pressure.

The subject was investigated in 40 rhesus monkeys. The perfusion pressure (PP) in the eye was altered by changing the blood pressure (BP) and/or intraocular pressure (IOP). Rapid orthograde axoplasmic transport was studied by intravitreal injection of 100 μCi of [³H]leucine and light microscopic autoradiography. The study was planned to determine whether the presence and degree of rapid orthograde axoplasmic flow blockage relate more closely to the level of IOP (suggesting a mechanical basis) or of PP (suggesting an ischemic mechanism). It proved technically impossible to maintain IOP and PP at desired levels for the necessary period ($\text{6–7}\text{1}\text{2}\text{hr}$)—this difficulty has never been admitted in the literature. Several biological and technical variables also confounded the interpretation of the data, e.g. non-availability of a reliable technique to ascertain the exact PP in the vessels of the optic nerve head, lack of information on the blood flow and the nutritive capability of a given PP, possible artefacts by the perfusion pump used to alter BP, and marked limitations of routine microscopic autoradiography in quantifying exactly the axoplasmic flow blockage. As a result an intensive and sustained study offers only inconclusive results, and cannot resolve the controversy as to whether the axoplasmic flow blockage is due to ischemia or mechanical compression of the nerve fibers in the optic nerve head. Although no single observation is absolutely decisive one way or the other, the sum total of the various evidence put together is somewhat suggestive that the axonal blockage is probably mediated by ischemic mechanism.     

灯盏细辛对眼压已控制青光眼患者视野的保护作用

-目的:探讨灯盏细辛对眼压已控制青光眼患者视野的保护作用。方法:选择有青光眼视野缺损,眼压控制在18mmHg以内的原发性青光眼患者24例40眼。按随机、双盲法予药物口服,药物分别为灯盏细辛片和安慰剂。患者每日口服3次,每次2片。2mo为一疗程,连续3个疗程,每2mo随访1次。试验结束由药物提供方拆盲并反馈信息。结果:①用药前后各疗程对照组和治疗组收缩压、舒张压、脉搏、眼压、C/D、视力均无显著性统计学差异(P>0.05),且所有患者用药过程中无明显不良反应。②治疗组用药6mo后的平均缺损(MD)、平均敏感度(MS)与用药前的MD、MS相比,差异有显著性意义(P<0.05)。③中晚期治疗组用药2,4,6mo后的MD、MS分别与用药前MD、MS相比差异均有显著性意义(P<0.05)。结论:降低眼压对部分青光眼患者的视功能有保护作用。灯盏细辛对原发性青光眼患者的视功能有一定的保护作用,且应用疗程越长,视野缺损改善越明显;而对于原发性中晚期青光眼患者,灯盏细辛改善视野更显著。灯盏细辛对血压、脉搏、眼压、视力、C/D比均没有影响。     

青光眼昼夜眼压波动

病理性高眼压和较大的昼夜眼压波动是青光眼视神经损害进展的重要危险因素.眼压具有波动性,正常人眼压波动的峰值多出现于凌晨,这种波动与体位、眼灌注压、眼轴等有关.正常眼压性青光眼患者眼压波动是视野进展的重要危险因素,且经24小时眼压监测发现大部分眼压是存在异常的,因此需根据其昼夜眼压曲线明确诊断和针对性治疗;原发性开角型青光眼患者昼夜眼压波动规律与正常人相似,眼压高峰多在夜间,但波动范围可能比正常人大,且双眼的波动呈明显的一致性;激光周边虹膜切开术后的慢性闭角型青光眼患者的昼夜眼压波动较大,其眼压波动与基线眼压和房角粘连程度呈正相关.与激光和药物相比,小梁切除术更有利于控制长期和昼夜的眼压波动.抗青光眼药物中前列腺素类药物是控制昼夜眼压波动效果最好的滴眼剂.     

Effects of chronic elevated intraocular pressure on parameters of optical coherence tomography in rhesus monkeys

AIM: To determine the progression of parameters from optical coherence tomography (OCT) in chronic elevated intraocular pressure (IOP) monkeys. METHODS: A chronic elevated IOP model of rhesus monkeys was induced by laser photocoagulation. Representative OCT parameters, including the average and four-quadrant retinal nerve fiber layer (RNFL) thickness, and parameters from optic nerve head (ONH) analysis were collected before and after laser treatments biweekly for up to 28wk. The performance of each parameter for early progression detection was analyzed. The progressive trends toward elevated IOP were analyzed using a linear mixed-effects model. RESULTS: There were 10 successfully maintained high IOP eyes in 7 monkeys. The follow-up time was 24±5.37wk. With cumulative IOP elevation, the cup area, rim area and C/D area ratio were statistically significantly changed as early as 2wk after elevated IOP induction (P<0.05). The quadrant RNFL thickness changed at 6wk after high IOP induction, and the superior and inferior RNFL thicknesses exhibited more obvious reductions than other quadrants. The average RNFL thickness was the last one to show a significant decrease at 8wk. CONCLUSION: The parameters of ONH are most sensitive to elevated IOP in a primate glaucomatous model. These findings suggest that we should focus on those parameters instead of RNFL thickness in patients with elevated IOP, as they might present with earlier glaucomatous changes.     

改良小梁切除术治疗青光眼高眼压持续状态的临床分析

目的:回顾性分析改良小梁切除术治疗青光眼持续高眼压状态的临床资料,评价改良小梁切除术的效果。方法:青光眼持续高眼压状态患者100例100眼,分为治疗组55例55眼和观察组45例45眼。治疗组采用改良小梁切除术,观察组采用常规小梁切除术。改良措施主要有:术前星状神经节阻滞降眼压,采用表面麻醉和20g/L利多卡因棉片巩膜面浸润麻醉;用隧道刀做板层巩膜瓣;在小梁切除部位做前房穿刺,慢放房水;术中置巩膜瓣调整缝线;术中散瞳,术毕睫状肌麻痹剂应用。结果:治疗组手术并发症发生率低于观察组,两组比较差异有统计学意义(P<0.05)。随访3mo,治疗组眼压低于观察组,两组比较差异有统计学意义(t=9.1535,P<0.05)。治疗组平均住院天数少于观察组(t=39.8010,P<0.01)。治疗组平均住院费用低于观察组(t=11.3219,P<0.01)。结论:改良小梁切除术治疗青光眼持续高眼压状态不仅挽救部分视功能,减少手术严重并发症发生率,术后眼压控制更好,还可以缩短平均住院日,减少医疗费用,从而提高了患者满意度。     

Age and intraocular pressure in murine experimental glaucoma

Age and intraocular pressure (IOP) are the two most important risk factors for the development and progression of open-angle glaucoma. While IOP is commonly considered in models of experimental glaucoma (EG), most studies use juvenile or adult animals and seldom older animals which are representative of the human disease. This paper provides a concise review of how retinal ganglion cell (RGC) loss, the hallmark of glaucoma, can be evaluated in EG with a special emphasis on serial in vivo imaging, a parallel approach used in clinical practice. It appraises the suitability of EG models for the purpose of in vivo imaging and argues for the use of models that provide a sustained elevation of IOP, without compromise of the ocular media. In a study with parallel cohorts of adult (3-month-old, equivalent to 20 human years) and old (2-year-old, equivalent to 70 human years) mice, we compare the effects of elevated IOP on serial ganglion cell complex thickness and individual RGC dendritic morphology changes obtained in vivo. We also evaluate how age modulates the impact of elevated IOP on RGC somal and axonal density in histological analysis as well the density of melanopsin RGCs. We discuss the challenges of using old animals and emphasize the potential of single RGC imaging for understanding the pathobiology of RGC loss and evaluating new therapeutic avenues.     

Experimental detection of retinal ganglion cell damage in vivo

In vivo detection of retinal ganglion cell (RGC) damage should have experimental and clinical relevance. A number of experimental models have been recently described to visualize RGCs in vivo. With retrograde injection of fluorescent tracers into the superior colliculus, lateral geniculate body, or optic nerve, RGCs can be detected in vivo with confocal laser scanning microscopy, fluorescent microscopy, or confocal scanning laser ophthalmoscopy. Although the resolution of these imaging techniques is limited to detecting only the cell bodies, the addition of adaptive optics has allowed in vivo visualization of axonal and dendritic processes. An ideal experimental model for detection of RGC damage should be non-invasive and reproducible. The introduction of a strain of transgenic mice that express fluorescent proteins under the control of Thy-1 promoter sequence has offered a non-invasive approach to detect RGCs. Long- term serial monitoring of RGCs over a year has been shown possible with this technique. In vivo imaging of RGCs could provide crucial information to investigating the mechanisms of neurodegenerative diseases and evaluating the treatment response of neuroprotective agents.     

川芎嗪对慢性高眼压大鼠视网膜神经节细胞凋亡的影响

目的 研究川芎嗪对慢性高眼压大鼠视网膜神经节细胞(retina ganglion cells,RGC)凋亡的保护作用.方法 健康SD大鼠30只,随机分为正常组、高眼压组和川芎嗪治疗组,每组10只.高眼压组和治疗组分别采用上巩膜静脉烧灼法制作高眼压模型,正常对照组不做处理.造模成功后7d治疗组给予川芎嗪腹腔注射,正常组与高眼压组给予等量的生理盐水腹腔注射.造模3周后取三组大鼠眼球,行视网膜HE染色、TUNEL原位细胞凋亡检测和Bcl-2免疫组织化学检测.结果 造模后高眼压组、川芎嗪治疗组各时间点眼压与正常组比较,差异均有统计学意义(均为P ＜0.05),而两组间各时间点比较,差异均无统计学意义(均为P＞0.05).正常组、高眼压组、川芎嗪治疗组大鼠视网膜RGC层TUNEL阳性细胞数分别为0、9.00±0.89、3.50±1.89,三组间差异有统计学意义(F =86.160,P=0.000);三组间两两比较,差异均有统计学意义(均为P =0.000).三组大鼠RGC层Bcl-2阳性表达光密度值分别为0.373±0.090、0.375±0.146、0.390±0.268,三组差异有统计学意义(F=84.658,P=0.000);三间组两两比较,差异均有显著统计学意义(均为P=0.000).结论 川芎嗪通过上调Bcl-2蛋白的表达可明显抑制慢性高眼压大鼠RGC凋亡,从而发挥RGC保护作用.