Protective limb remote ischemic post-conditioning against high-intraocular-pressure-induced retinal injury in mice

INTRODUCTION

The retina is an extension of the brain tissue and also is the highest oxygen-consuming organ in the body,with high sensitivity to ischemia. Retinal ischemia can lead to functional and morphological changes culminating in blindness. Several ophthalmic diseases are related to retinal ischemia, including glaucoma, obstructive retinopathy,ischemic optic neuropathy, carotid artery occlusive disease,and diabetic retinopathy. Thus far, retinal ischemic injury is still an issue for treatment. Current neuroprotective agents offer incomplete protection, whereas others generate nonspecific effects/risks or toxicity. Therefore, safe and effective alternative therapeutic interventions are required.

LRIC was performed after completing high-IOP procedures.A tourniquet (5 mm) was tightened around the right proximal thigh for three cycles; a cycle comprised of a 5min occlusion phase and a 5min release phase. When the pulse disappeared,skin temperature in the distal limb was decreased and the skin cyanosed, indicating the femoral artery was occluded. LRIC was conducted every day thereafter. Sodium pentobarbital(30 mg/kg) was intraperitoneally applied before LRIC treatment. The same dose of pentobarbital dose was used to treat the sham or control group.

All data were expressed as the mean±standard error of the mean (SEM). Differences among groups were statistically analyzed using one-way ANOVA. A0.05 value was considered statistically significant. Statistical analyses were performed using Sigma Stat 3.5.

MATERIALS AND METHODS

Procedures relating to animal performance and surgery were approved by the Committee of Medical Ethics and Welfare for Experimental Animals, Henan University School of Medicine (Ref no. MEWEAHUM 2014-0001). Strict efforts were made to reduce animal suffering in accordance with the Association for Research in Vision and Ophthalmology (ARVO) on the use of animals.

C57BL6 mice (8-12-week-old and weight range 18-28 g) were purchased from Zhengzhou University(Henan, China) and housed in individually ventilated cages.Environmental conditions were maintained over a 12h/12h light/dark cycle, with humidity at 60%±5% and temperature at 22℃±3℃. Food and water were freely accessible.Animals were initially divided randomly into three or four groups. LRIC in control animals (=18) did not affect retinal histology when compared with sham animals (sham group,=4). Subsequently, three animal groups were used. Animals undergoing high-IOP treatment in the eyeball were designated as the high-IOP group (=18). Animals undergoing high-IOP +LRIC were the high-IOP + LRIC group (=18).

原始社會(huì)生產(chǎn)資料公有，生產(chǎn)力低下，沒(méi)有文字，也無(wú)學(xué)校，更無(wú)學(xué)科教學(xué)。人們的教育活動(dòng)是緊密結(jié)合著人們的生產(chǎn)勞動(dòng)和社會(huì)生活，在一種自然狀態(tài)下，根據(jù)個(gè)人的需要進(jìn)行一對(duì)一的個(gè)別教學(xué)。教學(xué)方法多是以講述、問(wèn)答、示范、練習(xí)為主。

The mice were anaesthetized by using a pentobarbital (100 mg/kg)intraperitoneal injection, and 0.5% proparacaine hydrochloride was used as topical anesthesia in the cornea. Next, 1%tropicamide was applied externally applied onto the cornea to dilate the pupils. Anaesthetized mice were laid down on their left side under a stereo microscope. A 30-gauge needle connected to an NS bag was inserted horizontally into the anterior chamber of the right eye. Then, the bag was raised to approximately 150 cm. When the anterior segment of the globe got whitening (vessels were blocked), ischemia conditioning could be complete. The high-IOP was then maintained for 50min and, then returned to normal-IOP by removing the needle. Usually, the whitened anterior eyeball was restored with blood supply. For the sham group, a needle was inserted into the cornea but without elevated IOP. Eyeballs and retinas were collected for analysis at 1, 3, and 7d post high-IOP.

Ischemic conditioning was initially discovered by Murry. Later, it was used to intermittently block remote organs(, limbs) which were relatively ischemia-tolerant. We refer to this as limb remote ischemic post-conditioning (LRIC).Ischemic conditioning may be divided into pre-, per-, and postconditioning types. Since retinal ischemia onset is usually unpredictable, we used limb ischemic post-conditioning as a clinical strategy. In recent years, extensive research has shown that LRIC has been comprehensively developed from a basic research tool to a complex clinical technique, with its application potential constantly evolving. LRIC as a safe(non-invasive), economical, and effective adjuvant intervention has provided protective roles towards the heart, brain, lung,kidney, liver, and intestine. The retina is the most important tissue in the eye and extends from the central nervous system.In terms of increased oxygen consumption and metabolic activity, the retina is more sensitive to hypoxia and ischemia.However, LRIC-based treatment studies on retinal ischemic injury are limited. Zhangreported that middle cerebral and pterygopalatine artery occlusion induced retinal ischemic injury, and was ameliorated by an LRIC intervention. In our study, we investigated the role of LRIC on high intraocular pressure (IOP)-induced retinal ischemia injury in mice, which is a commonly used animal model without large surgery stress. Also, the pathological process is closer to real ischemia conditions. Furthermore, we assessed if plasma aliquots from LRIC-treated animals could induce protective effects on retinal ischemic injury. We sought to identify possible endogenous humoral molecules released into the circulation to promote LRIC-protective effects. This work provides a research basis for exploring effective molecules induced by LRIC (Figure 1).

Mouse plasma from LRIC (5 min occlusion/5 min release over three cycles) treated animals (30min later) was collected by intracardiac bleeding.Blood collected in anticoagulation tubes was used to prepare plasma by centrifugation at 1000 g. Plasma aliquots were stored at -80℃. Plasma was then systemically intravenously injected (150 μL/injection) into micethe tail.

合理配比原材料，也能夠避免發(fā)生裂縫現(xiàn)象。其中，混凝土強(qiáng)度等級(jí)與原材料的配比是否準(zhǔn)確息息相關(guān)，其會(huì)直接引起混凝土結(jié)構(gòu)出現(xiàn)一定的變動(dòng)，因此在實(shí)際施工前，必須根據(jù)現(xiàn)場(chǎng)施工條件和情況，對(duì)混凝土配比進(jìn)行科學(xué)、合理地設(shè)計(jì)，保證參數(shù)符合施工要求。除此之外，在一些路橋施工過(guò)程中，由于受原材料、環(huán)境及施工企業(yè)等影響，導(dǎo)致實(shí)際情況與規(guī)劃出現(xiàn)一定的偏差，這就需要施工企業(yè)在施工過(guò)程中通過(guò)具體施工情況對(duì)原材料配比進(jìn)行有效的優(yōu)化。

Malondialdehyde (MDA) levels and super oxide dismutase (SOD) enzyme activities were measured by commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Retinas were rapidly isolated from eyeballs with pigmentary epithelium discarded. After this, retinal tissues were homogenized in RIPA buffer (Beyotime, China). Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and gels transferred to nitrocellulose membranes (Millipore, MA, USA). These were blocked in 3% bovine serum albumen and incubated overnight at 4℃ with primary antibodies (CHOP, Beyotime,1:2000;Iba-1, Abcam, 1:5000; caspase 9, Boster, 1:1000). The next day, a secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit) was added and incubated for 1h at room temperature on a shaker. Protein band optical densities were semi-quantitatively measured by Image J v2.1. β-actin (1:400,Boster) was used as a loading control.

Horizontal retinal paraffin-embedded slides (5 μm in thickness around the optic disc) were deparaffinized in xylene and rehydrated in decreasing ethanol concentrations. Retinal sections were stained by hematoxylin and eosin. To examine retinal thickness, three measurements were averaged for each retina from the peripheral to the center. For immunofluorescence staining, retinal sections were washed in 0.01 mol/L phosphate buffered saline (PBS, pH 7.4) and incubated with antigen retrieval buffer (Boster, China) according to manufacturer's instructions. After this, sections were blocked in 10% normal goat serum (Boster, China) for 30min and incubated overnight at 4℃ with primary antibodies (anti-NeuN 1:400, Boster,China; anti-Iba-1, 1:1000, Abcam, USA, and anti-CHOP 1:1000, Beyotime, China; and anti-caspase 9 1:100, Boster,China). The following day, after washing with PBS, sections were incubated with relevant secondary antibodies (1:500,Beyotime) for 1h at room temperature. After washing twice in PBS (5min each), section images were observed using an optical or fluorescence microscope. For dihydroethidium(DHE) staining, eyeballs were embedded into compound tissue-tek (SaKura Finetec, USA) and fixed in liquid nitrogen.Retinal cryosections (5 μm) were incubated with DHE(40min at room temperature). Images were observed under a fluorescence microscope and the fluorescence intensity was then calculated.

骨髓抑制是細(xì)胞毒化療藥物的常見(jiàn)不良反應(yīng)之一，是大多細(xì)胞毒藥物的劑量限制性毒性?；熕幬锓N類(lèi)、劑量強(qiáng)度、白蛋白、腎功能、既往化療放療等是影響骨髓抑制的重要因素[2]。本組患者發(fā)生Ⅳ度骨髓抑制大多為既往接受過(guò)多次化療或放療，共99例/次（75.3%），此類(lèi)患者機(jī)體骨髓儲(chǔ)備能力降低，比既往未接受放化療患者更易發(fā)生Ⅳ度骨髓抑制。出現(xiàn)Ⅳ度骨髓抑制時(shí)若不予以積極干預(yù)，不僅影響化療方案的實(shí)施及臨床治療效果，甚至引起嚴(yán)重感染、貧血、出血及心力衰竭等并發(fā)癥，相前研究表明與Ⅳ度骨髓抑制有關(guān)的治療相關(guān)性病死率達(dá)4%-12%[3]。

然而，該方法仍存在一定的缺陷：對(duì)于體積較大的BPH，由于前列腺側(cè)葉表面黏膜切開(kāi)范圍相對(duì)較大，而腺體尿道黏膜血供較豐富，切開(kāi)時(shí)容易使出血較多，造成視野不清，進(jìn)而延長(zhǎng)手術(shù)時(shí)間。本研究用小能量高頻率的激光(1.5 J/50 Hz)可在一定程度上改善止血效果。此外，過(guò)大的前列腺尖部區(qū)域往往超過(guò)一個(gè)鏡野，初學(xué)者手術(shù)經(jīng)驗(yàn)欠缺，不易準(zhǔn)確判斷黏膜切開(kāi)線的位置，也使該方法的應(yīng)用范圍受到了一定的限制。

RESULTS

To explore whether endogenous protective molecules induced by LRIC exerted remote protective effects against retinal ischemic injury, LRIC-treated plasma was transfused into high-IOP animals. Our results indicated that application of LRIC-treated plasma appeared to inhibit high-IOP-induced increase of caspase 9, a key apoptosis enzyme (Figure 6). Furthermore, we also evaluated reaction oxygen species (ROS) levels in retina tissue, by using a ROS fluorescent DHE probe. The results showed that LRIC treatedplasma inhibited the enhanced ROS fluorescence intensity induced by high-IOP (＜0.05; Figure 7). Taken together, LRIC treated-plasma may exert protective effects in high-IOP retinas through anti-apoptosis and anti-oxidative stress. The effective endogenous component maybe came from the endocrine of LRIC treated limb.

CHOP is an endoplasmic reticulum stress marker.Western blotting and immunohistochemical staining analysis indicated that high-IOP induced increases in CHOP expression could be inhibited by LRIC treatment (＜0.05; Figure 4A,4B). MDA levels and superoxide dismutase (SOD) activities were evaluated as oxidative stress markers. As shown (Figure 4C, 4D), MDA levels were increased, whereas SOD activity was significantly decreased at day 1 after high-IOP (＜0.05).In contrast, the increases in MDA levels and the decreased in SOD activities were inhibited significantly by LRIC treatment.Retinal ischemic injury usually lead to strong microglia activation.Immunohistochemical staining (Figure 5A) and Western blot analyses (Figure 5B) were used to evaluate Iba-1 expression(a microglia and macrophage marker). Our data indicated that low Iba-1 levels were detected in the normal retina. However,at 1d after high-IOP, Iba-1 levels were abundantly expressed in the ganglion cell layer and inner plexiform layer. However, in the high-IOP + LRIC group, increased Iba-1 levels induced by high-IOP were significantly reduced when compared with the high-IOP group (＜0.05; Figure 5).

To evaluate the effects of plasma based endogenous factors induced by LRIC, the animals were divided into control(=12), high-IOP + normal saline (NS) group (=12), and high-IOP + plasma (=12) groups. The right eye was typically chosen to perform for high IOP procedures.

LRIC treatment in the normal animals did not affect retinal histomorphology. Thus, the sham group and LRIC control group were combined as the control group subsequently. In the high-IOP group, retina cross-sections were disorganized, edema and thickened 1d after application of high-IOP (Figure 2A). In the 7day after high-IOP, whole retina thickness and inner plexiform layers were significantly decreased since loss of neurons (Figure 2B). These histological changes induced by high-IOP were relieved by the LRIC treatment significantly (＜0.05; Figure 2). Immunofluorescence staining for neuN in retinal cross-sections revealed the positive signals were neuron cells (Figure 3). Significant neuronal loss in the ganglion cell layer was observed 7d after high-IOP, however,this loss was remarkably inhibited by LRIC (＜0.05). These data suggested LRIC may have protected the retina against high-IOP induced injury.

DISCUSSION

Our study suggested that LRIC generated retinal protective roles against high-IOP induced injury through the inhibition of CHOP, Iba-1 and oxidative stress levels. In addition, we observed that LRIC-treated plasma decreased caspase-9 levels and ROS formation in high-IOP retinas. Thus, endogenous factors induced by LRIC may release into the circulatory system and exerted remote protective roles.LRIC is a potent endogenous protection system which potentially triggers a series of endogenously active biological factors to exert protective effects against retinal ischemic injury. It applies intermittent blood flow blocking to an organ (typically the limbs) which in turns exerts protective roles toward the organ experiencing ischemic injury. Typically, three LRIC approaches are available (pre-, per- and post-conditioning)for practical applications. In reality, ischemic events cannot be predicted, therefore, post-conditioning may be considered a therapeutic intervention for multi-organ protection. A previous study reported that LRIC exerted protective roles against cerebral artery occlusion induced by retinal ischemia injury. Our study revealed that high-IOP in a mouse model treated with LRIC induced retinal protection. The surgical approach generating high-IOP was less invasive than other vascular occlusion surgery, therefore it may be more practical for exploring underlying LRIC mechanisms. High IOPinduced retinal ischemia is commonly used to generate retinal ischemic reperfusion in mouse models. Because the mouse lens is relatively larger than humans, it is easier to oppress backwards and induce acute ischemia under acute ocular hypertension conditions. The mouse model reflects the same pathological changes as seen in human conditions, including acute angle-closure glaucoma and retinal vessel occlusion.Similar to previous investigations, high-IOP stress led to changes in retinal histology. During the preliminary stages (24h) of retinal ischemic-reperfusion injury, the main pathological changes were vacuolation, edema, and increased thickness of the ganglion cell layer, inner nuclear layer and the whole retina. In later stages (7d), retinal thickness reduced, and ganglion cell numbers decreased significantly. LRIC at 5min and 3d after reperfusion ameliorated these histological changes induced by ischemic reperfusion injury. These protective roles were consistent with previous post-conditioning mouse models.

Ischemic-reperfusion injury is a main source of free radical generation, which if in excess, induce oxidative stress damage towards proteins, lipids, and nucleic acids. We showed that LRIC inhibited increased MDA levels and decreased SOD activity. MDA is a product of lipid peroxidation and is an indicator to assess oxidative stress damage and severity.Increasing evidence has identified associations between remote ischemic conditioning and antioxidant activity. CHOP is an endoplasmic reticulum (ER) stress-related protein and is used as an ER stress marker. Increased retinal CHOP levels are observed in many conditions, including ischemic reperfusion injury, diabetic retinopathy, and other diseases. ER homeostasis may be dysregulated by hypoxia, oxidative stress,and inflammation. The ER stress and oxidative stress always interact to generate apoptosis and even cause tissue injury.In our study, LRIC in ischemic reperfusion animals reduced CHOP expression, consistent with the ROS formation.

Ischemia/hypoxia induces direct retinal damage during high-IOP conditions. In addition, blood reperfusion induced leukocyte infiltration (including monocytes/macrophage)induces inflammatory response cascades which may induce more subsequent injury. Therefore, LRIC could alter these systemic responses and decrease the extent of nervous injury by decreasing leukocyte accumulation and inflammatory factor expression. Our results indicated LRIC inhibited high-IOP induced Iba-1 expression in the retina, which was in accord with previous report. Iba-1 is a macrophage/microglia marker.It was not very easy to distinguish Iba-1 positive microglia and infiltrated monocytesour morphology staining. From Ha'sstudy, we were aware of the Iba-1 positive staining including both types of the cellular during retinal ischemic reperfusion injury status. Thus, LRIC appeared to reduce infiltrated monocytes and microglia activation, which may contribute to retinal protection against ischemic reperfusion injury.

我院通過(guò)對(duì)2010年2月～2018年4月我院收治的56例股骨頸骨折患者進(jìn)行研究，得出觀察組患者的護(hù)理總有效率為96.43%，而對(duì)照組護(hù)理總有效率為64.29%，觀察組患者的護(hù)理效果明顯優(yōu)于對(duì)照組；觀察組的護(hù)理滿意度為100%，明顯高于對(duì)照組的護(hù)理滿意度（60.71%%），組間差異明顯，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。

(2)抗風(fēng)險(xiǎn)能力不足。湖北省零部件產(chǎn)業(yè)雖然規(guī)模較大，但產(chǎn)業(yè)中單個(gè)企業(yè)的規(guī)模普遍較小，在投資力度、技術(shù)水平方面均相對(duì)落后，而且生產(chǎn)的產(chǎn)品以勞動(dòng)密集型為主，具有技術(shù)含量的產(chǎn)品較少，具有自主品牌和核心競(jìng)爭(zhēng)力的產(chǎn)品不足，因此在對(duì)整車(chē)供應(yīng)中并不占優(yōu)勢(shì)，這將導(dǎo)致企業(yè)利潤(rùn)被擠壓，由此影響汽車(chē)零部件產(chǎn)業(yè)的抗風(fēng)險(xiǎn)能力。

Plasma transfusion are effective interventional methods and have been demonstrated for several disease conditions including cardioprotection, severe sepsis, Alzheimer's disease,and age related impairment. In our study, LRIC plasma was transfused into high-IOP animals, and showed that the apoptotic signal enzyme, caspase 9 and ROS formation in retinas were significantly reduced. Studies have reported that remote ischemic condition protection is generated by the release of multiple humoral factors into the circulation,including heat shock proteins, extracellular vesicles,adenosine, nitric oxide, and glutamate. Thus, we also preliminarily verified the protective effects of LRICtreated plasma by intravenous injection. Further research is required to fully clarify the essential humoral factors required for LRIC protection and related neuroregulatory mechanisms.In conclusion, since remote ischemic condition was proposed in 1986, extensive research and clinical transformational studies have emerged. We demonstrated that LRIC protected high IOP-induced retinal injuryantioxidant and ER stress inhibition. Reduced monocyte infiltration and microglia activation may have played an important role in this protection. The present study demonstrated that LRICplasma transfusion exerted protective effects from ischemic reperfusion injury by inhibiting caspase 9 and ROS production in high-IOP animals. However, LIRC humoral or/and neural mechanisms require further investigation, therefore, future studies need to determine and identify key factors released into the circulation. Ultimately, this effective, low cost, low risk therapy could be optimally developed for improved applications in clinical medicine.

三是圍繞變化環(huán)境下的風(fēng)險(xiǎn)規(guī)避與防災(zāi)減災(zāi)，積極為極端和突發(fā)水事件的應(yīng)急響應(yīng)提供有效支撐。10年來(lái)，適應(yīng)全球氣候變化和經(jīng)濟(jì)社會(huì)快速發(fā)展導(dǎo)致的極端和突發(fā)水事件頻發(fā)的情勢(shì)需求，水利科技創(chuàng)新突破，在應(yīng)對(duì)汶川特大地震、舟曲泥石流和玉樹(shù)地震等自然災(zāi)害的過(guò)程中，對(duì)堰塞湖處置、災(zāi)情判別和水情預(yù)報(bào)、飲用水應(yīng)急處理、城鄉(xiāng)供水恢復(fù)、水工程安全評(píng)估與除險(xiǎn)等防災(zāi)減災(zāi)救災(zāi)中起到了基礎(chǔ)性科技支撐作用。

Zhu QJ and Wang J designed,performed the experiments and drafted the manuscript; Zhang L, Lyu SY, and Cui ZJ analysed part of the data, provided some technical guidance for experiment; Jiang ES provided intellectual input, supervised the research and edited the manuscript. All authors reviewed and approved the final manuscript for submission.

Supported by the National Natural Science Foundation of China (No.31300884; No.81803573).None;None;None;None;None;None.