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。化療藥物種類(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。……