Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats

INTRODUCTION

Glaucoma is the second-leading irreversible blinding eye disease in the world. In recent years, many studies have focused on retinal ganglion cell (RGC) death

. The survival of retinal nerve cells is closely related to the microenvironment around them. The blood-retinal barrier (BRB) is essential in maintaining the retinal homeostasis of the microenvironment

.Structurally, the BRB is composed of two distinct barriers:the outer blood-retinal barrier (oBRB), consisting of retinal pigment epithelium that regulates transport between the choriocapillaris and the retina, and the inner blood-retinal barrier (iBRB), which regulates transport across retinal capillaries

. The local microenvironment of RGCs is mainly protected by iBRB

. The iBRB is not an absolute barrier because substances from the blood can cross it by two distinct mechanisms, caveola-mediated transport (transcellular) and paracellular transport

. Paracellular transport is strictly dependent on tight junctions (TJs), such as claudins and zonula occludens (ZO); adherens junctions (AJs), such as vascular endothelial cadherin (VE-cadherin); and gap junctions (GJs),such as connexin 43 (Cx43). Studies have shown that ZO-1 is a marker of BRB integrity, and the loss or reduction of ZO-1 is related to an increase in barrier permeability

.

父親與母親的結(jié)合正是如此舊道德婚姻形態(tài)下的一個縮影，帶有十分濃烈的悲劇色彩。人類作為一種情感動物，特別是在物質(zhì)匱乏的時候，對于精神方面的需求會急劇上升。然而，父親與母親卻不曾相愛。兩人在物質(zhì)上或許相互扶持，在精神上卻是無法互相慰藉。就好像母親知道父親的羅曼史，卻始終沒有嘗試去解開他的心結(jié)。在雙方的精神世界里，本應(yīng)最親密的枕邊人反而是局外人。

Many diseases can cause retinal ischemia-hypoxia

, such as diabetic retinopathy, age-related macular degeneration, retinal detachment, and partial acute glaucoma. The damage to BRB under hypoxia-ischemia conditions is one of the causes of the degeneration of retinal nerve cells

. The acute high intraocular pressure (HIOP) animal model is a kind of acute experimental glaucoma model

. Previous studies have found that BRB breakdown occurs after acute HIOP in rats

, and hypoxiainducible factor-1 alpha (HIF-1α) plays an important role in this process

. Elevated intraocular pressure can induce upregulation of HIF-1α protein and its target gene,

-

,but another study found up-regulation of only vascular endothelial growth factor (VEGF)-165b, one subtype of VEGF-A in the same model

. VEGF-A is the principal angiogenic factor that increases microvascular permeability during physiological and pathological angiogenesis

. VEGF-A is generated by alternative splicing of 8 exons to produce two subtypes, proangiogenic VEGF-165a and anti-angiogenic VEGF-165b

. It has been reported that VEGF-165b was cytoprotective and antiangiogenic in the retina

. However,the role of VEGF-165b in iBRB breakdown after acute HIOP has not yet been clarified. In this study, the rat acute HIOP model was established before and after intravitreous injection of anti-VEGF-165b antibody. Immunohistochemistry, Western blotting and Evans blue (EB) staining were performed to elucidate the role of VEGF-165b in BRB injury in the rat acute glaucoma model.

MATERIALS AND METHODS

Animal scarification and tissue collection were approved by the Animal Ethics Committee of Hainan Medical University and were performed according to the guidelines of the “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985). The SD rats we used in this study strictly followed the ARVO statement for the use of animals in ophthalmic and vision research.

目前我國競技體育領(lǐng)域仍施行“舉國體制”，在今后相當(dāng)長一段時間里，該體制仍將延續(xù)。隨著我國市場經(jīng)濟的推進，社會各行各業(yè)面臨改革，政府如何做好退役運動員安置工作，最大限度地發(fā)揮退役運動員的價值，實現(xiàn)其社會角色的成功轉(zhuǎn)型，這是政府和體育相關(guān)部門必須面對的重大挑戰(zhàn)，關(guān)系到我國體育強國建設(shè)，并直接影響到我國競技體育長盛不衰的長久大計。

Adult male SD rats weighing 250-300 g (Changsha Tianqin Biotechnology Co. Ltd.,Standard: GB14924.3-2010 Nutritional Composition of Mixed Feed for Laboratory Animals. Changsha, Hunan Province,China) were housed in a standard rat cage with unlimited access to water and food with a 12-hour continuous light supply every 24h. Rats were randomly divided into three groups: a control group, a 12h group and a 3d group. Each group was composed of 6 rats. Previous studies

found that BRB injury was more obvious in the early stage (within 24h)and gradually recovered in the late stage (1-7d). Therefore, in this study, the 12h time point was chosen to represent the early stage of injury, and the 3d time point was chosen to represent the late stage of injury. The control group did not have their intraocular pressure raised by inserting needles into the anterior chamber. The rats were sacrificed after 12h and 3d survival following the acute HIOP.

原句：Suddenly,in the middle of the night,she went berserk and hell broke out.

The animal model of acute HIOP was established as previously described

. Briefly, animals were anesthetized by intraperitoneal injection of 2% pentobarbital solution(40 mg/kg). A sterile disposable intravenous infusion needle connected to an instillation instrument filled with normal saline was inserted into the anterior chamber. The intraocular pressure of the two eyes was gradually elevated to 120 mm Hg and maintained for 60min, then slowly lowered to the normal level. A drop of levofloxacin eye drops was administered to the conjunctival sac for infection prevention. Rats with cataract or eye inflammation were excluded as unsuccessful models.

你是一個擁有清新脫俗氣質(zhì)的人，和你相處的人，都會有一種耳目一新的感覺。單純的你喜歡遐想，常常會有一些不太切合實際的想法，不過你的這種性格卻會激起人的保護欲，讓人情不自禁地想照顧你、呵護你。其實，你是一個蠻能吃苦的人，抗壓能力很強，只是不太喜歡復(fù)雜的人際關(guān)系，相比于那些虛情假意的奉承，你更希望人和人之間還是簡單一點的好。雖然說你比較單純，但是你不會輕易向別人訴說你的心事，只會對那些你信任的人敞開心扉。

The retinal tissue preparation was performed as previously described

. Briefly, the rats were anesthetized by intraperitoneal injection of 2% pentobarbital solution (40 mg/kg) and sacrificed with excessive dose (80 mg/kg).For immunofluorescence staining, rats were transcardially perfused with normal saline followed by paraformaldehyde (PF)solution. After perfusion, the eyeballs were dissected out. The eyecups were gradually dehydrated and subsequently embedded in Tissue-Tek optimal cutting temperature medium. Using a cryomacrotome (Thermo Electron Corporation, Cheshire, UK),10-μm-thick cryosections were obtained, and stored at 4℃.

After sealing with anti-fade mounting medium (E675011, Sangon Biotech, China) under dark conditions, EB red fluorescent spots in the retina were observed by confocal microscopy (Olympus FV1000, Japan)at an excitation wavelength of 654 nm, and the central and peripheral parts of the retina were photographed. The relative fluorescence intensity of each group was analysed by Image J software (National Institutes of Health, Maryland, USA) and normalized to the control group.

由以上Ⅰ～Ⅳ，我們可……