Accepted Manuscript The elevation of intraocular pressure is associated with apoptosis and increased immunoreactivity for nitric oxide synthase in rat retina whereas the effectiveness of retina derived relaxing factor is unaffected Dr. Selçuk Takır, Ebru Gürel-Gürevin, Ayça Toprak, Cihan Demirci-Tansel, B.Sönmez Uydeş-Doğan PII:
S0014-4835(16)30032-X
DOI:
10.1016/j.exer.2016.03.002
Reference:
YEXER 6876
To appear in:
Experimental Eye Research
Received Date: 29 May 2015 Revised Date:
5 February 2016
Accepted Date: 2 March 2016
Please cite this article as: Takır, S., Gürel-Gürevin, E., Toprak, A., Demirci-Tansel, C., Uydeş-Doğan, B.S., The elevation of intraocular pressure is associated with apoptosis and increased immunoreactivity for nitric oxide synthase in rat retina whereas the effectiveness of retina derived relaxing factor is unaffected, Experimental Eye Research (2016), doi: 10.1016/j.exer.2016.03.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: The elevation of intraocular pressure is associated with apoptosis and increased immunoreactivity for nitric oxide synthase in rat retina whereas the effectiveness of retina
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derived relaxing factor is unaffected
Authors: Selçuk Takıra,c*, Ebru Gürel-Gürevinb, Ayça Topraka, Cihan Demirci-Tanselb, B. Sönmez Uydeş-Doğana a
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Department of Pharmacology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey c Department of Medical Pharmacology, Section of Internal Medical Sciences, Faculty of Medicine, Giresun University, Giresun, Turkey
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b
Meeting Presentation: Part of this study was presented in 22th National Congress of Pharmacology (Turkish Pharmacological Society) Antalya/Rixos Hotel 4-7 November 2013.
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Financial Support: This study was supported by Istanbul University, Scientific Research Projects Unit under grant number 10608. None of the authors has any proprietary/financial interest to disclose. Conflict Of Interest: The authors declare that there is no conflict of interest. The authors thank to G. Zeynep Ortaköylü for her excellent assistance.
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Running Head: Elevation in IOP increased apoptosis and NOS immunoreactivity in retina but unchanged the response to RRF
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Address for Reprints:
* Corresponding author (): Dr. Selçuk Takır Giresun University, Faculty of Medicine, Department of Medical Pharmacology, Section of Internal Medical Sciences 28200, Giresun, TURKEY Tel: +90 4544101600/3015 Fax: +904544101699 E-mail:
[email protected]
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ACCEPTED MANUSCRIPT Abstract Glaucoma is a progressive ocular disease that stands in the upper rank for the cause of blindness in worldwide. In the present study, we aimed to elucidate the possible disturbances occurred in the layers of retina due to an increase in intraocular pressure (IOP) and to verify the effectiveness of retina derived relaxing factor, i.e., RRF in this pathologic
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condition. The increase in IOP was induced by cauterization of the three of episcleral veins simultaneously in rats. After 8 weeks period, the retinas excised from the vein cauterized eyes were evaluated for the possible histopathological and ultrastructural alterations as well as for the relaxing effects on isolated bovine retinal and rat mesenteric arteries, in
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comparison with the retinas obtained from contralateral sham-operated eyes. In the retinas of IOP-elevated eyes, profound morphological deteriorations were determined in the ganglion and outer nuclear cell layers which were associated with an increased number of TUNEL
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positive cells in the ganglion and inner nuclear cell layers. Increased immunohistochemical stainings for three isoforms of nitric oxide synthase (NOS) were defined in almost all layers of the retinas of IOP-elevated eyes, in which eNOS was abundant particularly in the inner plexiform and ganglion cell layers. An irregular basal folding of retinal pigment epithelium (RPE) and an increased inter lamellar space of photoreceptor cell layer furtherly
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characterized the prominent degeneration of those layers in the retinas of IOP-elevated eyes. On the other hand, the relaxing effects of the retina obtained from IOP-elevated eyes were determined to be unchanged on the retinal and mesenteric arteries precontracted either with prostaglandin F2α (PGF2α, 30 µM) or potassium chloride (K+, 100 mM), when compared
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with the relaxations of control retina obtained from contralateral sham-operated eyes. Overall, these findings suggested that the elevation of IOP induces prominent structural changes in rat retina particularly in the ganglion and inner layers that is associated with
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marked apoptosis and increased immunoreactivity for NOS, while the functional effectiveness of retina derived relaxing factor, i.e., RRF is unaffected.
Key words: Intraocular pressure, glaucoma, apoptosis, NOS, retinal relaxing factor, retinal artery, mesenteric artery, retina
1. Introduction Glaucoma, which stands in the upper rank for the cause of total visual loss (blindness) in worldwide, is a progressive ocular disease associated with an excavation in 2
ACCEPTED MANUSCRIPT optic nerve head (ONH) and a degeneration in retinal ganglion cells (RGC) (Tham et al., 2014). An increase in the intraocular pressure (IOP) is suggested to induce the excavation in ONH, which is a well-recognized major clinical feature in glaucoma. This disturbs the axonal transport and triggers the degenerative processes in ganglion cells via apoptosis; whereas, the exact mechanism is still poorly understood (Almasieh et al., 2012; Quigley et
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al., 1995). In regard to the degeneration of RGC, different mechanisms have been reported to mediate this impairment which is reliably verified in several experimental studies (Chen et al., 2011; Guo et al., 2005; Quigley et al., 1995). However, other studies have implicated a damage in the inner layers of the retina, as well (Pelzel et al., 2006; Wygnanski et al., 1995).
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A decrease in the diameter of retinal vessels is also proposed to be a contributory defective mechanism in glaucoma (Wang et al., 2007), which probably induces homeostatic changes in the retinal blood flow (Grieshaber and Flammer, 2005; Venkataraman et al.,
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2010). In relation, the retinal arterial tone is suggested to be altered due to an imbalance between several vasoactive factors released from the retina and/or the endothelial layer of the retinal vessels (Grieshaber and Flammer, 2005; Venkataraman et al., 2010). Among these substances, endothelin-1, nitric oxide (NO), N-methyl-D-aspartate (NMDA), adenosine and calcitonin gene related peptide (CGRP) were reported to play a crucial role in
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the local regulation of ocular blood flow as well as in the maintenance of IOP (Ghanem et al., 2011; Grieshaber and Flammer, 2005; Venkataraman et al., 2010). A novel relaxing factor derived from the retina namely, retinal relaxing factor (RRF), has recently been identified to influence the retinal arterial tone by producing a substantial relaxation and
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thereby, decreasing the contractions to various spasmogens (Delaey and Van de Voorde, 1998; Takir et al., 2011; 2015). Inspite of several experimental studies that attempted to describe its nature and mechanism of action, the vascular effectiveness of RRF in
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pathological conditions which can unfavourably affect the retina and/or the retinal circulation is still scarce. Of note, among the ocular diseases, glaucoma is proposed to develop morphological and functional abnormalities in the retina as well as in the retinal vasculature (Grieshaber and Flammer, 2005; Venkataraman et al., 2010). Accordingly, in the present study, we aimed to evaluate the probable differences in the vasorelaxant influence of retina obtained from the eyes in normal and disease conditions, particularly related to the elevation of IOP by the episceral vein cauterization model in rats. We also attempted to investigate the retinal degeneration initiated by the elevation of IOP via determining the
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ACCEPTED MANUSCRIPT alterations occurred in the histological and ultrastructural pattern of retinal layers as well as specifying the contribution of apoptosis and nitric oxide synthase (NOS).
2. Material and methods Male Wistar albino rats with an average age of 10-12 weeks (200-250 g) were used
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in the experiments. Animals were housed under standard temperature of 20±2 oC and humidity of 55-60% on a 12:12 hour light/dark cycle. Rats were prepared for the experimentation by daily handling for at least one week before starting to the surgical procedures and measurements of IOP. Overall, 51 rats were used in the experiments. 4 of the
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rats were used in the preliminary experiments to set the sustained increase in IOP with the episcleral vein cauterization method. While, 5 of the rats could not achieved a sustained elevation in the IOP for 8 weeks period and thus excluded from the experiments. 14 of the
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rats were used in histological, immunohistochemical and electron microscopy analyses and 28 of the rats were used in functional experiments performed in the myograph. All experimental and animal care procedures utilized were in accordance with the European Community and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and approved by
Decision No: 36).
2.1.
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Local Animal Experimentation Ethics Committee of Istanbul University (16/02/2010,
Induction of ocular hypertension in rats
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Induction of ocular hypertension (glaucoma) was performed on the right eyes of the rats that were anaesthetized with intraperitoneal injection of xylazine hydrochloride (Rompun®, Bayer, Turkey, 5 mg/kg) and ketamine hydrochloride (Ketalar®, Pfizer, Turkey,
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50 mg/kg). After a 2-3 mm of conjunctival incision, one temporal and two dorsal episcleral veins located near the superior and lateral rectus muscles of the eyes were cauterized by a hand-held low temperature cautery (Advanced Meditech International, New York, USA) (Shareef et al., 1995). Care was taken to avoid thermal damage to the sclera. The contralateral left eyes were sham-operated and served as the corresponding control. Both eyes were flushed with saline and treated with ophthalmic gentamisin (Genta®, İ.E. Ulagay, Turkey, 0.3%) drop after the surgery. The mechanism of pressure elevation induced by episcleral veins cauterization involves obstruction of aqueous humour outflow and in part congestion in ocular vasculature, which might disturb overall venous circulation and cause 4
ACCEPTED MANUSCRIPT the deterioration of choroidal, retinal as well as ONH perfusion (Goldblum and Mittag, 2002; Grodzanic et al., 2003; Morrison et al., 2015; Pang and Clark, 2007). Although, the retinal blood vessels were reported to appear normal following to the cautery procedure as assessed by funduscopic examination (Sawada and Neufeld, 1999), it is uncertain to what degree the retinal pathology may be affected by the vascular congestion and/or decreased
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blood flow, apart from the elevation of IOP in this experimental model. On the other hand, the advantages of the episceral vein cautery model is that the increase in IOP is rapid, stable, and long lasting, and the procedure is less invasive, efficacious, easy accessible, and induces no complications in the anterior chamber (Grodzanic et al., 2003; Ischikawa et al., 2015;
2.2.
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Neufeld, 1999; Urcola et al., 2006).
Measurement of IOP
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IOP was measured in both eyes of the rats under anaesthesia. A drop of proparacaine HCl (0.5%) was applied to each eye to desensitize the cornea before performing the measurements by a calibrated hand-held tonometer (Tono-Pen Avia, Reichert Technologies, New York, USA). To eliminate the probable influence of anaesthesia on IOP, the measurements were performed within 2 minutes. The tonometer was applied perpendicularly
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to the cornea. Five consecutive readings were taken for each eye and then averaged. The initial IOP values in rats eyes were established immediately before and after the cauterization or sham-operation process. Thereafter, the successive IOP measurements in both cauterized and sham-operated eyes were performed on the 1st, 4th and 7th days after the
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operation and then, once a week for the following 8 weeks (56 days) period. All measurements were performed between 10:00-12:00 a.m. in order to avoid a circadian
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variation in IOP readings.
2.3.
Histological studies
The rat retinal tissues obtained from both the normal (control) and vein cauterized
eyes were used for the histological evaluations. At the end of 8 weeks (56 days) period, the retinas were carefully excised from the control (sham-operated) and vein cauterized eyes, which displayed a sustained increase in IOP, then fixed in 10% neutral formalin for 24 hours, dehydrated through graded alcohol and finally embedded in paraffin blocks. The paraffin-embedded sections (4µm in thickness) were stained with Haematoxylin and Eosin (H&E) and further examined under a light microscope (Leica Microsystems, Mannheim, 5
ACCEPTED MANUSCRIPT Germany). Histological images were obtained by using the Kameram 390 CU Software (Mikro Sistem Ltd. Sti, Turkey).
2.4.
In situ detection of apoptosis by the TUNEL assay
The paraffin-embedded retinal tissues from the control and vein cauterized eyes were
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cut into 4 µm-thick sections. TdT-mediated dUTP-biotin nick end labelling (TUNEL) analysis was carried out for determining the apoptosis by using the In Situ Cell Death Detection Kit (POD, Roche, Germany) according to the manufacturer’s protocol. The colour reaction was developed with diaminobenzidine (DAB) for 3-6 minutes and the sections were
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counterstained with 5% methyl green prior to analysis by light microscopy. For the negative control, sections were incubated in 50 µL/well of Label Solution (without terminal transferase) instead of the TUNEL reaction mixture. All the sections were examined under
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Kameram 390 CU software. TUNEL-positive cells were quantified as the percentage of TUNEL-positive to the total number of cells in the retinal ganglion cell layer and expressed as apoptotic index (Yang et al., 2010).
2.5.
Immunohistochemical studies
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The localization of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) in the retinas of the control and the vein cauterized eyes were examined immunohistochemically. For this purpose, the formalin-fixed rat retinal tissues in 4 µm thicknesses were deparaffinised in xylene and
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dehydrated in descending series of ethanol. The tissue sections were treated with antigen retrieval in a microwave oven (700 Watts) buffered with citrate buffered solution (pH 6.0) for 7-8 minutes. Then, the tissue sections were left to cool down for 20 minutes at room
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temperature and rinsed with distilled water. Endogenous peroxidase activity was eliminated by the incubation of tissue sections for 10 minutes in 3% H2O2 (Merck, 1.08597) containing phosphate-buffered saline (PBS; 0.01 M; pH 7.4). Then, tissue specimens were washed and blocked by incubating specimens at room temperature with a blocking serum (Thermo Scientific, TA-125-UB) for 10 minutes. Afterwards, the tissue specimens were incubated with eNOS (RB-9279-P), iNOS (RB-1605-P) or nNOS (Invitrogen, 61-7000) antibodies overnight (18 hours) at 4°C. eNOS, iNOS, and nNOS, antibodies were diluted in a large volume of UltrAb Diluent (Lab Vision, TA-125-UD) at 1:100. After rinsing with PBS for three times, each for 5 minutes, the specimens were incubated at room temperature with 6
ACCEPTED MANUSCRIPT biotinylated goat anti-rabbit antibodies (Thermo Scientific, TP-125-BN) for 30 minutes. The specimens were rinsed with PBS and incubated with streptavidin peroxidase label reagent (Thermo Scientific, TS-125-HR) at room temperature in a humid chamber for 30 minutes. The coloured product was developed by incubation with AEC (3-amino-9-ethyl carbazole) substrate kit (Thermo Scientific, TA-004-HAC) for 10 minutes. The specimens were rinsed
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with distilled water, counter-stained with Mayer’s Haematoxylin (Thermo Scientific, TA125-MH) for 45 seconds and mounted in vision mount (Thermo Scientific, TA-060-UG). The reaction was observed with a Leica compound light microscope and photographed with Kameram 390 CU. Control procedures were performed on adjacent sections of the
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specimens. No immunolabelling was detected when the primary antibody was omitted or
2.6.
Electron microscopy
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replaced with PBS.
The retinal samples were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and kept overnight at 4 °C. The retinal samples were then post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer for 45 minutes. After washing in phosphate buffer, the samples were dehydrated with alcohol series followed by propylene oxide and embedded in
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araldite. Ultrathin sections were cut with a diamond knife on the ultra-microtome and mounted on copper grids. The contrast of ultrathin sections was enhanced with 5% uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). The sections were examined by
2.7.
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JEOL JEM 1011 (JEOL Ltd., Tokyo, Japan) transmission electron microscope.
Myograph experiments
Due to the narrow internal diameter (< 50µm), it is not practically easy to isolate rat
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retinal artery and to study the functional reactivity in an in vitro experimental setup. Thus, in order to evaluate the relaxing effects of rat retinas obtained from the control and vein cauterized eyes, we used bovine retinal artery and rat mesenteric artery as the detector tissues, which both have much larger internal diameters (250-350µm) and easy to handle in the myograph system. At the end of 8 weeks after the operation, rats that displayed a sustained increase in IOP were sacrificed. Then, the retinas of the control (left) and vein cauterized (right) eyes as well as the mesenteric arteries were carefully excised from rats and placed in cold Krebs Ringer-bicarbonate solution of the following composition (mM): NaCl, 118, KCl, 4.7, 7
ACCEPTED MANUSCRIPT KH2PO4, 1.2, NaHCO3, 25, MgSO4.7H2O, 1.2, CaCl2, 2.5, glucose, 10 and disodium EDTA, 0.026. In parallel, bovine eyes (Brown Swiss [Montafoner], either sex, 2-4 years of age) were freshly obtained from a local slaughterhouse and carried to the laboratory in ice-cold Krebs Ringer-bicarbonate solution. The second order of the rat mesenteric arteries from ileal track and the first branch of bovine central retinal arteries were carefully dissected under a
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stereomicroscope (Zeiss, Model 2000, Oberkochen, Germany). Thereafter, the isolated mesenteric and retinal arteries were cleaned of the surrounding tissues and mounted in a multi-chamber wire myograph system (Model 610M, DMT, Aarhus, Denmark). Two stainless steel wires 40 µm in diameter were treaded into the lumen of the arteries and then fixed to the mounting devices of the force transducer and the micrometer. The arteries were
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equilibrated for 1h at 37oC, gassed with 5%CO2+95%O2 (pH=7.4) and set to a normalized internal circumference L1 (0.9L100) in accordance to the passive wall tension–internal
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circumference relationship under a passive transmural pressure of 100mmHg. Normalized arteries were contracted twice with potassium chloride (K+; 100 mM) plus noradrenaline (NA; 100 µM) to check the viability and for standardization of the preparations. Each artery specimen was subjected to different experimental protocol and preparations which developed a tension less than 0.5 mN/mm were discarded. Functional endothelial and
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smooth muscle relaxation capacities of the arteries were checked by acetylcholine (Ach: 10 nM-100 µM) and the nitrovasodilator, sodium nitroprusside (SNP: 10 nM-100 µM) respectively, on prostaglandin F2α (PGF2α: 30 µM) precontracted arteries. In a group of experiments, the retinal and mesenteric arteries were deendothelized mechanically by
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forcing a piece of hair into the lumen of the artery. Removal of endothelium was checked by the absence of relaxation to Ach (100 µM) on PGF2α (30µM) precontracted arteries.
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The relaxing effects of rat retinas obtained from the control (left) and vein cauterized (right) eyes were investigated concurrently on rat mesenteric and bovine retinal arteries that mounted parallel in the myograph chambers and precontracted with either PGF2α (30 µM) or K+ (100 mM). Retinal relaxation was determined by placing of the retinal tissue (235.45±4.32 mm2; n=28) on top of the precontracted arteries within close proximity, as previously described in detail (Boussery et al., 2002; Delaey and Van de Voorde, 1998; Takir et al., 2011; 2015). The relaxing responses of rat retinal tissues from both the control and vein cauterized eyes were determined on isolated retinal and mesenteric arteries in a consecutive manner. In time-match control experiments, we defined that the contractions
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ACCEPTED MANUSCRIPT induced by PGF2α (30 µM) or K+ (100 mM) were stable enough for the period required to complete the retina derived relaxations. The possible role of endothelial vasodilators, namely, nitric oxide (NO) and prostaglandins, in the relaxant effects of the retinal tissues from the normal and vein cauterized eyes were ascertained on isolated mesenteric and retinal arteries that removed of
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endothelium or pretreated with the combination of the known effective concentrations of NO synthase inhibitor, L-NOARG (100µM), guanylate cyclase inhibitor, ODQ (10µM) and cyclooxygenase inhibitor, indomethacin (10µM) for 30 minutes (Takir et al., 2011).
Chemicals
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2.8.
All drugs used in the experiments were purchased from Sigma Chemical Co. (Taufkirchen, Germany) except for ODQ (Tocris, Bristol, United Kingdom). The stock
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solution of ODQ was prepared in dimethyl sulfoxide (DMSO) whereas, indomethacin in 5% (w/v) sodium bicarbonate solution. Noradrenaline was dissolved in 0,001N hydrochloric acid and ascorbic acid (1mg/ml) was added to prevent oxidation. All other drugs were dissolved in distilled water. DMSO concentration achieved in the myograph chamber was 0.1% (v/v) at the highest and determined to have no direct influence on the contractile or
2.9.
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relaxant responses.
Statistical analyses
Data are presented as “mean±s.e.m” while, “n” is the number of retinas or the
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arteries (mesenteric and retinal) used in the experiments. The contractile responses to each spasmogen were expressed as “mN/mm” whereas, the relaxant responses to Ach, SNP and the retinal tissue were expressed as percent decreases of the precontractile tone. Emax was
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indicated as the maximum relaxations to vasodilators and the retinal tissue in the experiments. Statistical analyses were determined by Student’s paired and unpaired t-tests using GraphPad Prism (GraphPad Prism Version 5.00 Software Program, San Diego, CA). A “p” value less than “0.05” was considered statistically significant.
3. Results 3.1.
Cauterization of episcleral veins induced a sustained increase in IOP
Cauterization of three episcleral veins simultaneously in the right eyes of rats rapidly increased IOP whereas, in sham-operated left eyes, the IOP was unchanged (before 9
ACCEPTED MANUSCRIPT cauterization, IOP: 16.73±0.69 mmHg vs after cauterization IOP: 34.11±1.25 mmHg, n=42, p0.05, respectively). This significant increase in the IOP of the cauterized eyes was also verified on the 1st
day after the operation (cauterized eyes:
29.47±1.36 mmHg, vs control eyes: 18.85±0.86 mmHg, p0.05).
Placing the retinas of sham-operated (control) and vein cauterized eyes
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consecutively, on top of PGF2α (30 µM) precontracted rat mesenteric arteries produced rapid and stable relaxations. The maximal relaxations achieved by the retinas of the control and vein cauterized eyes were comparable (Retina of control eye, Emax: 78.74±2.92% and Retina
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of vein cauterized eye, Emax: 80.67±3.69%, n=10; p>0.05). Similar results were obtained when the retinas were placed on isolated bovine retinal arteries that precontracted with PGF2α (30 µM) (Retina of control eye, Emax: 82.35±9.63% and Retina of vein cauterized eye, Emax: 87.12±2.63%, n=10; p>0.05) (Fig. 6A).
On the other hand, moderate relaxations were determined when the retinal tissues
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were placed on K+ (100mM) precontracted mesenteric (Retina of control eye, Emax: 24.92±2.63% and Retina of vein cauterized eye, Emax: 31.60±5.54%, n=7; p>0.05) and retinal arteries (Retina of control eye, Emax: 49.55±6.25% and Retina of vein cauterized eye, Emax: 43.97±6.05%, n=6; p>0.05), which displayed similarity in between the control and
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IOP elevated groups (Fig. 6B).
Reversing the placing order of the retinas from the control and vein cauterized eyes
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on the precontracted mesenteric and retinal arteries did not induce any significant difference in the retinal relaxation responses (Fig. 6C, 6D).
3.7. The effects of deendothelization and pretreatment with the inhibitors of endothelial vasodilators on the relaxation induced by retina Incubation of rat mesenteric and bovine retinal arteries with the combination of L-
NOARG, ODQ and indomethacin for 20 minutes did not significantly modify the relaxations to retinas obtained from the control or vein cauterized eyes. Comparably, removal of the endothelium in both of the detector arteries did not significantly change the relaxing effects of the retinas from the control and vein cauterized eyes (Fig. 7). 12
ACCEPTED MANUSCRIPT 4. Discussion In the present study we aimed to identify the possible disturbances in rat retina, which subjected to chronic elevation of IOP due to the cauterization of episcleral veins, by analyzing the morphological and functional properties. We determined prominent structural
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changes, marked apoptosis and an increased immunoreactivity for all NOS isoforms, which displayed a diverse intensity within different layers, in the retinas of the vein cauterized eyes in comparison with that of control retinas from sham-operated eyes. While, the functional relaxing effects of the retinas (i.e. RRF) from the vein cauterized eyes on isolated bovine
retinas from sham-operated (control) eyes.
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retinal and rat mesenteric arteries were found comparable to the relaxations elicited by the
Elevated IOP is the most common clinical feature for the primary open angle
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glaucoma. Various animal models have been developed to ascertain the underlying pathophysiology of this ocular disease. Among these, episcleral vein cauterization is a well confirmed and widely used model of glaucoma in rats (Bai et al., 2014), which targets to increase the IOP and RGC death, similar as in human glaucoma, via obstructing the uveoscleral outflow of aqueous humour in the anterior chamber of the eye (Shareef et al.,
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1995) and also partially via vascular congestion (Grodzanic et al., 2003, Pang and Clark 2007). In this context, cauterization of three of episcleral veins simultaneously in rat rapidly increased the IOP in the right eye, compared to the sham-operated left eye, and sustained elevated for the following 8 weeks period (Fig.1). In parallel to the significant increase in
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IOP, the histopathological and ultrastructural deteriorations observed in the retina also supported the reliable induction of experimental glaucoma in rats. Regarding to this, in the light microscopy analysis, an apparent damage was detected in the ganglion and
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photoreceptor cell layers of the retinas obtained from eyes with increased IOP (Fig. 2). Likewise, in the electron microscopic analysis, irregularities in the photoreceptor cell layer were determined in the retinas of the eyes with increased IOP. This data reasonably suggested the loss of photoreceptor cell layer in the retina due to the elevation of IOP. Indeed, the damage on the RPE cell layer, which is known to be important for the local homeostasis and maintenance of the extra-photoreceptor matrix in the retina, may explain the deficiency in photoreceptor cell layer (Zigler et al., 2011). Thus, the decrease in the amount of RPE cells could be associated with the malnutrition of photoreceptor cell layer which in turn induces cell loss (Panda and Joanas, 1992). Notably, the loss of ganglion cells 13
ACCEPTED MANUSCRIPT and their axons due to the damage of inner retinal layers have been documented in various glaucoma models by use of conventional histological methods and apoptotic markers (Chen et al., 2011; Guo et al., 2005; Quigley et al., 1995; Shareef et al., 1995). However, the issue of whether the photoreceptor cells are also affected additional to the inner retinal neurons is critical and still under debate. Several studies have provided a clear evidence for the death of
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RGC and their axons, whereas, no other neurons were visibly affected (Chen et al., 2011; Guo et al., 2005; Quigley et al., 1995; Shareef et al., 1995). In contrary to the aforementioned studies, Bai et al. (2014) suggested that high IOP is not a condition that only damages the ganglionic cell layer (GCL), but also harmful to the whole retina. Likewise,
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Wang et al. (2002) reported that glaucoma damages both the ganglion cells and some of the neurons in the inner nuclear cell layer of the retina. It was also reported that TUNEL positive neurons were observed not only in the GCL, but also in the inner and outer nuclear
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cell layers of the glaucomatous retina even at 7th day after the cauterization of the veins (Wang et al., 2000). In the present study, the results of TUNEL assay, which stains apoptotic cells, showed an increased staining in the ganglion as well as in the inner and outer nuclear cell layers of the retina obtained from the vein cauterized eyes (Fig. 2D).This suggested that the elevation of IOP not only influences GCL but also affects the inner and outer nuclear cell
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layers of the retina. Accordingly, the apoptotic index was found significantly higher in the retinas of IOP-elevated eyes than the control (sham-operated) eyes. Moreover, supporting the findings of TUNEL assay, the electron microscopy analysis clearly demonstrated the formation of the phagosomes in the inner nuclear and ganglion cell layers of the retinas of
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IOP-elevated eyes (Fig. 4 and 5).
The activation of NOS has been reported as another potential mechanism of the damage induced by glaucoma in the retina and optic nerve (Ghanem et al., 2011; Grieshaber
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and Flammer, 2005; Venkataraman et al., 2010). Actually, NO regulates the baseline ocular blood flow rate in response to physiological stress and so, the suppression of NO production reduces the choroidal blood flow (Grieshaber and Flammer, 2005; Venkataraman et al., 2010). However, activation of NOS also plays an important role in the formation of free radicals, and in fact, glaucoma induced neuropathy is evidently considered as a type of oxidative stress. In a rat model of glaucoma, where the IOP was elevated by thermal blockade of the perilimbal/episcleral drainage vessels and anterior angle via the laser irradiation, NO levels were found to be increased in the retina, suggesting the activation of NOS-2 isozyme (Siu et al., 2002). In other study, the total NO level was also found 14
ACCEPTED MANUSCRIPT consistently high in the retina of IOP-elevated eyes and thereby, the retina was suggested to be at risk for a significant oxidative damage (Siu et al., 1998). In the present study, we observed a weak immunoreactivity for all NOS isoforms (eNOS, nNOS and iNOS) in the ganglion and inner plexiform layers of the control retina (Fig.3). However, in the retinas obtained from IOP-elevated eyes, a strong immunolabeling was determined for eNOS;
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especially in the inner plexiform and ganglion cell layers, also for iNOS; in the ganglion, inner nuclear and photoreceptor cell layers as well as for nNOS; in the ganglion, inner plexiform and inner nuclear cell layers (Fig. 3B, 3D, 3F). In fact, this immunohistochemical data is in line with the findings obtained in histopathological analysis, where the
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degeneration of retina due to the elevation of IOP was reflected especially on the ganglion, inner nuclear and photoreceptor cell layers. Indeed, the increased immunoreactivity of NOS may correspond to the excess NO production within the retina, and thereby, support the
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aforementioned studies implicating the role of NO in retinal damage due to the elevation of IOP. Thus, the excess NO formation might possibly initiated the degeneration within different layers of retina in this experimental glaucoma model consistent with a previous study by Neufeld et al. (1999) which implicated a pathological role for NO in the RGC damage induced by the cauterization of episcleral vein. Accordingly, a significant decrease
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in the rate of RGC death was defined after the administration of iNOS inhibitor, aminoguanidine to rats (Neufeld et al. 1999). In relation, iNOS could be a reliable candidate for the abundant formation of NO to initiate the degeneration in photoreceptor cell layer. Retinal circulation is maintained locally by the vasoactive substances released from
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the retina as well as the endothelium of the retinal arteries (Grieshaber and Flammer, 2005; Venkataraman et al., 2010). Among these, a novel mediator, namely, retina derived relaxing factor (RRF) was suggested to contribute to the modulation of the retinal arterial tone, by
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decreasing the contraction to spasmogens and producing a substantial relaxation on the underlying retinal artery (Boussery et al., 2002; Delaey and Van de Voorde, 1998; Takir et al., 2011; 2015). However, the effectiveness of RRF in a pathological condition of the eye affecting the retina and/or the retinal circulation is still unknown. This is the first study that aimed to investigate the possible influences of disease conditions, in particular related to eye, on the vasorelaxing effect of this putative retinal factor. In parallel to previous in vitro studies (Boussery et al., 2002; Delaey and Van de Voorde, 1998; Takir et al., 2011; 2015), placing the retinal tissue on top of PGF2α precontracted bovine retinal or rat mesenteric arteries produced rapidly occurring, complete relaxations. The relaxation response was 15
ACCEPTED MANUSCRIPT reversible upon removing the retinal tissue away from the artery and generally displayed a biphasic characteristic, similar as previously observed (Takir et al., 2011). Indeed, this substantial relaxation response of the retina determined in the mesenteric artery supports the suggestion that RRF could be a more general relaxing substance (Maenhaut et al., 2007). Herein, the induction of experimental glaucoma in rats by increasing the IOP via
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cauterization of episcleral veins did not alter the maximum and the biphasic profile of the relaxing response to retina, i.e.,RRF, on PGF2α precontracted retinal and mesenteric arteries, when compared to the relaxant responses of retinas obtained from control (sham-operated) eyes (Fig. 6). In parallel, the retinas excised from IOP-elevated and control (sham-operated)
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eyes also displayed similar relaxations on K+ precontracted arteries, though, the maximum of retinal relaxations were much lower than that achieved in the presence of PGF2α. Indeed, the modest relaxation response to RRF was previously indicated when K+ was used as the
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contractile agent (Delaey and Van de Voorde, 1998; Takir et al., 2011; 2015). Taken together, we determined that the relaxation response to retina, i.e., RRF, that obtained from IOP-elevated eyes is unchanged on either retinal or mesenteric vasculature, when compared to the retinal responses of control (sham-operated) eyes in this rat model. The neural retina is known to release various molecules of which some of have vasoactive properties. In
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previous studies these substances were evaluated as potential candidates for being RRF. Among these, the neurotransmitters; glutamate and glycine, which are well known to be present in retina, have taken the initial attention (Delaey and Van de Voorde, 1998). Notably, glutamate excitotoxicity was recently implicated in glaucoma and the blockade of
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NMDA receptors was shown to prevent ONH astrocyte dysfunction in glaucomatous neurodegeneration (Ju et al., 2015). However, glutamate, and glycine were shown to be unrelated to RRF (Delaey and Van de Voorde, 1998). Furthermore, adenosine, which was
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determined to reduce vascular resistance, increase the blood flow in retina and optic nerve head, and decrease the RGC death (Zhong et al., 2013), was also excluded from being the RRF (Delaey and Van de Voorde, 1998; Maenhaut et al., 2009) although, it was shown to potentiate the relaxing response of RRF (Maenhaut et al., 2009). Actually, the inhibition of neurotransmitter release from the retinal neurons by the use of tetradotoxin unchanged the relaxing response to retina and thus, suggested that RRF is released from the glial cells rather than neuronal cells in the retina (Delaey and Van de Voorde, 1998). Indeed, one may suggest that the unchanged relaxing effect of RRF derived from the retina of IOP-elevated eyes might be related to a continuous release from the glial cells which merits further 16
ACCEPTED MANUSCRIPT investigation. However, the unknown origin of the cell type releasing RRF currently limits the evaluation of its potential importance in glaucoma. Endothelium derived vasodilator substances, in particular, NO (Grieshaber and Flammer, 2005; Venkataraman et al., 2010) and prostaglandins (Nielsen and Nyborg, 1990) are known to contribute to the regulation of retinal arterial tone. In the present study, neither
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the presence of the inhibitors (L-NOARG, ODQ and indomethacin) of endothelial vasodilators nor the removal of endothelium significantly influenced the RRF response derived from the retinas of normal and IOP-elevated eyes on isolated bovine retinal and rat mesenteric arteries. This data is in line with that of previous studies documenting the
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relaxing characteristics of retinal tissue from different species (Boussery et al., 2002; Delaey and Van de Voorde, 1998; Takir et al., 2011; 2015). Reinforcing the lack of a role for NO, retinas obtained from nNOS knockout mice were reported to display similar relaxation
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responses when compared to the retinas obtained from wild type mice (Boussery et al., 2002). The current findings furtherly supported that the possible candidates, NO and vasodilator prostanoids are unlikely to mediate the RRF response even that of originated from the retinas of IOP–elevated eyes.
As it is known, studies conducted in animals give very valuable information to
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understand the related conditions in humans. However, considering the differences in anatomical and physiological properties it is reasonable to suggest that findings in rodents do not necessarily fit to humans. Notably, it is previously documented that rat eyes display various structural and functional differences from human eyes (Brown, 1965) Thus, the
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characteristics and action mechanism of retina derived relaxing factor, i.e. RRF, particularly on the underlying retinal artery, could be different in the normal and pathological conditions of the human eye. This is an important limitation of the current research which merits further
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investigation. However, as the availability of human eye tissues is very limited, the rat model of elevated IOP could be a promising option in determining the pathological and functional changes in the retinal tissue due to glaucoma.
5. Conclusion Experimental glaucoma induced by the elevation of IOP via the cauterization of episcleral veins in rats, produced prominent histopathological and ultrastructural changes particularly in the ganglion, inner and photoreceptor cell layers, evoked marked apoptosis and an increased the immunoreactivity for all NOS isoforms (eNOS, nNOS and iNOS) in 17
ACCEPTED MANUSCRIPT the retina. On the other hand, the vascular effects of retina derived relaxing factor, i.e., RRF, remain unchanged in this experimental glaucoma model. These results suggest that the chronic elevation of IOP would lead to an apparent damage in the retina particularly at the cellular level which possibly involves apoptosis and presence of excess NO. Whereas, considering the response to RRF, the functional regulatory role of retina particularly on the
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retinal vasculature is likely to be preserved in IOP-elevated eyes. In fact, this could be due to the lack of a detrimental influence on the retinal cells that is responsible for the release of RRF, an issue which is already unknown. Of note, RRF has yet only been demonstrated on isolated dissected tissues, and thus, its physiological significance in vivo is not evident at
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this stage. Accordingly, the importance of RRF, particularly in the paracrine regulation of retinal circulation, could be reliably understood when its chemical structure is identified, the mechanism of synthesis is specified, and a specific blocker is determined. Current findings
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can be considered as a kind of indirect indication that there is some physiological role to it, while remain to be established in future research.
6. Acknowledgements
Part of this study was presented in 22nd National Congress of Pharmacology (Turkish
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Pharmacological Society) Antalya/Rixos Hotel 4-7 November 2013. This study was supported by Istanbul University, Scientific Research Projects Unit under grant number 10608. The authors thank to G. Zeynep Ortaköylü for her excellent assistance.
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7. Conflict of interest
The authors declare that there is no conflict of interest.
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Figure 1: Intraocular pressure (IOP) values measured in the control (left) and episcleral veins cauterized (right) eyes of the rats. As presented, IOP measurements were performed by
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tonopen on the 1st ,4th and 7th days after the cauterization or sham-operation process and then, once a week for the following 8 weeks (56 days) period. * p0.05, compared to corresponding controls.
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Figure 7: Bar graphics showing the maximum relaxant effects of the retinas obtained from the control and vein cauterized eyes in PGF2α(30 µM) precontracted bovine retinal and rat mesenteric arteries that were either pretreated with the combination of inhibitors, namely, NO synthase inhibitor, L-NOARG (100 µM), guanylate cyclase inhibitor, ODQ (10 µM), and cyclooxygenase inhibitor, indomethacin (10 µM) for 20 minutes or deendothelized.
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n= 6-8; p>0.05 compared to corresponding controls. (-) inhibitors: in the absence of inhibitor
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combination, (+) inhibitors: in the presence of inhibitor combination.
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ACCEPTED MANUSCRIPT Highlights • Cauterization of episcleral veins of rat eyes induced sustained increase in intraocular pressure (IOP). • Morphological deteriorations were determined in the ganglion and outer nuclear cell
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layers of the glaucomatous retinas. • Increase in IOP induced apoptosis and NOS immunoreactivity in inner layers of retina. • The relaxing influence of retina was similar both in control and glaucomatous retina.
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• Elevation in IOP induces structural damages prominently in the ganglion and inner layers of the retina that is associated with an increase in NOS immunoreactivity while,
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the responsiveness to RRF was unaffected.
