Experimental animal models of induced intraocular hypertension

2014/2015 Miguel Trigo Coimbra Experimental animal models of induced intraocular hypertension março, 2015 Mestrado Integrado em Medicina Área: Fisiologia Tipologia: Monografia Trabalho efetuado sob a Orientação de: Doutor Amândio António Rocha Dias de Sousa Trabalho organizado de acordo com as normas da revista: Experimental Eye Research Miguel Trigo Coimbra Experimental animal models of induced intraocular hypertension março, 2015 Review Experimental animal models of induced intraocular hypertension M. Coimbra a,*, A. Moleiro a, A. Rocha-Sousa a,b a Laboratory of Physiology, Faculty of Medicine, University of Porto, Portugal b Department of Senses Organs, Faculty of Medicine, University of Porto, Portugal * Correspondence: Miguel Trigo Coimbra, Department of Physiology and Cardiothoracic Surgery, Faculty of Medicine, University of Porto Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal. Telephone: +351 924124243 E-mail: [email protected] 2 Abstract As the ethiologic and pathologic processes of glaucoma disease remain largely unknown, the benefit of animal models that effectively mimic the condition is crucial, and therefore they are increasing in popularity. The aim of this paper is to provide a descriptive summary for each of the included intraocular hypertensive methods and the results obtained, along with some of the advantages and limitations considering the models used. A systematic revision of studies published between 1957 and 2014 in English in MEDLINE was performed. Search words used included: intraocular pressure, glaucoma, animal, model, laser photocoagulation, trabecular meshwork, episcleral vein, cauterization, ligation, microsphere, microbead, glycosaminoglycan, cellulose, steroid, cannulation, Morrison, monkey, rat, rabbit, cat, mouse, pig. The models described promote impairment in the “conventional” aqueous humor drainage pathway with elevation of intraocular pressure, one major risk factor for glaucoma. The intraocular hypertensive mechanism was classified as pre-trabecular, if it simulates excessive aqueous humor inflow, or post-trabecular if a decreased aqueous humor outflow is obtained downstream from the trabecular meshwork, associated with vein congestion. Trabecular mechanisms induce obstruction or destruction of the trabecular meshwork itself. Many efficient and reproducible methods were performed in monkeys, rats, mice, rabbits, cats, pigs, sheep, bovine and ferrets. Induced intraocular hypertension animal models are very promising in terms of a better understanding of the pathophysiologic processes of glaucoma, as well as in the discovery of novel therapies for this condition. Keywords: Glaucoma, intraocular pressure, animal models. 3 1. Introduction Elevated intraocular pressure (IOP) is nowadays accepted as the only known treatable major risk factor for glaucoma. It has been shown to affect the prevalence, incidence and development of this condition (Paulavičiūtė-Baikštienė et al., 2013), even though the underlying pathophysiologic mechanisms still remain uncertain (Wang et al., 2012). Nevertheless, changes in human glaucomatous eyes have been recently described in the trabecular meshwork, including excessive production of extracellular matrix by increased TGF-β2, impaired phagocytosis and increased contractility of trabecular meshwork cells. These changes lead to partial obliteration of the spaces between collagen beams, increasing the resistance to aqueous outflow, and ultimately increasing IOP (Paulavičiūtė-Baikštienė et al., 2013). Animal models over the decades have proven to be crucial to understanding numerous diseases. Indeed, in terms of glaucoma research, a wide variety of animals of different species have been employed, and researchers took into account the animal size, cost and the amount of morphologic and physiologic overlap with human eye characteristics. The intraocular hypertensive animal models considered to be more suitable to study glaucoma neuropathy should induce sustained moderately high IOP levels during a reasonable period. These models allow examination of both the precise onset and the progression of pathological changes of glaucomatous disease in a controlled and reproducible way (Vecino and Sharma, 2011). The pathophysiologic changes observed in the retinal ganglion cell (RGC) layer should be similar to those in human individuals, and optic disk cupping should be observed. They are also important to evaluate novel therapies in an organized manner (Samsel et al., 2011). Genetic animal models of glaucoma impose practical difficulties to many researchers worldwide, including availability and cost, and they were not included in this review. Normal tension glaucoma models were also not included. 2. Methods A systematic revision of studies published between 1957 and 2014 in English in MEDLINE was performed. Search words used included: intraocular pressure, glaucoma, animal, model, laser photocoagulation, trabecular meshwork, episcleral vein, cauterization, ligation, microsphere, microbead, glycosaminoglycan, cellulose, steroid, cannulation, Morrison, monkey, rat, rabbit, cat, mouse, pig. 4 3. The aqueous humor outflow pathway In humans, the aqueous humor is produced by the ciliary epithelium at the ciliary processes in the pars plicata of the ciliary body, and is secreted into the posterior chamber. It flows between the lens and the iris, through the pupil and into the anterior chamber. From the anterior chamber angle, the aqueous humor is drained by a “conventional” pathway through the trabecular meshwork and into the Schlemm’s canal, draining collector channels, radial aqueous veins and episcleral veins by this order. However, it can also be drained by a “nonconventional” pathway through the ciliary muscle anteriorly, uveal meshwork, suprachoroidal space and finally the sclera. Some authors consider this pathway to be analogous to lymphatic drainage (Johnson and Erickson, 2000), it decreases with age and is responsible for approximately 40 to 50% of aqueous humor drainage. However, it is independent from IOP level variation, and when aqueous humor production surpasses its drainage through the “conventional” pathway, an elevation of IOP occurs (Goel et al., 2010). The models described in this section are aimed at artificially impairing the “conventional” aqueous humor drainage pathway with elevation of IOP levels. For reasons of convenience, we decided to categorize the models according to the site of intervention mechanism relatively to the trabecular meshwork, as listed in table 1. 3.1. Chymotrypsin Injection The long-term ocular hypertension induced by alpha-chymotrypsin is believed to result from an increased aqueous humor inflow by disruption of the blood-aqueous barrier, rather than trabecular blockage by lysed zonular material (Melena et al. 1999; Chee and Hamasaki, 1971; Anderson, 1971). 3.1.1. Monkey Several authors described cupping of the optic disk and atrophy of the optic nerve as early as 3 days of sustained rise in IOP after injecting 75 and 750 units (respectively) of chymoptrypsin in the posterior chamber of monkeys (Kalvin et al., 1966; Lessell and Kuwabara, 1969). However, the subjects with higher doses of chymotrypsin often underwent corneal perforation. 3.1.2. Rabbit Based on many authors (Sears and Sears, 1974; Gelatt, 1977; Vareilles et al., 1979), Fernandez-Durango et al. (1991) injected unilaterally 0.86 mg of α-chymotrypsin diluted in 0.13 mL of saline solution into the posterior chamber of New Zealand albino rabbits. They found increased IOP as soon as 3 days in 82 % of the animals, which maintained during 40 days, with mean values of 28.4 mmHg in treated eyes compared to 13.1 mmHg in contralateral control 5 eyes. This is a well-established chronic intraocular hypertensive model, with relevant similarities to human glaucoma (Percicot et al., 1996). 3.2. Manometric Intraocular cannulation 3.2.1. Monkey Anderson and Hendrickson (1974) originally describe a method in which they manometrically control the IOP in owl monkeys. A 27 gauge needle is inserted obliquely through the peripheral cornea and into the posterior chamber. It is connected to an external saline container, which content is varied according to the systemic blood pressure, measured by femoral artery cannulation, in order to maintain a constant perfusion pressure (PP), measured as the difference between mean arterial pressure and IOP, during eight hours. The authors observed that at slightly elevated levels of IOP (PP of 60 mmHg) there was a partial obstruction of axoplasmic transport, which became more obvious at moderate levels of IOP (PP of 35 to 45 mmHg). Severe obstruction to axonal transport was achieved at higher IOP levels (PP = 25 mmHg) with greater axonal dilation. However, with a PP under 25 mmHg, there was a complete absence of axoplasmic transport in the ganglion cells. According to Anderson and Davis (1975), it is at a perfusion pressure under 15 mmHg that most permanent retinal changes occur, namely partial necrosis of iris, stroma and ciliary processes, macroscopic lesions around the disc and in the retinal periphery, possibly due to a nonischemic pressure-induced mechanism in chronic glaucoma. 3.2.2. Cat In adult cats Grehn et al. (1984) inserted a Ringer solution injecting cannula (20 U/mL) and a pressure transducing cannula in the anterior chamber through the limbus. The perfusion pressure was calculated as the difference between mean blood pressure and IOP. The authors confirmed that PP is the main ischemic factor, since animals with higher mean blood pressure also tolerate higher IOP levels, and they observed that total suppression of retinal neurons transmission is only observed when PP reaches critical levels below 20 mmHg in cats. The authors also mention that short-term IOP elevation by cannulation at critical PP levels preferentially causes functional retinal impairment by anoxia and can be reversible up to 100 minutes, whereas a long-term approach with moderate PP levels (50 mmHg) causes mechanically induced optic disk excavation. 12 3.8.1. Rabbit Bonomi et al. (1978) believed that the chronic effect of high local corticosteroid doses spreading systemically caused organic side effects associated with heavy weight loss, considerable IOP fluctuations and often death to the subjects. Therefore, they proposed a model of 3 unilateral subconjunctival injections in rabbits, once per week, with 4mg of a repository betamethasone preparation containing hydroxiethylcellulose. From baseline IOP levels of 18 mmHg, a mean peak of 27 mmHg was reached at the third week in 96% of the animals, but the contralateral control eye also reached a peak of 22 mmHg. Nevertheless, significant elevated IOP levels were maintained until 5 weeks. An improved mortality rate of 3.12% was also described. Knepper et al. (1978), however, have witnessed differently. With 1 drop of topical dexamethasone at 0.1% every 6 hours, they concluded that at 4 weeks only young rabbits aged between 8 and 10 weeks were responsive, possibly due to an age-related difference in the ratio of keratan sulfate to uranic acid containing glycosaminoglycans in the trabecular meshwork. It represents a clear contrast with human glaucoma, which is progressively induced, preferably in older individuals. 3.8.2. Cat Zhan et al. (1992) applied a 10 L solution of dexamethasone sodium phosphate (at 1.0%) to the cornea 3 times daily during a month to normotensive cats. However, only a group of cats which underwent an interval of 7 days without treatment and repeated the dexamethasone sodium phosphate (1%) twice daily for another month showed a fairly significant IOP elevation. When applying a solution containing 10 L of 1% prednisolone acetate topically twice a day with a 3 to 5 minute interval to ocular normotensive cats, a slight but significant increase in IOP was observed after 22 days and up to 60 days. Increasing the frequency of administration did not further significantly increase the IOP. The authors emphasize that the feline eye resembles the human eye at a greater extent in terms of intraocular corticosteroid target sites than the rabbit eye. 3.8.3. Rat Young Wistar rats were given topical ocular dexamethasone 4 times daily for 4 weeks (Sawaguchi et al., 2005). IOP was increased slightly but significantly since the second week of treatment. 13 3.8.4. Bovine Gerometta et al. (2004) provided 12 cows with unilateral topical ocular prednisolone acetate drops at 1% initially and 0.5% after the first week, 3 times daily during 49 days. There was a significant IOP variation from 7 to 15 mmHg between hypertensive and control eyes and up to 10 weeks. 3.8.5. Sheep 18 sheep received unilateral topical ocular prednisolone acetate (0.5%) drops 3 times daily during 4 weeks (Gerometta et al., 2009). All developed increased mean IOP values of 23 mmHg in the second week and 27.5 mmHg in the third week, compared to contralateral values of 11.2 to 11.7 mmHg. Therefore, they concluded that the results in sheep and bovine steroid models are more enticing than in other animals. 3.9. Laser Photocoagulation of limbal venous plexus and episcleral veins 3.9.1. Rat WoldeMussie et al. (2001) used blue-green Argon laser photocoagulation on the limbal venous plexus and episcleral veins within 0.5 to 0.8 mm from the limbus. 130 to 150 laser burns, with 0.05mm width, a power of 1 Watt and 0.2 seconds duration were used. A second treatment was performed 1 week later. An increase of 60% and 100% from baseline IOP values was accomplished after the first and second treatments, respectively, and maintained significantly high for 2 months. Considering a slowed RGC loss rate from 12% to 2% per week between the third week and two months, the authors defend a direct correlation with IOP levels during this period. 3.9.2. Mouse Identically, Gross et al. (2003) described a laser photocoagulation model for C57BL/6J mice. Episcleral and limbal veins were photocoagulated within 1 mm from the limbus, using a laser with 50 µm width, 0.1 seconds duration and a power between 80 and 110 mW. At 4 weeks, 90% had a mean IOP value of 20 mmHg versus baseline values of 13 mmHg, and a RGC loss of 22.4%. However, the C57BL/6J mouse strain used develops anterior segment anatomic abnormalities that might interfere with IOP values. 3.10. Episcleral Vein Cauterization In this model, IOP elevation is likely to result from vein congestion and reduction of aqueous humor outflow combined (Naskar et al., 2002). 14 3.10.1. Rats Shareef et al. (1995) describe a model of unilateral cauterization of a variable number of deep episcleral veins. After dissecting the conjunctiva at the limbal periphery and exposing the extraocular muscles, a suture underneath would anchor them and reveal the adjacent deep episcleral veins. Once the vein is visually detached from the overlying conjunctiva and muscle, an ophthalmic cautery is applied to the episcleral trunk to fully interrupt the drainage (Shareef et al., 1995). Alternatively, Sharma (2003) argue that cauterization to the junction of aqueouscontaining radial veins and ciliary veins is also effective. Shareef et al. (1995) observed that if 2 or more veins were cauterized, a rise of at least 90% was observed in IOP. However, after 1 week, IOP returned to values slightly higher than baseline. In contrast, Naskar et al. (2002) cauterized 2 episcleral veins in adult Sprague-Dawley rats and observed a 1.6 fold increase in IOP levels up to 3 months. When Shareef et al. (1995) cauterized 3 veins, IOP increased from 13.2 mmHg to 53 mmHg, stabilizing at 29 mmHg after 2 months. Little to none decompensation symptoms were found in this group. When all four veins were cauterized, a stable raised IOP level of 60 mmHg was maintained until 1.5 weeks, but most eyes showed proptosis, exposure keratopathy, corneal edema and corneal endothelial malfunction (Shareef et al., 1995), and Sharma (2003) observed a high risk of necrosis of the eye within 1 week. This method is reproducible and excellent for the study of primary open angle glaucoma, as well as to test for possible neuroprotective agents (Naskar et al., 2002; Shareef et al., 1995; Sharma, 2003). However, practice is necessary (Sharma, 2003), since initial trials only led to a success rate of 25%, compared to 85% later on. They also observed that leaking from the cauterization site was associated with neovascularization in up to 15%, and hence they suggest ligating the vein before cauterization. 3.10.2. Pig Ruiz-Ederra et al. (2005) cauterized unilaterally 3 episcleral veins of adult pigs. Since the third week and during 21 weeks, an increased mean IOP value of 20.8 mmHg was witnessed (versus control levels of 15.6 mmHg). The significant RGC death in the mid-peripheral and peripheral retina observed resembles the human pattern of RGC degeneration in glaucoma. 3.10.3. Mouse According to Aihara et al. (2003) the mouse eye anatomy is too small and the outflow vessels form a thin plexus rather than largely visible episcleral veins. Moreover, the mouse sclera is too thin and a high incidence of perforation may result. In fact, Ruiz-Ederra and 15 Verkman (2006) excluded 12 out of 35 mice due to scleral complications and episcleral vein leakage from cauterization of 3 deep episcleral veins. Nonetheless, they achieved an IOP elevation success rate of 87% and a maximum peak of 28 mmHg up to nine days after the procedure. The results were variable, but 94% showed significantly increased IOP until 4 weeks. They also observed aqueous outflow resistance 2.5 times higher than in control eyes, and a RGC loss of 20%. 3.11. Episcleral Vein Ligation 3.11.1. Rats Yu et al. (2006) described a method of unilateral ligation of 3 deep episcleral veins after their exposure in the respective eye quadrants, by dissecting through the overlying conjunctiva and Tenon’s capsule. A 10-0 nylon was used. 40.8% of treated eyes had a mean IOP value of 24.7 mmHg for 7 months. In 59.2%, however, there was a decrease below 25 mmHg until 4 weeks, and another intervention with ligation of collateral neovasculature was performed. Optic disk escavation and a RGC loss of 1.3% weekly were also reported. 3.11.2 Rabbit As originally described by Huggert (1957), Zhu and Cai (1992) observed that the ligation of three vortex veins in adult pigmented rabbits induced a mild mean value of 27.1 mmHg (versus baseline levels of 19 mmHg) during approximately 11 days only. These results could be explained by collateral circulation and an enlargement up to three times higher of the unoperated vortex vein. Retinal hemorrhage was often found, while hyperemia and corneal edema were sometimes present. The authors concluded this method is not adequate for a chronically induced rise in IOP. 3.12. Saline Injection in Episcleral Veins 3.12.1. Rat The limbal vasculature, Schlemm’s canal and trabecular meshwork of the rat eye are mostly analogous to primates, and a “conventional route” in rats is identically responsible for only part of the aqueous humor outflow (Morrison et al., 1995). Morrison et al. (1997) induced sclerosis of episcleral veins in Brown Norway rats. After a lateral canthotomy and the dissection of the conjunctiva with exposure of one radial aqueous vein, 50 µL of hypertonic saline solution at 1.65 or 1.75 M are injected causing whitening of the vessel. If IOP fails to rise significantly until 2 weeks, a second cannulation is performed in an opposite radial aqueous vein. A success rate of 80% was achieved. IOP increase was variable 16 between 7 and 28 mmHg during 200 days. However, inflammation occurred often, and sometimes sclerosis of the trabecular meshwork with anterior synechia was visible at the iridocorneal angle. They witnessed an increased superior temporal susceptibility to axonal degeneration and a pressure-related deposition of extracellular matrix components in the optic nerve head fibers, both analogous to human glaucomatous eyes. 3.13. External oculopression 3.13.1. Rat Sellés-Navarro et al. (1996) described a mechanical method of increasing the IOP by external oculopression. In Sprague-Dawley rats, two 6-0 silk sutures were placed unilaterally around the bulbar conjunctiva on either sides of the corneoscleral limbus and were pulled on opposite directions. The eye was kept above systolic arterial pressure levels during the ischemic period. Interruption of blood flow duration varied from 30 to 120 minutes. During 30 days, only periods of transient ischemia longer than 45 minutes induced retinal RGC death, which occurred as early as 3 hours and became more severe with longer ischemic intervals. Periods of ischemia of at least 90 minutes cause the death of approximately 50% of the RGC population after 5 days and 95% after 30 days. Mild edema of the conjunctiva and cornea were commonly present, and sometimes retinal or vitreous hemorrhages were also found. 3.14. Laser Photocoagulation of trabecular meshwork and episcleral veins 3.14.1. Rat In Wistar rats, Levkovitch-Verbin et al. (2002) combined 60 to 80 unilateral laser deliveries to the trabecular meshwork, mostly through the cornea, and also 15 to 20 laser spots to episcleral veins. Using a diode laser at 532 nm wave-length through a slit-lamp mechanism, they determined that a laser power of 0.4 Watt with 0.2 second duration is more efficient, witnessing a mean IOP of 25.5 mmHg versus 19.8 mmHg in control eyes during 9 weeks. Since 75% of the animals returned to baseline IOP levels mostly after 3 weeks, they required another intervention. The authors argue that the extent of RGC loss in the retina is associated with higher IOP levels. They concluded that their model is still expensive, yet reproducible, relatively simple to perform and has many similarities with human glaucoma. 17 4. Conclusion This paper is an attempt to summarize most intraocular hypertension animal models currently used by the scientific community, especially for a better insight into glaucoma processes (Table 1). Recent investigation in glaucoma therapy is directed to the risk factors instead of the etiology and pathophysiology of the disease, and current clinical practice focuses on the reduction of aqueous humor formation or increasing the outflow, and thus lowering IOP (Paulavičiūtė-Baikštienė et al., 2013; Cioffi,2011). As such, establishing an effective and reproducible induced intraocular hypertension animal model is essential. Not forgetting that the pathophysiologic changes witnessed in these models should ideally be similar to those found in humans, comparison must be careful and generalizing from a certain animal and method should be avoided. In the meanwhile, as more technology is being developed in this field, we hope to achieve considerable progresses in prevention as well as treatment of glaucoma. Level of aqueous flow modification Procedure Animal Author Pre-trabecular a Chymotrypsin Monkey Kalvin et al. (1966); Lessell and Kuwabara (1969) Rabbit Fernandez-Durango et al. (1991); Percicot et al. (1996) Manometric Intraocular Cannulation Monkey Anderson and Hendrickson (1974) Anderson and Davis (1975) Cat Grehn et al. (1984) Rat Büchi et al. (1991) Trabecular b Laser Photocoagulation of trabecular meshwork Monkey Gaasterland and Kupfer (1974) Quigley and Hohman (1983) Rat Ueda et al. (1998) Mouse Aihara et al. (2003) Mabuchi et al. (2003) Latex Microspheres Monkey Weber and Zelenak (2001) Polystyrene Microspheres Rat Sappington et al. (2010) Mouse Sappington et al. (2010) Cone et al. (2012) Magnetic microspheres Rat Samsel et al. (2011) Conjunctival Cells Ferret Fujishiro et al. (2014) Glycosaminoglycans Monkey Schubert et al. (1984) Rabbit Harooni et al. (1998) Equi et al. (1997) Rat Benozzi et al. (2002) Cellulose derivatives Rabbit Zhu and Cai (1992) Törngren et al. (2000) Corticosteroids Rabbit Bonomi et al. (1978) Knepper et al. (1978) Cat Zhan et al. (1992) Rat Sawaguchi et al. (2005) Bovine Gerometta et al. (2004) Sheep Gerometta et al. (2009) 18 Post-trabecular c Laser Photocoagulation of limbal venous plexus and episcleral veins Rat WoldeMussie et al. (2001) Mouse Gross et al. (2003) Episcleral Vein Cauterization Rat Shareef et al. (1995) Naskar et al. (2002) Sharma (2003) Pig Ruiz-Ederra et al. (2005) Mouse Ruiz-Ederra and Verkman (2006) Episcleral Vein Ligation Rat Yu et al. (2006) Rabbit Huggert (1957) Zhu and Cai (1992) Saline Injection in Episcleral Veins Rat Morrison et al. (1997) External oculopression Rat Sellés-Navarro et al. (1996) Trabecular b and Post-Trabecular c Laser Photocoagulation of trabecular meshwork and episcleral veins Rat Levkovitch-Verbin et al. (2002) Table 1: List of intraocular hypertensive animal models categorized according to the level at which the impairment in aqueous humor flow occurs. aPre-trabecular refers to models which simulate mostly an unbalanced increase in aqueous humor inflow. bTrabecular refers to models which induce obstruction or destruction of the trabecular meshwork. cPost-trabecular indicates models which preferentially decrease aqueous humor outflow through the “conventional” pathway downstream from the trabecular meshwork, causing vein congestion. 19 References Acott, T.S., Westcott, M., Passo, M.S., Van, Buskirk, E.M., 1985. Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye. Invest. Ophfhalmol. Vis. Sci. 26, 1320-q. Aihara, M., Lindsey, J.D., Weinreb, R.N., 2003. Experimental mouse ocular hypertension: establishment of the model. Invest. 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Johnson, M., Erickson, K., 2000. Mechanisms and routes of aqueous humor drainage, in: Albert, D.M., Jakobiec, F.A. (Eds.), Principles and Practice of Ophthalmology. WB Saunders, Philadelphia. Kalvin, N.H., Hamasaki, D.I., Gass, J.D., 1966. Experimental glaucoma in monkeys. I. Relationship between intraocular pressure and cupping of the optic disc and cavernous atrophy of the optic nerve. Arch. Ophthalmol. 76(1): 82-93. Kerrigan-Baumrind, L.A., Quigley, H.A., Pease, M.E., Kerrigan, D.F., Mitchell, R.S., 2000. Number of ganglion cells in glaucoma eyes compared with threshold visual field tests in the same persons. Invest. Ophthalmol. Vis. Sci. 41(3): 741–748. Knepper, P.A., Breen, M., Weinstein, H.G., Blacik, J.L., 1978. Intraocular pressure and glycosaminoglycan distribution in the rabbit eye: effect of age and dexamethasone. Exp. Eye Res. 27(5): 567-75. Knepper, P.A., Goossens, W., Palmberg, P.F., 1996. Glycosaminoglycan stratification of the juxtacanalicular tissue in normal and primary open-angle glaucoma. Invest. Ophthalmol. Vis. Sci. 37(12): 2414-25. Lanza, R., 2014. Principles of Tissue Engineering, fourth ed. Edited by Robert Lanza, Robert Langer, and Joseph Vacanti. Academic Press. Lessell, S., Kuwabara, T., 1969. Experimental alpha-chymotrypsin glaucoma. Arch. Ophthalmol. 81(6): 853-64. Levkovitch-Verbin, H., Quigley, H.A., Martin, K.R., Valenta, D., Baumrind, L.A., Pease, M.E., 2002. Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats. Invest. Ophthalmol. Vis. Sci. 43(2): 402-10. AUTHOR INFORMATION PACK 15 Mar 2015 www.elsevier.com/locate/yexer 2 ABSTRACTING AND INDEXING . BIOSIS Chemical Abstracts Current Contents/Life Sciences MEDLINE® EMBASE Research Alert Excerpta Medica Scopus EDITORIAL BOARD . Editor-in-Chief Joe Hollyfield, Cleveland Clinic Foundation, Cleveland, Ohio, USA Aqueous Humor and Blood Flow Section Editors Abbot Clark, University of North Texas, Fort Worth, Texas, USA Ernst Tamm, University of Regensburg, Regensburg, Germany Cornea and Ocular Surface Section Editors David Birk, University of South Florida (USF) College of Medicine, Tampa, Florida, USA Shukti Chakravarti, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Lens Section Editors Frank Giblin, Oakland University, Rochester, Michigan, USA Roy Quinlan, Durham University, Durham, UK Retina and Choroid Section Editors Steven Fliesler, The State University of New York at Buffalo, Buffalo, New York, USA Joe Hollyfield, Cleveland Clinic Foundation, Cleveland, Ohio, USA Special Issues and Reviews Editor S.J. Fliesler, The State University of New York at Buffalo, Buffalo, New York, USA Executive Editors M.R. Al-Ubaidi, University of Oklahoma College of Medicine, Oklahoma City, Oklahoma, USA B. Anand-Apte, Cleveland Clinic Foundation, Cleveland, Ohio, USA J. Ash, University of Florida, Gainesville, Florida, USA C. Belmonte, Consejo Superior de Investigaciones Científicas (CSIC), Sant Joan d'Alacant, Spain D.G. Birch, Retina Foundation of te Southwest, Dallas, Texas, USA M.F. Cordeiro, Institute of Opthalmology, London, U.K. M.J. Costello, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA W.J. Dupps Jr., MD, PhD, Cleveland Clinic Foundation, Cleveland, Ohio, USA C.R. Ethier, Georgia Institute of Technology, Atlanta, Georgia, USA D. Ferrington, University of Minnesota, Minneapolis, Minnesota, USA I.K. Gipson, Harvard Medical School, Boston, Massachusetts, USA M. Gorin, Dept of Opthalmology, Jules Stein Eye Institute J. Graw, Helmholtz Zentrum München, Oberschleissheim, Germany D.S. Gregerson, University of Minnesota, Minneapolis, Minnesota, USA C. Grimm, Universität Zürich, Zurich, Switzerland D.R. Hyde, University of Notre Dame, Notre Dame, Indiana, USA M. Iuvone, Emory University, Atlanta, Georgia, USA J. Kiel, University of Texas Health Sciences Center at San Antonio, San Antonio, Texas, USA G.W. Laurie, The University of Virginia, Charlottesville, VA A. Lewin, University of Florida College of Medicine, Gainesville, Florida, USA G. Lewis, University of California at Santa Barbara, Santa Barbara, California, USA A.V. Ljubimov, UCLA School of Medicine, Los Angeles, California, USA M.C. McGahan, North Carolina State University, Raleigh, North Carolina, USA N.S. Peachey, Cleveland Clinic Foundation, Cleveland, Ohio, USA J.S. Penn, Vanderbilt University, Nashville, Tennessee, USA W.M. Petroll, University of Texas Southwestern Medical Center, Dallas, Texas, USA N. Philp, Thomas Jefferson University, Philadelphia, Pennsylvania, USA U. Schloetzer-Schrehardt, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany S.L. Semple-Rowland, University of Florida College of Medicine, Gainsville, Florida, USA AUTHOR INFORMATION PACK 15 Mar 2015 www.elsevier.com/locate/yexer 3 D. Stamer, Duke University School of Medicine, Durham, North Carolina, USA M.A. Stepp, Ph.D., George Washington University, Washington, District of Columbia, USA D.A. Thompson, University of Michigan Medical School, Ann Arbor, Michigan, USA V. Vasiliou, PhD, Yale School of Public Health, New Haven, Connecticut, USA G.J. Wistow, National Institutes of Health (NIH), Bethesda, Maryland, USA T. Young, Duke University Medical Center, Durham, North Carolina, USA J.D. Zieske, Harvard Medical School, Boston, Massachusetts, USA S. Zigler, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA AUTHOR INFORMATION PACK 15 Mar 2015 www.elsevier.com/locate/yexer 4 GUIDE FOR AUTHORS . INTRODUCTION The goal of Experimental Eye Research is to publish original research papers on all aspects of the cell biology, physiology, genetics, biochemistry, biophysics, molecular biology, biophysics, pharmacology, developmental biology, microbiology, and immunology of the eye. The journal is subdivided into four sections; Aqueous Humor and Blood Flow, Cornea and Ocular Surface, Lens and Retina and Choroid, each with their own section editors. Short Communications, Letters to the Editor, Methods in Eye Research; individual Review Articles or collections of Review Articles specifically commissioned by the Journal are also published. Research areas include: Production and circulation of ocular fluids and the dysfunction of these pathways underlying ocular disease Angiogenesis, neovascularization and regulation of blood flow in the eye in health and disease Cell biology, molecular biology, biochemistry, and biophysics of the eye or eye tissueDevelopmental and regenerative biology of the eyeHuman and molecular genetics studies of inherited eye diseasesGene therapy and neuroprotection targeted at preventing inherited ocular diseasesNeural and general physiology of the visual process Types of communications 1. Research Articles :Original Research Articles describing the results of experimental studies that address fundamental biological issues on vision, the eye, or specific ocular tissues constitutes the majority of communications published in Experimental Eye Research. Detailed instructions for formatting regular research articles are provided below under the subheading “Preparation”. 2. Letters to the Editor:Letters to the Editor should provide substantive comment(s) on a publication in this Journal or an eye research article published elsewhere; or on issues of broad interest to the eye and vision research community. A Letter should be concise, to the point (generally no more than 750 words), contain only text (no abstract, figures, tables, acknowledgments, or reference list), and be written in continuous narrative style (no headings/subheadings). The Editor-in-Chief or a designated member of the Editorial Board will be responsible for reviewing Letters. Receipt of a Letter does not guarantee that it will be accepted for publication. In the event that the Letter challenges some aspect of a prior publication, a complete citation of the publication in question should be fully spelled out in the body of the text. The authors of the publication in question will be given the opportunity to respond to the comments made, and the two Letters (if accepted) will be published sequentially in the same issue of the Journal. 3. Short Communications: Short Communications are intended for preliminary reports of original, significant research results that are limited in scope and, thus, do not warrant publication in the form of a regular Research Article. Communications should be no longer than 4,500 words (generally not to exceed 4 printed pages in the Journal), inclusive of all literature citations, and should contain no more than two Figures (which may be multi-panel) and/or Tables;“Supplementary Data” is not permitted. The word count pertains only to the main body of text, excluding the title, author/institution details, abstract, figures/tables, figure legends, and acknowledgments; the Abstract should not exceed 250 words. Communications should not contain headings/subheadings (i.e.,Introduction, Materials and Methods, Results, Discussion), other than References, but otherwise should follow the rules pertaining to the preparation, text-formatting and submission of Research Articles for this Journal. 4. Focus on Molecules:Focus on Molecules articles are no longer accepted by Experimental Eye Research. 5. Methods in Eye Research:These feature articles provide a detailed overview of a specific method or technique used in experimental research of the visual system. This contribution should contain sufficient information to allow successful reproduction of the experimental method/ technique in another laboratory. Each article should contain the following headings (the first four being numbered):IntroductionMaterials and SuppliesDetailed MethodsPotential Pitfalls and Trouble ShootingReferencesArticle Specification: The article should not exceed 12 published pages in length including equivalent space for figures. For members of ISER, colour figures will be printed without charge. Include enough detailed information to allow researchers in independent laboratories to successfully reproduce this method AUTHOR INFORMATION PACK 15 Mar 2015 www.elsevier.com/locate/yexer 5 Please highlight the potential "problem areas" for the method and provide "trouble shooting" solutions Please ensure you select the correct article type (Methods in Eye Research) when uploading your article via http://ees.elsevier.com/yexer. If you would like to submit an unsolicited Methods In Eye Research article for consideration, or if you have any editorial queries, please , please contact the Methods in Eye Research Editor, Dr. Abe Clark, at [email protected]. 6. Special Issues: Periodically, Experimental Eye Research will publish a special issue containing review articles that cover selected topics in depth relevant to eye research. While the breadth and scope of these special issues can vary widely, they are intended to contain in a single issue the state of the art in specific areas of eye research. Up to four color plates will be published free of charge in each review articles commissioned by the journal. If you are interested in developing a special issue, please contact the Editor-in-Chief, or the Special Issues and Review Articles Editor, Dr. Steven J. Fliesler, at: [email protected]. Each review included in a special issue will undergo peer review before being accepted for publication. 7. Review Articles: Single review articles are periodically published in Experimental Eye Research. Most published review articles are solicited, but the Editor-in-Chief is always willing to consider new topics for a review. Prior to preparing a review article it is important to first contact the Editor-in-Chief, or Dr. Steven J. Fliesler, Special Issues and Review Articles Editor, ([email protected]) as to whether such a review would be appropriate for publication consideration. No reviews will be published without full peer review. We want all reviews to be succinct and pithy. While the length of a review will be governed by the scope of the topic covered, we suggest to authors that the length be approximately 6000 words, including space for tables, figures and references. Up to four color plates will be published free of charge in each review articles commissioned by the journal. Contact details for submission Experimental Eye Research Editorial Office, 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA Tel.: (619) 699-6278; Fax: (619) 699-6850; E-mail: [email protected] BEFORE YOU BEGIN Ethics in publishing For information on Ethics in publishing and Ethical guidelines for journal publication see http://www.elsevier.com/publishingethics and http://www.elsevier.com/journal-authors/ethics. Human and animal rights If the work involves the use of animal or human subjects, the author should ensure that the work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans http://www.wma.net/en/30publications/10policies/b3/index.html; EU Directive 2010/63/EU for animal experiments http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm; Uniform Requirements for manuscripts submitted to Biomedical journals http://www.icmje.org. Authors should include a statement in the manuscript that informed consent was obtained for experimentation with human subjects. The privacy rights of human subjects must always be observed. Conflict of interest All authors are requested to disclose any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work. See also http://www.elsevier.com/conflictsofinterest. Further information and an example of a Conflict of Interest form can be found at: http://help.elsevier.com/app/answers/detail/a_id/286/p/7923. Submission declaration and verification Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a published lecture or academic thesis or as an electronic preprint, see http://www.elsevier.com/sharingolicy), that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder. To verify originality, your article may be checked by the originality detection service CrossCheck http://www.elsevier.com/editors/plagdetect. 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Regardless of the application used other than Microsoft Office, when your electronic artwork is finalized, please 'Save as' or convert the images to one of the following formats (note the resolution requirements for line drawings, halftones, and line/halftone combinations given below): EPS (or PDF): Vector drawings, embed all used fonts. AUTHOR INFORMATION PACK 15 Mar 2015 www.elsevier.com/locate/yexer 11 TIFF (or JPEG): Color or grayscale photographs (halftones), keep to a minimum of 300 dpi. TIFF (or JPEG): Bitmapped (pure black & white pixels) line drawings, keep to a minimum of 1000 dpi. TIFF (or JPEG): Combinations bitmapped line/half-tone (color or grayscale), keep to a minimum of 500 dpi. Please do not: • Supply files that are optimized for screen use (e.g., GIF, BMP, PICT, WPG); these typically have a low number of pixels and limited set of colors; • Supply files that are too low in resolution; • Submit graphics that are disproportionately large for the content. Color artwork Please make sure that artwork files are in an acceptable format (TIFF, EPS or MS Office files) and with the correct resolution. If, together with your accepted article, you submit usable color figures then Elsevier will ensure, at no additional charge, that these figures will appear in color on the Web (e.g., ScienceDirect and other sites) regardless of whether or not these illustrations are reproduced in color in the printed version. For color reproduction in print, you will receive information regarding the costs from Elsevier after receipt of your accepted article. Please indicate your preference for color in print or on the Web only. There is no charge for colour in print for members of ISER, or for invited Reviews. For further information on the preparation of electronic artwork, please see http://www.elsevier.com/artworkinstructions. Please note: Because of technical complications which can arise by converting color figures to "gray scale" (for the printed version should you not opt for color in print) please submit in addition usable black and white versions of all the color illustrations. Figure captions Ensure that each illustration has a caption. Supply captions separately, not attached to the figure. A caption should comprise a brief title (not on the figure itself) and a description of the illustration. Keep text in the illustrations themselves to a minimum but explain all symbols and abbreviations used. Tables Please submit tables as editable text and not as images. Tables can be placed either next to the relevant text in the article, or on separate page(s) at the end. Number tables consecutively in accordance with their appearance in the text and place any table notes below the table body. Be sparing in the use of tables and ensure that the data presented in them do not duplicate results described elsewhere in the article. Please avoid using vertical rules. References Citation in text Please ensure that every reference cited in the text is also present in the reference list (and vice versa). Any references cited in the abstract must be given in full. Unpublished results and personal communications are not recommended in the reference list, but may be mentioned in the text. If these references are included in the reference list they should follow the standard reference style of the journal and should include a substitution of the publication date with either 'Unpublished results' or 'Personal communication'. Citation of a reference as 'in press' implies that the item has been accepted for publication. Reference links Increased discoverability of research and high quality peer review are ensured by online links to the sources cited. In order to allow us to create links to abstracting and indexing services, such as Scopus, CrossRef and PubMed, please ensure that data provided in the references are correct. Please note that incorrect surnames, journal/book titles, publication year and pagination may prevent link creation. When copying references, please be careful as they may already contain errors. Use of the DOI is encouraged. Web references As a minimum, the full URL should be given and the date when the reference was last accessed. Any further information, if known (DOI, author names, dates, reference to a source publication, etc.), should also be given. Web references can be listed separately (e.g., after the reference list) under a different heading if desired, or can be included in the reference list.