Increased episcleral venous pressure in a mouse model of circumlimbal suture induced ocular hypertension☆
Graphical abstract
Introduction
Glaucoma is a multifaceted neurodegenerative condition characterised by progressive retinal ganglion cell loss and optic nerve degeneration. Elevated intraocular pressure (IOP) is the most well-defined and only modifiable risk factor in glaucoma (Cook and Foster, 2012; Kass et al., 2002; Zhan et al., 2002). Glaucoma is characterised by retinal ganglion cell dysfunction and retinal nerve fibre layer thinning as has been documented in both laboratory and clinic studies (Fortune et al., 2004; Pollet-Villard et al., 2014; Saleh et al., 2007; Viswanathan et al., 2001). Rodent models are commonly used in glaucoma research as they are inexpensive and suitable for longitudinal assessment. Mice models also have distinct advantages in terms of the availability of genetic tools to probe our understanding of disease pathogenesis. Mice show IOP levels similar to those seen in humans (Johnson and Tomarev, 2010; Martinez-Aguila et al., 2016), and have similar aqueous humor dynamics relative to human eyes (Johnson and Tomarev, 2010; Pang and Clark, 2007). Because of these similarities and advantages, mice are increasingly used by glaucoma researchers, notwithstanding differences in optic nerve structure (Fischer et al., 2009; Jeon et al., 1998).
There are a variety of rodent glaucoma models, each with distinct advantages and disadvantages. The circumlimbal suture model (He et al., 2018; Lee et al., 2019; Liu et al., 2017a, 2017b; Zhao et al., 2017, 2019) employed in this study produces sustained mild and stable IOP elevation, preferential loss of retinal ganglion cells, clear optics to facilitate in vivo imaging and ease of IOP normalization (suture removal) without potential confounds associated with pharmacological intervention (He et al., 2018; Liu et al., 2017a; Zhao et al., 2017, 2019). However, the mechanism of IOP elevation in the circumlimbal approach has not yet been examined. This knowledge would better inform the use of this model for glaucoma research.
The level of IOP in the eye is determined by a balance between a range of factors. Aqueous humor (AH) is produced by the ciliary body and drains out of the eye through the ‘conventional route’ (trabecular meshwork) or ‘unconventional’ route (uveoscleral and uveovortex pathways) (Johnson et al., 2017). The trabecular meshwork pathway (otherwise known as the pressure sensitive pathway) involves aqueous draining through the trabecular meshwork into the Schlemm's canal which feeds into the episcleral venous network (Gong et al., 1996; Johnson et al., 2002; Tamm, 2009). The majority of aqueous outflow occurs through this pathway, but a certain proportion also flows out the uveoscleral pathway. The uveoscleral route (or pressure-insensitive pathway) involves AH drainage between the iris and ciliary muscle bundles which is then absorbed by orbital blood vessels surrounding these muscles. A fraction of this AH is taken up by the choroidal vessels via osmosis and drained into the vortex veins (uveovortex outflow). The pressure gradient that exists between the higher-pressure anterior chamber (IOP) to the lower pressure episcleral veins (EVP) facilitates aqueous outflow. The relationship between IOP and AH dynamic parameters can be described by a modified Goldmann equation (Becker and Neufeld, 2002; Brubaker, 2004; Goldmann, 1951; Mermoud et al., 1996; Millar et al., 2011) where Fin is the AH production rate, Fu is the uveoscleral outflow rate, C is the outflow facility and EVP is the episcleral venous pressure (Equation (1)).
The mechanism of IOP elevation varies depending on the type of glaucoma. In angle closure glaucoma, aqueous outflow is compromised through physical obstruction of the drainage pathways. In chronic primary open angle glaucoma, it has been shown that increased outflow resistance in the trabecular pathway contributes to elevated IOP (Allingham et al., 1996; Alvarado and Murphy, 1992; Becker and Hahn, 1964; Jackson et al., 2006; Tamm and Fuchshofer, 2007) and some studies indicate a role for elevated EVP (Greenfield, 2000; Greslechner and Oberacher-Velten, 2019; Jorgensen and Guthoff, 1988; Selbach et al., 2005), however the exact mechanisms still remain unclear. In terms of animal studies, current rodent glaucoma models target the trabecular meshwork or episcleral vein outflow routes to elevate eye pressure. For example, the microbead injection model (Chen et al., 2011; Smedowski et al., 2014) and laser photocoagulation model (Kwong et al., 2011; Levkovitch-Verbin et al., 2002) aim to clog and scar the trabecular meshwork which should lower outflow facility (C), and other models like the episcleral vein cauterization (Anders et al., 2017; Liu et al., 2017b; Ruiz-Ederra and Verkman, 2006; Shareef et al., 1995) and ligation models (Dey et al., 2018; Yu et al., 2006) scar and tie off episcleral veins, thus an elevation of EVP would be expected. The circumlimbal suture model involves attaching and tightening a thin thread behind the limbus to apply inward compression of the globe and surrounding episcleral veins (He et al., 2018). However, the exact mechanism by which this model induces chronic IOP elevation has not yet been examined.
Section snippets
Animals
In total 24 adult male C57BL/6J mice aged 3 months old (Animal Resource Centre, Caning Vale, WA, Australia) were used in this study. All procedures were conducted in accordance with the National Health and Medical Research Council of Australia guide for the use of animals in research and conformed with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animal ethics was approved by the Howard Florey Animal Ethics Committee (ethics number: 13-068-UM). Mice were housed
IOP elevation on sutured eyes
Fig. 3 shows the IOP measured in awake and anesthetized mice in naïve, sham and OHT groups. In Fig. 3A–D groups are illustrated separately, in Fig. 3E and F naïve and sham groups are combined given at the chronic end-point no statistical differences were found between these groups. IOP levels were similar between all groups at baseline (Fig. 3A). An IOP spike after circumlimbal suture implantation was evident in both sham and OHT groups (Fig. 3B sham: 40.2 ± 2.1 mmHg, n = 9, vs. OHT:
Episcleral vein dilation is an objective method to estimate EVP
We used two methods to estimate EVP in this study. Currently, direct observation of blood reflux into Schlemm's canal during gradual intracameral pressure lowering is the most well-established method to estimate EVP in murine models (Aihara et al., 2003b; Millar et al., 2011; Sit and McLaren, 2011). In a normal eye, aqueous humor is constantly generated by the ciliary body and drained into Schlemm's canal which feeds into the episcleral venous network. This outflow is driven by the pressure
Conclusions
This is the first study to investigate the mechanism of IOP elevation induced by circumlimbal suture in mice during the chronic phase of IOP elevation. We found that sham mice who experienced an acute IOP spike (suture removed after 2 days) without any long term IOP elevation exhibited the same aqueous dynamic profiles as naïve mice. We also showed that there were no signs of angle closure in OHT mice compared with controls. We determined that EVP was higher in the OHT group compared with
Supported by
Australian Research Council Linkage Grant LP160100126 (C. T. Nguyen, B. V. Bui), Melbourne Research Fellowships (C.T. Nguyen).
Declaration of competing interest
The authors declare they have no competing interests.
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Vickie H.Y. Wong and Da Zhao contributed equally to the work and should therefore be regarded as equivalent first authors.