Introduction
In the previous chapter, the potential of the therapeutic downregulation of TJ components to treat elevated IOP in POAG was investigated in wildtype mice, showing an increase in conventional outflow facility and a decrease in IOP in these normotensive animals. In order to better characterise the efficacy of this treatment, it was desired to examine the effect of this same treatment in animals in a state of ocular hypertension, representing a better model of POAG. The proposed siRNA-based treatment functions to increase the permeability of the SC inner wall through destabilisation of TJ mediated paracellular clefts, leading to the opening of paracellular pores. It has been shown previously that, while transcellular pore density is unaffected by pressure, paracellular pores are responsive to pressure, with pore density increasing at the inner wall in a pressure dependent manner, implying a pressure driven mechanical opening of these pores (Ethier et al. 1998). It is therefore reasonable to infer that siRNA mediated downregulation of TJ components at the SC inner wall, shown in the previous chapter to increase the number of open clefts in normotensive mice, would have an even greater effect on the number of open pores in murine models of ocular hypertension.
Selecting a murine model of ocular hypertension
While there are many different murine model systems utilised in POAG research, it is important to carefully consider the suitability of different model systems to a particular approach. As glaucomatous optic neuropathy represents the primary concern in the development and progression of POAG, a significant proportion of murine models used in POAG research are concerned with retinal degeneration alone. While ocular hypertension is commonly used as a means to induce this degeneration, the means by which IOP is elevated is not of relevance to the study of optic neuropathy . As a result, the mechanisms by which ocular hypertension arises in these models does not well represent that seen in POAG, discounting the use of these models in investigations involving AH dynamics and modulation of the outflow pathways. For
this reason it was necessary to implement a murine model of ocular hypertension that occurred through increased resistance in the inner portion of the conventional outflow pathway, at the JCT or SC inner wall. While ocular hypertension induced retinal damage is clearly an important factor in POAG, for the purpose of the validation of this approach IOP reduction and conventional outflow facility increase were viewed as primary experimental goals, and glaucomatous optic neuropathy were not essential in the selected murine model.
The DBA/2J mouse
The DBA/2J strain of mouse represents a spontaneous genetic model of ocular hypertension and glaucomatous optic neuropathy (John et al. 1998). These mice exhibit significantly elevated IOP at around 6 - 8 months of age, with axonal loss in the optic nerve observed soon after the development of ocular hypertension, while RGC loss occurring in a retrograde fashion from the damaged ONH axons in the months following this (Libby et al. 2005; Schlamp et al. 2006). DBA/2J have homozygous mutations in two genes that are responsible for the observed IOP elevation, Tyrp1b, a melanosomal structural protein, and GpnmbR150X, a transmembrane glycoprotein also expressed in melanosomes, the latter mutation being a premature stop codon, with DBA/2J mice homozygous for wildtype forms of these genes reported as having no glaucoma symptoms (Howell et al. 2007). Mechanistically, IOP is elevated through pigment dispersion from the iris into the AH, leading to the accumulation of pigment in the iridocorneal angle, blocking the outflow of AH. As such, these mice represent a model of pigmentary glaucoma. There is also evidence that pigment dispersion alone may not be the only factor in the development of glaucoma in these mice, as the replacement of DBA/2J bone marrow with that of mice with wildtype versions of the above genes also prevents disease development, indicating a systemic or immune role of these genes in neurodegeneration as well as a local role in IOP elevation (Anderson et al. 2005). As the increased resistance to AH outflow in these mice is caused by physical blocking of the outflow pathways they were not considered as a potential model.
Induced models of ocular hypertension
Aside from spontaneous mouse models of ocular hypertension, an elevation in IOP can be induced in mice through various means, all requiring an increase in the resistance to AH outflow. One approach to increasing this resistance involves targeting of the episcleral veins, these being the downstream drainage site of the conventional outflow pathway. Through occlusion of some of these veins, outflow resistance is increased by reducing the potential outflow routes for AH from the lumen of SC. This episcleral vein occlusion can be created through cauterisation, or laser photocoagulation (Ruiz-Ederra & Verkman 2006; Gross et al. 2003; Ji et al. 2005). Laser energy has also been used to induce ocular hypertension through applying burns directly to the trabecular meshwork (Aihara et al. 2003b). While these laser induced methods are effective in elevating IOP, as they take effect in the distal outflow regions of the conventional pathway, or in the case of TM burns, the outer regions of the TM, they do not well represent the increase in resistance at the JCT and SC inner wall as observed in POAG.
Elevated IOP can also be induced in mice through intracameral injection of 10 µM polystyrene microbeads. These beads accumulate at the iridocorneal angle in a manner similar to pigment particles in the DBA/2J mice, blocking the passage of AH in to the outflow pathways, thereby increasing outflow resistance (Chen et al. 2011). Greater increases in IOP and retinal damage are attained through the injection of a mixture of 6 µM and 1 µM microbeads, together with a viscoelastic binding material (Cone et al. 2012). As this process obstructs the passage of AH upstream of the site of therapeutic activity of the siRNA approach being assessed, these models were also not deemed suitable. A further mechanism to induce an elevation in murine IOP involves the placement of a circumlimbal suture, providing continuous inward pressure on the limbus (Liu et al. 2017). This likely causes IOP elevations through compression of the ciliary muscle, blocking the unconventional outflow pathway, and as such is an inadequate model of POAG.
Models of ocular hypertension with increased outflow resistance in the trabecular meshwork
Two mouse models of ocular hypertension exist that closely resemble the increase in ocular hypertension seen in human POAG, with the AH outflow resistance increase
occurring primarily in the JCT in both cases. The first of these models came arose from the study of a Mendelian form of human POAG, which arises from mutations in the myocilin gene (MYOC), previously known as trabecular meshwork induced glucocorticoid response gene (TIGR). While wildtype MYOC is secreted from TM cells, in its mutant forms it instead accumulates in the TM, with over 90 % of identified human mutations occurring in the olfactamedin domain, and being predicted to affect protein structure (Resch & Fautsch 2009; Jacobson et al. 2001). Expression of human mutant MYOCY437H, a mutation found in juvenile onset POAG patients, via an adenoviral vector transducing the iridocorneal angle was shown to cause ocular hypertension (Shepard et al. 2007). Subsequently, a transgenic mouse model was generated, Tg-MYOCY437H, which showed accumulation of the mutant protein in the TM, along with ocular hypertension, RGC loss, and axonal degeneration at the ONH. Ocular hypertension manifested in these animals from three months of age. Reduction of endoplasmic reticulum (ER) stress in the TM, through treatment with phenylbutyric acid, was associated with an amelioration of glaucomatous optic neuropathy, linking ER stress to disease pathology in human patients (Zode et al. 2011). These mice also exhibit reduced AH outflow facility, and abnormal intracellular accumulation of ECM components in the TM, while cultured TM cells expressing the mutant gene showed a reduction in active MMP-2 and MMP-9 (Kasetti et al. 2016). This murine model of ocular hypertension and POAG therefore represents an acceptable replication of the human condition.
The second murine model that has an IOP elevating site of action in the TM is the glucocorticoid induced ocular hypertension model. Glucocorticoids (GCs) are a widely prescribed class of steroids used in the treatment of inflammation and immune related disorders, which, while extremely effective, have significant ocular side effects, chief among these the elevation of IOP, leading to GC-induced glaucoma (Overby & Clark 2015). GC-induced glaucoma presents clinically in the same manner as POAG, with elevated IOP in patients with open iridocorneal angles, requiring a known patient history of GC use for diagnosis. While ocular hypertension desists after GC treatment is discontinued, any resultant glaucomatous optic neuropathy is irreversible. GC treatment does not give rise to ocular hypertension in all patients, with affected patients referred to as ‘steroid responders’, and from 11 – 79 % of patients in one meta-analysis
that 45 % of eyes receiving long term GC implantation treatment required surgical intervention to address the resultant GC-induced glaucoma (Bollinger et al. 2011). Analysis of post mortem human eyes diagnosed with GC-induced glaucoma showed an increased deposition of the ECM components collagen IV, heparan sulfate proteoglycan, and fibronectin in the JCT (Tawara et al. 2008). In perfused human organ culture, ECM accumulation is seen ultrastructurally in steroid responder eyes, with narrowing of intercellular spaces in the JCT, while non-responder eyes showed no change (Clark et al. 1995). GC treatment also modifies the cytoskeleton of TM cells, causing the formation of cross-linked actin networks (CLANs), potentially increasing resistance to outflow through alterations of the mechanical properties of these cells (Overby & Clark 2015). As is seen in humans, GC-induced hypertension also occurs in mice, with long-term subcutaneous delivery of dexamethasone (DEX) by osmotic mini- pump shown to elevate IOP significantly within 2 weeks of treatment, with a concomitant reduction in conventional outflow facility of 50 % (Whitlock et al. 2010; Darryl R. Overby, Bertrand, Tektas, Boussommier-Calleja, Schicht, Ross Ethier, Woodward, Daniel Stamer & Lütjen-Drecoll 2014). Mice treated with DEX are also known to have a similar increase in ECM deposition in the TM, with increased levels of collagen I, fibronectin, and a-smooth muscle actin reported (Patel et al. 2017). Also, of particular relevance to our TJ modulation based approach, GC treatment of cultured human SC endothelial cells has been shown to significantly increase ZO-1 expression, and to increase the number of TJ-containing intercellular contacts (Underwood et al. 1999). Murine GC-induced ocular hypertension therefore represents a further acceptable model of ocular hypertension caused by increased resistance to AH outflow in the JCT. As such, this model represents a suitable means to assess the modulation of outflow facility through the siRNA based approach previously described, acting as a model of POAG in general, and more specifically, of human GC-induced glaucoma.
While both of these models are suitable for evaluation of the effect of siRNA mediated downregulation of TJ transcripts on IOP and outflow facility in ocular hypertensive mice, for the purpose of this investigation, the murine GC-induced ocular hypertension model was used, as IOP elevation was seen in this model within 2 weeks in wildtype mice, while the use of MYOCY437H, which is in fact also being currently pursued, required first the establishment of an in-house breeding colony, followed by a delay of three months before animals are expected to show significant IOP increases.
Aims
The aims of this thesis research chapter were to implement a DEX induced murine model of ocular hypertension, and to investigate the effect of siRNA mediated downregulation of selected TJ proteins at SC inner wall on IOP and outflow facility in these hypertensive mice. This would enable analysis of whether higher IOP prior to siRNA treatment would result in a greater decrease in IOP and increase in outflow facility post-treatment, as would be expected by pressure driven opening of weakened paracellular pores at the inner wall of SC.
Results
Evolution of elevated intraocular pressure in dexamethasone treated mice
Wildtype C57BL/6J were implanted subcutaneously with osmotic mini-pumps delivering DEX at 2 different dosages, a low dose of 0.13 mg/kg/day (N = 6), and a high dose of 2 mg/kg/day (N = 14). Two mice in the high-dose treatment group were excluded from all analysis, one due to GC side effect related death, and the second due to congenital ocular malformation. A third mouse in this group was sacrificed prior to the final IOP measurement due to GC induced illness, but is included in the preceding IOP time points. IOP was measured by rebound tonometry in one eye and mice were weighed one day prior to pump implantation, and these measurements were repeated weekly thereafter for 4 weeks. Figure 4.1 shows the recorded IOP measurements for low dose and high dose DEX treated mice.
Figure 4.1: Evolution of intraocular pressure in dexamethasone treated mice
Weekly IOP measurements in low dose (left) and high dose (right) DEX treated mice. Box plots represent 25th to 75th percentile, centre line of box plots represent the median IOP, while whiskers show the 5th and 95th percentile. * represents statistical significance ( p ≤ 0.05, t-test).
As is clear from this figure, baseline IOP measurements prior to pump implantation are exceptionally high, with these high initial IOP values masking any ocular hypertension inducing effects of DEX treatment, potential causes of this are discussed further below. For the low dose group, baseline IOP was measured as 20.10 [17.65,
High dose DEX
basel ine Wk1 Wk2 Wk3 Wk4 8 12 16 20 24 28 32 IO P ( m m Hg )
Low dose DEX
basel ine Wk1 Wk2 Wk3 Wk4 8 12 16 20 24 28 32 IO P ( m m Hg ) * *
22.55] (mean [95% confidence interval]) mmHg (N = 6), while at week 4, the mean IOP for this group was 16.35 [13.24, 19.46] mmHg (N = 6). For the high dose treatment group, baseline IOP was recorded as 17.75 [15.34, 20.16] mmHg (N = 12), while at week 4 the mean IOP for the group was measured as 16.75 [13.74, 19.75] mmHg (N = 11). If the anomalous high baseline IOP readings are excluded from analysis, a significant increase in IOP is detected from week 1 to week 4 in both low dose and high dose treatment groups, fig. 4.1. The mean increase in IOP was 3.12 [6.18, 0.07] mmHg (p = 0.046) in the high dose treatment group and for the low dose group the mean increase was 3.48 [7.00, 0.03] mmHg (p = 0.05).
Figure 4.2: Intraocular pressure change in low dose and high dose dexamethasone treated mice.
The final IOP readings for low dose and high dose DEX treated mice (left) showed no significant difference in IOP between dosage groups, while a significant increase in IOP from week 1 to week 4 was observed in both groups (right). Box plots represent 25th to 75th percentile, centre line of box plots represent the median IOP, while whiskers show the 5th and 95th percentile. * represents statistical significance from no change to IOP (p ≤ 0.05, one sample t-test, theoretical mean of 0).
Interestingly, when analysing IOP measurements in this manner, no significant difference was detected between the effects of low dose and high dose DEX in mean IOP at week 4, with a mean difference in IOP at week 4 between the two groups of 0.40 [-4.76, 3.97] mmHg, (p = 0.85, unpaired t test), fig. 4.2. Similarly, while both groups
IO P a t W k 4 (m m Hg ) Low dose DEX High dose DEX 10 15 20 25 30 Ch an g e in IO P (m m H g ) Low dose DEX High dose DEX -5 0 5 10 15 * *
the level of IOP elevation between groups 0.36 [-4.17, 4.88] mmHg (p = 0.87, unpaired t test). This is useful for future studies, as the lower DEX dosage is better tolerated by mice.
Potential reasons for high baseline IOP readings
High baseline IOP readings are a confounding factor in this analysis of DEX induced IOP elevation. It was noticed after DEX treatment had been completed that temperatures within the animal facility that housed these mice had been unstable during the course of the study, fig. 4.3. In order to determine whether these temperature fluctuations could have impacted tonometric IOP readings, all IOP measurements carried out on untreated wildtype mice were collated and analysed with respect to the average temperature recorded in the room these mice were housed in on the day of IOP measurement.
Figure 4.3: Temperature fluctuations in animal housing facility
This plot shows the maximum (red) and minimum (blue) temperatures recorded in the room where mice were housed over the duration of their housing for this study. Black line represent the recommend temperature range for the animal facility
This analysis showed a correlation between housing temperature and measured IOP, R2 = 0.461, with higher IOP recorded at lower temperatures, fig 4.4. A similar effect
was seen in pooled IOP measurements taken in all DEX treated animals, R2 = 0.479, fig. 4.5. As baseline IOP measurements were obtained in early November, when housing temperature was notably lower than following weeks, this may account for observed differences between the baseline IOP readings and those measured in later in
15 17 19 21 23 25 27 29 24/10/16 26/10/16 28/10/16 30/10/16 1/11/16 3/11/16 5/11/16 7/11/16 9/11/16 11/11/16 13/11/16 15/11/16 17/11/16 19/11/16 21/11/16 23/11/16 25/11/16 27/11/16 29/11/16 1/12/16 3/12/16 5/12/16 7/12/16 9/12/16 11/12/16 13/12/16 15/12/16 17/12/16 Temp eratu re ( ºC) Max Min
the study. In addition to the effects of temperature fluctuation, it was also discovered that the cyclic lighting system in the animal facility had malfunctioned and lights had been on for 24 hours a day, as opposed to the usual 12/12 hour light/dark cycle. As IOP has a strong circadian component this likely also played a role in the IOP data reported.
Figure 4.4: Correlation between intraocular pressure and temperature in untreated and dexamethasone treated mice
The recorded tonometric IOP from untreated wildtype mice(top), not included in this study, and DEX treated mice (bottom) is shown here against the average temperature in the room they were housed in on the day of IOP measurement ((Max + Min)/2).
y = -2.3757x + 71.761 R² = 0.4609 6 8 10 12 14 16 18 20 22 24 26 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0
IO
P
(m
m
H
g
)
Average temperature (Cº)
y = -2.6582x + 77.998 R² = 0.47945 8 10 12 14 16 18 20 22 24 26 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0IO
P
(m
m
H
g
)
Average temperature (Cº)
Effect of siRNA treatment on intraocular pressure in dexamethasone treated mice
After DEX treatment, all mice were injected intracamerally with 1 µg each ZO-1 and tricellulin siRNA in one eye, while contralateral control eyes received NT siRNA injections, as in normotensive mice in chapter 3. At 48 hours post-injection, mice were anesthetised and IOP was measured again by rebound tonometry.
In low dose DEX treated mice, pre-injection IOP was measured as 16.45 [13.68, 19.22] mmHg, while at 48 hours it was 14.95 [10.06, 19.84] mmHg with no significant change in IOP detected, -1.5 [-6.13, 9.13] mmHg (p = 0.635, N = 6). For eyes injected with treatment siRNA, targeting ZO-1 and tricellulin, the mean IOP before the injection was 16.05 [13.21, 18.89] mmHg, while mean IOP at 48 hours was 13.55 [10.52, 16.58] mmHg, with no significant change in IOP detected again in this case, -2.50 [-2.50, 7.50] mmHg (p = 0.255, N = 6), fig. 4.5.
Figure 4.5: Effect of siRNA downregulation of ZO-1 and tricellulin on intraocular