Transiently Raised IOP Equivalent To That Experienced During Ocular Surgery Causes Moderate Inflammation But Does Not Affect Retinal Function Or Result In Retinal Ganglion Cell Loss In An Animal Model

Purpose: High intraocular pressure (IOP) is known to result in retinal ganglion cell (RGC) loss, both with chronically raised intraocular pressure (such as with glaucoma) and with acute raises in pressure (due to injury or acute angle closure). Because IOP is often raised during ocular surgery, the purpose of this study was to evaluate the effect of transient moderate IOP on retinal function, RGC survival and the expression of Connexin 43 (Cx43) and glial fibrillary acidic protein (GFAP), ubiquitously expressed central nervous system (CNS) proteins that are known to be elevated during the retinal inflammatory response to injury. Materials and Methods: Wistar rats were exposed to transient IOP at 40 mmHg for 5 or 30 minutes, and 60 mmHg for 5 minutes (via cannulation of the anterior chamber with a saline reservoir raised to a height corresponding to the desired IOP), mimicking potential IOP rises during surgery such as DSAEK and some laser procedures (LASIK and femtosecond laser cataract surgery). Separate groups of animals had IOP maintained at 10 mmHg for 5 or 30 minutes as cannulation controls, or 120 mmHg for 60 minutes as positive controls. Changes in the optic nerve and retina were assessed immunohistochemically for GFAP and Cx43 expression. Retinal function was assessed using electroretinography (ERG) recorded at baseline and 14 days after the IOP rise and compared with RGC counts. Results: Results showed that there was a differential GFAP labelling pattern observed in the anterior optic nerve in the 40 mmHg 30 minute and 60 mmHg 5 minute groups 4 hours after manipulation. Gap junction protein Cx43 was minimally up-regulated in the retina in the short-term. There was, however, minimal long-term effect on retinal function and no RGC loss. Conclusions: In conclusion, elevations of IOP that are short in duration such as those occurring during surgical procedures, do not cause significant changes long-term in retinal function or RGC survival. Key Messages: Cx43 and GFAP are known to be elevated during the retinal inflammatory response to injury. No previous study has explored the effect of moderate and relatively short increases in IOP on the initial inflammatory response. We observed a mild glial inflammatory response in the anterior optic nerve, but only a minimal upregulation of Cx43. However, transient and moderate IOP rises did not induce long term disruption to RGC function or number as measured by electrophysiology and RGC counts, respectively. This is applicable to clinical practice, as it means the IOP elevations that occur during some surgical procedures are unlikely to be causing long term damage in retinal function or RGC survival. DOI : 10.14302/issn.2470-0436.jos-17-1453 Corresponding author: Helen V. Danesh-Meyer, Department of Ophthalmology, Faculty of Medical and Health Sciences, University of Auckland, Private bag 92019, Auckland 1142, New Zealand Email: helendm@gmail.com Phone: (64) 212291840, Fax: (64) 9 3677173


Introduction
Transient and moderate increases in intraocular pressure (IOP) occur during many ophthalmic procedures.With Descemet's stripping endothelial keratoplasty (DSEK) IOP increases of 30-40 mmHg are seen for 5-10 minutes, [1][2][3].Other commonly performed ocular procedures produce shorter IOP rises, such as laser in-situ keratomileusis (LASIK) where the IOP increases to at least 65 mmHg for up to 45 seconds, and with femtosecond laser cataract surgery where increases in the IOP up to 40 mmHg for several minutes can occur, [4].
Despite the known common occurrence of such IOP elevations, the effects on the optic nerve and retinal function have been minimally investigated.Recently, the role of glial behaviour with increases in IOP has been investigated and is suggested to be involved in retinal ganglion cell (RGC) dysfunction, [5][6].A variety of proinflammatory and inflammatory protein markers that are induced by IOP elevations have been identified, [7].
We have focused on the expression of the gap junction protein Connexin43 (Cx43) and the glial fibrillary acidic protein (GFAP) to evaluate the initial inflammatory response.GFAP is a marker for astrocytes and activated Müller cells.Astrocytes are recognised to have a role in controlling the microenvironment and providing structural and metabolic support to the optic nerve and retina, [8][9].Cx43 gap junction signalling and especially hemichannel opening plays a role in propagation of cell death signals after injury, [10][11][12][13][14].Following injury to the CNS there is an astrocytic response that is well defined, where rapid proliferation is seen along with hypertrophy and changes in the expression of GFAP, as well as the upregulation of Cx43 expression, [15][16].
We have used electroretinogram (ERG) responses to measure retinal function.ERG responses in rats have been shown to be affected after IOP increase, with components of the ERG response showing differing levels of sensitivity to varying degrees of IOP elevation, [17][18].The negative and positive scotopic threshold response (nSTR and pSTR, reflecting retinal ganglion cell function) and photopic b-wave (bipolar cell function) were the most sensitive ERG components, being consistently reduced and delayed immediately after 75 minutes of IOP increase to 30 mmHg, and were completely abolished after 75 minutes of IOP at 70 mmHg, [17].The most resilient ERG component was the a-wave (photoreceptor function), being the only component still present after 75 minutes of IOP at 70 mmHg, [17].At IOPs above 70 mmHg, all components of the ERG were diminished, [17].Therefore an IOP increase seems to affect cell function in the inner retina first, at an IOP level of 30 mmHg, followed by cell function in the middle and outer retina at progressively higher IOP levels.
Transient IOP increases above the systolic blood pressure for more than 1 hour have been shown to result in retinal ganglion cell (RGC), inner retinal layer and outer nuclear layer cell apoptosis, [19] and RGC loss has been reported in several other raised IOP models, [20][21][22][23][24][25][26].The purpose of this study was to assess whether transient moderate elevated IOP relevant to those which patients are exposed to during ocular surgical procedures, modifies the optic nerve and retinal glial response, as well as RGC functional modification and RGC loss.Imaging was with a confocal laser scanning microscope (Olympus FV1000).Two fields in each quadrant of each retina were imaged using a 10x objective lens giving a total of eight images per retina and total linear area of 6.5 mm 2 per retina.

Animals
Quantification was performed using automated spot counts in NIH ImageJ software.RGC density was calculated as the number of RGCs per cm 2 .RGC density was compared to uninjured control and was analysed using Student's t-test with a significance level of 0.05.

Electroretinograms
Full field ERGs were recorded from both eyes simultaneously at baseline and 14 days after IOP elevation.Animals were dark-adapted overnight and prepared for ERG recording under dim red lighting.
Animals were anaesthetised with an intraperitoneal injection of ketamine (60 mg/kg) and medetomidine hydrochloride (0.4 mg/kg) and the cornea anaesthetised with oxybuprocaine hydrochloride (0.4%, Bausch & Lomb).The animal's body temperature was maintained using a heat pad.
ERGs were recorded in a Faraday cage (custommade) using a hand-held battery-operated ERG system (EPH-01, Ephios, Sweden) simultaneously from both eyes with silver / silver chloride electrodes (custommade using a previously described method by Bui and Fortune) placed at the apex of the corneas, [27].The EPH-01 ERG system incorporates a signal amplifier, a Analysis of Cx43 spot count (number of spots per 210 µm linear image) in the NFL/GCL/IPL showed that the degree of IOP elevation was a statistically significant predictor of Cx43 spot count (p<0.05,B=0.344) in a multiple linear regression analysis, whereas duration of IOP elevation (p=0.44) was not (Figure 2).However, no significant difference was found between any of the moderate IOP manipulation groups and uninjured retinas using a Student's t-test.A significantly higher Cx43 spot count was found in the 120 mmHg 60 minute group positive control compared to uninjured retinas (p<0.050,79 ± 9.7 vs. 42 ± 10) using a Student's ttest.The same analysis of Cx43 spot count in the INL/ OPL/ONL did not reveal significant differences.No significant differences were found in Cx43 spot count in the optic nerve (Supplementary figure 1).

Retinal Ganglion Cell Density
The transient periods of raised IOP at 40 and 60 mmHg did not affect RGC density at the end of 14 days (Figure 3).A significant decrease (p<0.001) in RGC density was found in the positive control group of animals where the IOP was raised to 120 mmHg for 60 minutes (115632 ± 6173 cells/cm 2 ) compared to uninjured (218771 ± 4582 cells/cm 2 ), 10 mmHg (normal IOP cannulation) for 5 minutes (228308 ± 26611 cells/ cm 2 , or 10 mmHg for 30 minutes (normal IOP cannulation) (232035 ± 7970 cells/cm 2 ).No significant decrease in other treated groups was found compared to uninjured, 10 mmHg for 5 minutes or 10 mmHg for 30 minutes.

Retinal Function
Retinal functional modifications were assessed 14 days after transiently raised IOP (Figure 4). Figure 4A gives examples of graphs that show how measurements No significant change in pSTR or b-wave implicit time was found in the manipulated eyes of any group.
There was also no significant change in nSTR, a-wave amplitude (Supplementary figure 4) or implicit time for any group compared to baseline.

Discussion
This study suggests that transient and moderate   no disruption to either anterograde or retrograde axonal transport at any time point beyond the acute injury event, or long term damage to RGCs, [32].In general there seems to be a threshold for permanent RGC functional loss in the rat at an IOP between 60-70 mmHg if sustained for 105 minutes or more, [33], or with shorter time periods at higher pressures (120 mmHg sustained for 60 minutes), [25].
No previous study has explored the effect of moderate and relatively short increases in IOP on the initial inflammatory response.We observed a mild glial inflammatory response in the anterior optic nerve.
Astrocytosis was identified in the anterior optic nerve in There is increasing evidence that glial cells play a pivotal role in modulating the microenvironment of the optic nerve.GFAP is recognised to be a sensitive marker of astrocyte activation in response to injury, [34] with activation of glial cells in the optic nerve head in experimental glaucoma models as well as in humans with glaucoma, [35][36][37][38].Previously, we have shown that ischemic IOP levels (120mmg, 60 minutes) led to activation of astrocytes and Müller cells in the retina at 8 hours and 3 days post-injury, [25,39].In that study, as in the present study positive control group, this is associated with subsequent RGC death.
Cx43 has been shown to be up-regulated in both the GCL and anterior optic nerve in human glaucoma retinas, [37] and in the GCL 3 days after animals were subjected to partial optic nerve transection, coinciding with the timing of RGC loss, [40].
An ischaemic IOP increase to 120 mmHg for 60 minutes resulted in Cx43 up-regulation in the GCL from 4 hours, co-localising with activation of glial cells and preceding RGC loss at 21 days, [25].To make comparisons between the human and rat eye, one should consider the differences in anatomy and morphology.In the rat, the diameter of the optic nerve head is smaller than in humans (about nine times smaller), [42].Like humans, the rat does have a lamina cribrosa-like structure, but the quantity of the lamina collagen, [43].Thus, there may be differences in the biomechanical properties of the lamina cribrosa between these species.However, a study done by one group looked at the changes in the optic nerve head in aged rats and found that the changes were similar to those found in the human lamina cribrosa, thus concluding that the rat model was useful for studying the influence of age on the onset of glaucoma, [42].The blood vessels (central retinal artery and vein) in rats enter the optic nerve head at the level of the sclera as opposed to retrolaminar in humans, predisposing the retinal blood supply to IOP rises more than in humans.The blood supply of the optic nerve in rats is via the central retinal artery and possibly the pia vessels, whereas in humans the blood supply is via the central retinal artery, pia vessels, and possibly via the choroid.These suggest that rat retinas may be more prone to the effects of transient IOP increases than human retinas, even though more experimental evidence is needed.Lastly the ganglion cell layer (GCL) morphology differs between humans and rats, with the average cell number being between 72,000 and 113,000 in rats and between 700,000 and 1,500,000 in humans, [42].
In humans ophthalmic surgery is usually carried out under local anaesthetic, so the impact of general anaesthesia on RGC survival should also be taken into Our laboratory is particularly interested in the role of connexins and astrocytic regulation after injury due to intraocular pressure increases, hence the focus on this marker of inflammation.However, future directions may also involve the examination of various other genes (and their proteins), such as the proinflammatory and inflammatory genes Bcl2, Birc4/XIAP, Cat and SAA1 genes, whose transcription levels have been shown to be altered by elevations in IOP, [7].
A limitation of the study is the use of a semiquantitative grading system, even though a masked observer analysed the images collected in order to reduce subjectivity.Other molecular biology approaches including PCR and Western Blotting may be useful in studying connexin43 and GFAP gene and protein expression changes.
Future studies should aim to more clearly define the threshold of IOP elevation and/or ischemia that will induce permanent retinal changes and subsequent permanent vision loss.This should include aged animals with IOP levels and durations pertinent to cataract surgery (40-80 mmHg for 15-30 minutes).There have been reports of increased RGC susceptibility to permanent injury after IOP elevation and sham cannulation insult in older rat eyes, [47].
In conclusion, the range of transient moderate IOP changes in ophthalmic procedures such DSEK and LASIK may provoke a glial response in the optic nerve, All procedures were conducted in compliance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Ethics Committee of the University of Auckland.Ninety-six adult male Wistar rats weighing 250-300 g were obtained from the Vernon Janson Unit at the University of Auckland and housed in a 12 hour light / 12 hour dark cycle and received food and water (Continued on page 38) Freely Available Online www.openaccesspub.org| JOS CC-license DOI : 10.14302/issn.2470-0436.jos-17-1453Vol-1 Issue 3 Pg.no.-38 ad libitum.Light in the animal breeding unit was provided by white fluorescence light (Philips Master TLD 18W/965; Koninklijke Philips Electronics N.V., Shanghai, China).The light source covers broad band fluorescence, from 380 to 760 nm, and the average intensity at the top of the cage is 120 W/m2.Ninety animals had unilateral transient elevation of IOP to 10 mmHg for 5 or 30 minutes (cannulation controls, n = 10 per time point), 40 mmHg for 5 or 30 minutes (n = 20 per time point), 60 mmHg for 5 minutes, (n=20) and 120 mmHg for 60 minutes (n = 10).Uninjured animals (n = 6) were also used.Transient Raised IOP Model Animals were given an intraperitoneal injection of ketamine (60 mg/kg, Parnell Technologies, New Zealand) and medetomidine hydrochloride (0.4 mg/kg, Pfizer Animal Health, Australia) and the cornea anaesthetised with oxybuprocaine hydrochloride (0.4%, Bausch & Lomb, UK).The animal's temperature was maintained using a heat pad.The left anterior chamber was cannulated with a 30-gauge infusion needle connected by silicone tubing to a reservoir of sterile 0.9% saline (Baxter, Australia).Cannulation was performed using a stereotaxic manipulator arm to avoid injury to the corneal endothelium, iris or lens.The saline reservoir was raised to a height corresponding to intraocular pressures of 10 mmHg for 5 or 30 minutes (normal IOP, cannulation control), 40 mmHg for 5 or 30 minutes, 60 mmHg for 5 minutes and 120 mmHg for 60 minutes (positive control based upon previous studies, [

43 (
locations per retina.This method ensured similar locations were assessed between different eyes.At each location, two images were taken -one of the nerve fibre layer, ganglion cell layer and outer plexiform layer (NFL/ GCL/OPL), and one of the inner nuclear layer, outer plexiform layer and outer nuclear layer (INL/OPL/ONL).The same settings were used within imaging of each retina.Owing to the low level of Cx43 in the normal retinas, imaging of the INL/OPL/ONL required a z-stack of 6 optical slices to be imaged, taken at 1 μm increments.Quantification of Cx43 was performed using automated spot counts in ImageJ software version 1.National Institutes of Health, Bethesda, MD) as previously described,[26].GFAP intensity in the anterior optic nerve was analysed by binomial logistic regression using fluorescence ('strong' or 'normal') as the dependent variable and IOP and time of manipulation as covariates.A p-value less than 0.05 for each predictor indicates significance.A correlation test was also performed between fluorescence, IOP and time of manipulation.A p-value less than 0.05 for each pair tested indicated a significant correlation between the pair of variables.Cx43 spot counts were analysed using multiple linear regression with average Cx43 spot count as the dependent variable and IOP and time of manipulation as independent variables.Student's t-test was performed to further compare each experiment group to the uninjured group.Retinal Ganglion Cell CountsTo investigate the effects of transient raised IOP on RGC survival in the long term, whole mount immunohistochemical techniques were used.For details refer to,[25].Eyes were enucleated and the posterior eye cup was fixed in 4% paraformaldehyde in phosphate-buffered saline, (60 minutes, room temperature).The retina was then removed and permeabilised with 0.5% Triton X-100, (15 minutes, -80°C).Following thorough washing with PBS, free floating retinas were incubated overnight at 4°C in goat anti-Brn3a primary antibody (SC31984, Santa-Cruz Biotechnology, 1:100), 2% horse serum, 2% Triton X-100, PBS.After washing, a donkey anti-goat Cy3 (705-165147, Jackson Immuno Research, 1:500) secondary antibody solution was applied for 2 hours, room temperature.Nuclei were labelled with DAPI and retinas mounted onto SuperFrost-Plus slides (Thermo Scientific, US) using CitifluorTM mounting medium.

Freely 41 Figure 1 .
Figure 1.GFAP labelling (red) in the optic nerve 4 hours post IOP manipulation.Blue labelling is DAPI for cell nuclei.(A) An optic nerve from the uninjured (i.e.completely naïve) group showing uniform GFAP fluorescence in both the anterior (first 500 µm) and posterior optic nerve.(B) An optic nerve from the 120 mmHg 60 minute group showing much stronger GFAP labelling in the anterior part of the optic nerve compared to the posterior partthis sample was assigned to the 'strong' labelling group.(C) Optic nerves from the 40 mmHg 30 minute group and (D) 60 mmHg 5 minute group showing stronger GFAP in the anterior optic nerve compared to the posterior optic nerve.Scale bar = 500 µm.(E) Graph showing the percentage of animals with normal (pale grey) or strong (dark grey) GFAP fluorescence in the anterior optic nerve (first 500 µm) examined 4 hours after transiently raised IOP for different periods of time.n = 6, 5, 5, 10, 10, 10, 5, respectively, from left to right.
were read from an ERG recording and Figure4Bshows examples of ERG waveforms in the scotopic threshold range and the b-wave range.There was very limited evidence for a decrease in pSTR (-5.1 to -3.9 log cd.s.m -2 ) and b-wave (-3.05 to 2.0 log cd.s.m -2 ) amplitude with cannulation and after transient IOP rise to 40 and 60 mmHg regardless of the duration of the IOP rise (Figure 4C and D).A smaller b-wave amplitude with significant difference compared to the baseline was only found in the 120 mmHg 60 minute positive control group at -2.7, -2.2, -1.3, and 1.4 log cd.s.m -2 , including 2 animals out of 4 that completely lost their pSTR and bwave response from -5.1 to -2.7 log cd.s.m -2 .pSTR amplitude (Supplementary figure2) and b-wave amplitude (Supplementary figure3) of the contralateral eyes at 14 days were compared to their respective baselines (the same eyes recorded before manipulation), and no significant difference was found in any group.
increases in IOP (40-60 mmHg over several minutes), similar to those that occur during many ophthalmic procedures such as DSEK, LASIK and femtosecond laser cataract surgery,[1-4], do not result in permanent functional retinal damage.This study extends the work of other investigators studying the effect of acute IOP elevations on retinal ERG responses.We did not see any change in ERG responses at 14 days, except in the positive control where IOP was raised to 120 mmHg for 60 minutes.Other investigators have considered the 30 minute time point or longer and have not seen permanent damage with moderate increases in IOP.In one study the pSTR significantly changed one week Freely Available Online www.openaccesspub.org| JOS CC-license DOI : 10.14302/issn.2470-0436.jos-17-1453Vol-1 Issue 3 Pg.no.-43

Figure 2 44 Figure 3
Figure 2. Representative confocal microscope single optical slice images of retina sagittal sections labelled for Cx43 (green), GFAP (red), and DAPI for nuclei (blue) and graphed Cx43 spot counts.(A) An uninjured retina showing regular glial processes labelled with GFAP in the NFL and Cx43 dots with some co-localisation in the same layer (resulting in yellow label).(B) A retina 14 days after 120 mmHg 60 minute manipulation shows reactive glial processes (red) and an increased amount of Cx43 label.(C) Retinas 14 days after 40 mmHg 30 minute and (D) 60 mmHg 5 minute manipulations did not change the number of Cx43 immunoreactive labelling spots or cause astrocytosis in the retina after 14 days.Scale bar = 100 µm.(E) Graph showing Cx43 spot counts in the NFL/GCL/ IPL.n=6, 5, 5, 10, 10, 10, 5 for each group from left to right.

Figure 4 .
Figure 4. Full-field ERG findings 14 days after a transient IOP increase.(A) Graphs showing how the various measurements were read from an ERG recording.pSTR amplitude and implicit time were measured from the baseline (0 µV and 0 ms) to the peak.nSTR amplitude and implicit time were measured from the baseline to the trough.awave amplitude and implicit time were measured from the baseline to the trough.b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave.b-wave implicit time was measured from the baseline to the peak.(B) Examples of ERG waveforms in the scotopic threshold range (first 4 rows) and the b-wave range (last 8 rows).pSTR amplitudes (C), and b-wave amplitude (D) measured 14 days after transiently raised IOP for different durations.Data from all cannulated eyes recorded before cannulation was pooled to give a representation of baseline pSTR or b-wave amplitudes at different flash intensities (black lines).* indicates significant differences compared to baseline at the particular flash intensity at a significance level of 0.025.(C-D) 10 mmHg 5 min n=5, 10 mmHg 30 min n=5, 40 mmHg 5 min n=10, 40 mmHg 30 min n=10, 60 mmHg 5 min n=9, 120 mmHg 60 min n=4, Baseline n=43.Error bars represent SEM.

the 40 mmHg 30
minute and 60 mmHg 5 minute groups 4 hours after manipulation, but not in the 40 mmHg 5 minute group.Both the degree and duration of the IOP elevation were significant predictors of astrocytosis, and functional changes in RGCs were seen when the IOP was raised to higher levels in the positive control group (120mmHg for 60 minutes).However, as in the Abbott et al. study, transient and moderate IOP rises did not induce long term disruption to RGC function or number as measured by electrophysiology and RGC counts, respectively.

Freely
Available Online www.openaccesspub.org| JOS CC-license DOI : 10.14302/issn.2470-0436.jos-17-1453Vol-1 Issue 3 Pg.no.-47 cribrosa seems to be dependent on rat strain.Collagen types I, III, and IV are found in the lamina cribrosa.In rats the lamina cribrosa is composed of more type IV collagen fibres than humans.Type I and III fibres are interstitial collagens, whereas type IV is a basement photoreceptor death, suggesting that anaesthesia may provide neuroprotective effects in light induced cell damage,[46].With advances in technology it may be possible, in the future, to perform ERG studies in animals without requiring general anaesthesia and thus removing the confounding effect of it.