The authors have declared that no competing interests exist.
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.
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 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.
n 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.
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 up-regulation 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.
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,
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,
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,
Transient IOP increases above the systolic blood pressurefor more than 1 hour have been shown to result in retinal ganglion cell (RGC), inner retinal layer and outer nuclear layer cell apoptosis,
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
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,
Immunohistochemistry and confocal laser scanning microscopy (Olympus FV1000) were used to investigate the immediate effects of transient raised IOP on astrocytosis and Cx43 expression in the retina and anterior optic nerves 4 hours post IOP elevation. 4 hours after IOP elevation eyes were enucleated and fixed in 4% paraformaldehyde for 2 hours. The eyes were washed in PBS and transferred to sucrose prior to freezing in optimal cutting temperature compound (IA018, ProSciTech) using liquid nitrogen. For further details of methods refer to,
GFAP intensity in the anterior optic nerve (the first 500 µm from the anterior end of the longitudinal section) as compared to the posterior optic nerve was assigned the category of either ‘strong’ or ‘normal’ by a masked observer. GFAP intensity in the GCL/NFL/OPL was also compared to posterior optic nerve intensity and assessed in the same way. To assess changes in Cx43, three locations were imaged from both the superior and inferior retina, starting one microscope field peripheral to the border of the optic disc, giving a total of six 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 1.43 (National Institutes of Health, Bethesda, MD) as previously described,
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.
To investigate the effects of transient raised IOP on RGC survival in the long term, whole mount immunohistochemical techniques were used. For details refer to,
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 mm2per retina. Quantification was performed using automated spot counts in NIH ImageJ software. RGC density was calculated as the number of RGCs per cm2. RGC density was compared to uninjured control and was analysed using Student’s t-test with a significance level of 0.05.
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 (custom-made) using a hand-held battery-operated ERG system (EPH-01, Ephios, Sweden) simultaneously from both eyes with silver / silver chloride electrodes (custom-made using a previously described method by Bui and Fortune) placed at the apex of the corneas,
The scotopic threshold response (STR) was recorded at -5.1, -4.5, -4.2, and -3.9 log cd.s.m-2 with 10 flashes per light intensity with 2 second inter-stimulus intervals, and 10 second between-light intensity intervals,
For each transient IOP treatment, 14 day ERG data at each flash intensity was compared to baseline from the same eyes of the same animals using paired sample two-tailed Student’s t-test. Two dependent variables (amplitude and implicit time) were tested against an independent variable - manipulation group. Because two outcome measures were tested against one hypothesised predictor, a Bonferroni-adjusted significance level of 0.025 was calculated to account for the increased probability of type one error. pSTR amplitude, pSTR implicit time, b-wave amplitude, b-wave implicit time, a-wave amplitude, and a-wave implicit time were compared.
The optic nerve was labelled with GFAP and the pattern of labelling in each eye was analysed. A binomial logistic regression analysis of GFAP labelling intensity in the anterior (first 500 µm) and posterior optic nerve 4 hours after manipulation showed that both extent of IOP elevation (p=0.008, B=0.100, Exp(B)=1.105) and the duration (p=0.012, B=0.104, Exp(B)=1.110) were statistically significant predictors for a higher probability of strong GFAP labelling (
The transient periods of raised IOP at 40 and 60 mmHg did not affect RGC density at the end of 14 days (
Retinal functional modifications were assessed 14 days after transiently raised IOP (
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.
This study suggests that transient and moderate 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,
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 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
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,
Cx43 has been shown to be up-regulated in both the GCL and anterior optic nerve in human glaucoma retinas,
It is of note that cannulation alone can cause an inflammatory response,
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),
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 consideration. A number of studies have looked at the effect of general anaesthesia on retinal response and it has been found that there are differing effects on the ERG depending on the anaesthetic used. One group showed that ketamine/xyalazine with pancuronium was the best combination to minimise eye movement and maximise retinal function,
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 pro-inflammatory and inflammatory genes Bcl2, Birc4/XIAP, Cat and SAA1 genes, whose transcription levels have been shown to be altered by elevations in IOP,
A limitation of the study is the use of a semi-quantitative 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,
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, but no significant changes in Cx43 expression. We have yet to define how persistent this response might be. No significant retinal functional changes or reduction in RGC number was found. These surgical procedures therefore appear to be relatively safe.
The authors report no conflicts of interest.
This study was conducted with support from the Save Sight Society of New Zealand. The funding body had no involvement in the collection, analysis, and interpretation of data, in the writing of the report, or in the decision to submit the article for publication.