Retinal electrophysiologic response to IOP elevation in living human eyes

Abstract

Purpose:

The relationships between intraocular pressure (IOP), ocular perfusion pressure (OPP), retinal perfusion, and retinal electrophysiologic responses have been explored experimentally across several animal models. These studies have demonstrated that elevated IOP reduces OPP, and when this reduction in OPP exceeds the autoregulatory capacity of the retina vasculature, retinal perfusion and electrophysiologic responses are reduced. This study aimed to evaluate these interactions for the first time in the living human eye.

Methods:

Five eyes from three research-consented brain-dead organ donors underwent optical coherence tomography with angiographic (OCT/A; Spectralis, Heidelberg Engineering) and electroretinographic (ERG, Diagnosys LLC) measurements while IOP was manometrically-elevated stepwise to pressures of 10, 30 and 50 mmHg. Systemic blood pressure (BP) was monitored continuously during testing. Correlation analysis was applied to assess association between ERG and OPP changes. In a single eye, prolonged IOP elevation was induced with viscoelastic injection and serial ERG measurements were obtained.

Results:

Reductions in inner retinal function defined by photopic ERG were observed with elevation in IOP and concomitant reduction in OPP. Reductions, especially in b-wave, and photopic negative response (PhNR) amplitudes and implicit times were significantly correlated with elevation in IOP and reduction in OPP. There were more appreciable changes in perfusion and functional responses in eyes tested while systemic blood pressure was lower. With prolonged IOP elevation, selective loss of the PhNR response was observed.

Conclusions:

In the living human eye, retinal perfusion and inner retinal function are acutely impacted by elevation of IOP, and this impact is related to systemic BP and OPP. This novel approach provides a viable model to study the autoregulatory responses to IOP elevation in the living human eye.

Introduction

Glaucoma is a leading cause of irreversible blindness worldwide [1–4] and results in progressive loss of retinal ganglion cells (RGC) with characteristic alterations of the optic nerve head (ONH) and progressive loss in the visual field. While the pathogenesis of glaucoma is incompletely understood, mechanical, vascular, cellular, and biochemical mechanisms all likely play an interactive role. Despite this complexity, the level of IOP remains a primary risk factor for the development and progression of glaucoma, and reduction of IOP is the only proven treatment to prevent or halt progressive glaucomatous injury [5]. Elevated IOP has been studied both acutely and chronically, and in both cases, can result in tissue deformation within the ONH and retinal vasculature [5]. Moreover, changes in IOP can directly alter vascular perfusion within the ONH, inner retina, and choroid, affecting the function of the neural retina [6–16]. Vital work in a wide variety of animal models with experimentally induced acute IOP elevation has begun to uncover the complex relationships between IOP, ocular perfusion pressure, retinal perfusion, and retinal electrophysiologic responses [7, 9, 12, 17–30].

Across animal models, sensitivity to IOP-induced ERG changes appears tightly coupled to BP and is thought to be driven by ocular perfusion pressure (OPP) [6–16, 31]. Moreover, ERG components arising from the inner retinal RGCs are selectively impacted at lower IOPs whereas at higher pressures there is a more generalized loss of ERG response, including diminished a-waves and b-waves, due to more global loss of retinal perfusion [32]. For this reason, ERG measurements have been proposed to ensure that animal models of glaucoma are sub-ischemic [33]. While these models have provided valuable insights regarding relationships between retinal function, ocular perfusion, and increased IOP, there are important cellular, functional, and anatomic differences in the retinas of these animal models compared with the human retina.

We have developed a novel resource, the Living Eye Project, that affords the unprecedented ability for the examination, imaging, electrophysiologic testing, and IOP manipulation performed in the living human eye of research-consented brain-dead organ donors. Following in vivo testing, the tissues can be made immediately available for additional ex vivo studies. Here, we evaluated the direct impact of IOP on retinal perfusion using OCT/A and measure the electrophysiologic changes induced by both acute and prolonged (4 hour) elevation of IOP in the living human retina for the first time.

Methods

Donor Eye Acquisition and Screening.

The Living Eye Project is a collaboration between the UAB Department of Ophthalmology and Visual Science, the Legacy of Hope (LoH) Donor Recovery Center and Advancing Sight Network (formerly the Alabama Eye Bank). LoH developed and opened the regional Donor Recovery Center at UAB, as previously described [34]. For this study, we performed ERG testing and OCT/A imaging while adjusting IOP manometrically in five eyes of three organ donors consented for research and maintained on life-support while awaiting organ procurement. All components of this study adhered to the Declaration of Helsinki and were approved by the UAB Institutional Review Board and LoH Research Review Board.

The procedures for screening and selection of research donors have been described previously [34]. In brief, a certified tissue procurement technician conducted a structured interview with the next-of-kin to obtain the medical and ophthalmic history of donors consented for organ and tissue transplant and research. Donors with a reported history of retinal, ONH or central nervous system disease, or whose medical records revealed ocular pathology other than cataracts and/or cataract extraction, were excluded from the current study. Only donors over 18 years old were included and there were no exclusions based on gender, race or ethnicity. All the eyes were directly examined by a specialist in glaucoma and neuro-ophthalmology (CAG), including ocular fundus examination by binocular indirect ophthalmoscopy. Only eyes with clear media, no signs of agonal effects on the retina or ONH, and no signs of ONH edema were included. In addition, any eyes with signs of retinal or ONH edema evident on initial baseline OCT imaging were excluded. After donor selection and clearance as a non-medical examiner case (as vitreous samples within the chain of custody are required), the Advancing Sight Network also certified that the eyes in question were appropriate for collection for research.

Acute IOP Elevation.

Following the above selection process, the eyes were cannulated with a 25-gauge anterior chamber maintainer (Anodyne Surgical, Inc, O’Fallon, Missouri) through a 1 mm keratome incision in the peripheral cornea. This cannula is designed to be watertight through the 1 mm planar corneal incision. A digital manometer (XP2i, Crystal Engineering; San Luis Obispo, CA) was placed at the same height of the donor eye (by using a Self-Leveling Cross Line Laser, Bosch GmbH, Gerlingen, Germany), in line with the infusion into the anterior chamber to monitor inflow pressure. Inflow was regulated manometrically, and IOP was initially set to 10 mmHg and the eye let equilibrate for 5 minutes before testing. The cornea was lubricated with balanced salt solution and a rigid gas permeable contact lens was fitted on the cornea to prevent evaporation and enhance OCT image quality.

After baseline imaging and ERG (each described in the next paragraphs) were obtained at 10 mmHg, IOP was raised to 30 mmHg, and then to 50 mmHg using a switch actuator. Finer increments of IOP elevation were not attempted due to the limited testing window available to perform the invasive testing, which are interspersed with the interventions needed for the organ recover process. At each pressure step, OCT/A imaging was repeated following IOP adjustment. Following OCT/A imaging, ERG testing was performed at baseline and at each IOP level. Measurements began immediately (< 5 seconds) following elevations and were completed over 20 mins. Systemic BP in the supine position was constantly monitored via an arterial line and was recorded during every step of each experimental procedure. OPP was computed at each step of acute IOP elevation as mean arterial pressure (MAP) – IOP, and MAP is 2/3 diastolic BP + 1/3 systolic BP. The common correction factor for OPP measured in the seated position of 2/3 was used. While our subjects were supine, they were not completely flat. Our protocol does not currently allow us to significantly reposition the donor to adjust the relative positions of these two pressure measurements.

OCT/A Imaging.

OCT/A imaging was performed with a second-generation spectral-domain OCT angiography (Spectralis OCT2; Heidelberg Engineering Inc., Germany) with the imaging head mounted on a custom counterweighted support arm, allowing for six-axis fine manipulation (Spectralis Flex Module^™). A baseline high-resolution 20° (6mm) cube scan of the macula and ONH are performed which consists of 512×496×512 voxels, 11μm spacing, with a target of 5 images averaged/scan per B-scan. All follow-up scans were aligned to the baseline scan using the Spectralis’s internal “TruTrack” algorithm.

ERG measurements.

Following OCT/A imaging, the Diagnosys ERG system with a portable ganzfeld stimulator (Diagnosys LLC, Lowell, MA) was utilized to test each eye individually. Pupils were dilated with 1% Tropicamide and 2.5% Phenylephrine drops, and ERGs were recorded with DTL Plus silver-nylon thread electrode (Diagnosys, Lowell, MA) draped into the inferior cul-de-sac with gold-cup electrodes at the temple (reference) and forehead (ground). Three electrophysiologic stimuli were presented under photopic conditions using a handheld monocular Ganzfeld stimulator (Diagnosys LLC, Lowell, MA) and were recorded using an Espion E³ Desktop Visual Electrophysiology System (Diagnosys, LCC, Lowell, MA). Photopic conditions included full-field flash ERG (3.0 cd.s/m² “white” flash on a 30 cd.s/m² white background, W/W ffERG), measuring every 0.5 ms in 6 trials per run with 4 runs performed per eye in each condition, flicker ERG (30 Hz with 30 cd background), measuring every 0.5 ms in 30 trials with 4 runs performed per eye in each condition, and a modified full-field ERG using red stimuli (2 cd/m² red flash on a 10 cd/m² blue background, R/B ffERG), measuring every 0.5 ms in 100 trials with 4 runs performed per eye in each condition to enhance the photopic negative response (PhNR) per the protocols from the International Society for Clinical Electrophysiology of Vision protocol [35, 36]. The PhNR stimulus was delivered for a duration of 4 ms. The protocols were all performed without a 60 Hz filter applied to remove line noise.

All recording trials were used to produce an average waveform and amplitude and implicit times for a-wave, b-wave, and the PhNR were measured. A-wave amplitude and implicit time was measured at the base of the a-wave trough from baseline (0 μV). B-wave amplitude and implicit time was measured from the a-wave trough to the peak of the b-wave. PhNR amplitude and implicit time is measured from the maximum negativity relative to the baseline.

Prolonged IOP Elevation.

Prolonged IOP elevation was induced in only one eye of one donor to examine the effects of longer IOP exposure on the ERG. In this donor, the left eye was randomly selected following the cannula removal used for acute testing. Healon viscoelastic (Johnson & Johnson, North Jacksonville, FL) was gradually injected into the anterior chamber and IOP was measured with a handheld Tonopen tonometer (Lombart Instruments, St Paul, MN). Healon was gradually injected to raise IOP to maintain the pressure at approximately 30 mmHg to avoid ischemia. Following initial injection, the IOP measurements were repeated at hourly intervals, and ERG testing was repeated at four-hour intervals until organ procurement.

Statistical analysis:

The relationship between IOP, OPPs and ERG responses was determined using a linear mixed-effect model (LME) with a random intercept per study subject and eye. An LME model was used to fit each response variable of interest as they vary with IOP and OPP (dependent variables). For each statistical model a goodness of model fit metric was computed according to the method proposed by Nakagawa et al. [37] for R-squared in mixed-effect models. Statistical analyses were performed in R (R Foundation for Statistical Computing: Vienna, Austria; lme4 package). A p-value of < 0.05 was considered significant.

Results

The demographic ocular characteristics and quantitative results of IOP, BP, and ERG amplitudes and implicit times are shown in Table 1. The amplitudes measured are within the age-matched normative parameters using DTL electrodes (PhNR normative range is on average −31 μV +/− 10 μV) with the Diagnosys ERG (Lowell, MA). The a-wave, b-wave, average flicker, and PhNR-wave amplitudes for each eye measured are plotted against OPP in Figure 1, while the LME model results for the analysis of associations between ERG parameters and either IOP or OPP are shown in Table 2 and 3, respectively. The individual ERG tracing for each eye along with OCT/A en face images of the retinal vasculature for each eye at each pressure level used in acute testing are shown in Figure 2 for AOC039 and AOC040 and the remainder as supplementary figures (Figures S1–S3). Estimates from LME models did not show a significant decrease in the a-wave amplitude or change in implicit time with both elevations in IOP or concurrent reduction in OPP. B-wave amplitudes were significantly decreased and implicit times were significantly increased with increasing IOP and concurrent decreasing OPP. The PhNR wave, which is normally hyperpolarizing, exhibits a significantly reduced amplitude (i.e. more positive) during increased IOP and decreased OPP with no change in implicit time. Reduction in flicker amplitude was significantly associated with changes in IOP and OPP, although the association was weaker than that seen with the b-wave and PhNR parameter. The scatter plots showing the individual changes within the group (Figure 1) illustrate a greater amplitude change in those individuals with lower OPP and that the only PhNR amplitude reduced with IOP and OPP changes in each of the individual eye tested, regardless of BP. These results are also consistent with animal models indicating the inner retina is more susceptible to IOP-induced functional losses.

Table 1.

A-wave, b-wave, PhNR, and photopic negative response (PhNR) amplitudes and implicit times for each donor at different intraocular pressures (IOP), Central cornea thickness (CCT). Mean blood pressure (BP) for each donor was also recorded and mean ocular perfusion pressure (OPP) was calculated as a result of BP and IOP.

Donor ID	Age	Sex	Race	CCT (mm)	Axial Length	IOP mmHg	Mean BP	Mean OPP	a-wave amplitude (μV)	a-wave implicit time (ms)	b-wave amplitude (μV)	b-wave implicit time (ms)	PhNR amplitude (μV)	PhNR implicit time (ms)	Flicker amplitude (μV))	Flicker implicit time (ms)
AOC039 OD	28	M	White	489 ± 4.9	23.83	10	113/57	40.45	−62.88	17.56	184.65	36.13	−47	68.72	208.79	27.89
						30		20.45	−66.03	17.67	182.65	36.83	−45.36	71.54	213.05	29.29
						50		0.45	−42.55	22.69	57.33	44.44	−23.32	70.53	93.54	31.02
AOC040 OD	26	F	White	610 ± 3.1	23.97	10	163/108	74.22	−74.14	17.13	223.24	38.19	−41.35	61.75	264.28	25.76
						30		54.22	−66.84	16.56	212.81	37.81	−31.17	63.34	240.13	24.85
						50		34.22	−65.16	16.56	208.78	39.19	−21.49	65.53	239.80	24.83
AOC040 OS	26	F	White	625 ± 8.6	23.89	10	163/108	74.22	−59.26	16.25	207.24	38.5	−42.52	61.16	201.24	25.52
						30		54.22	−70.77	16.75	225.71	39.56	−25.32	62.75	246.20	26.09
						50		34.22	−57.17	16.81	203.56	38.81	−25.19	62.16	191.11	24.92
AOC041 OD	29	M	Black	593 ± 4.9	23.51	10	103/64	41.33	−39.85	16.25	168.33	36.69	−20.49	76.38	141.15	25.25
						30		21.33	−35.93	16.31	146.49	36.75	−23.18	76.81	131.88	24.28
						50		1.33	−32.16	19.75	44.4	43.81	−14.3	72	83.74	32.24
AOC041 OS	29	M	Black	612 ± 4.1	23.43	10	143/87	60.45	−39.51	18.19	132.28	35.13	−30.44	75.03	149.34	32.43
						30		40.45	−34	15.75	123.86	36	−16.15	75.38	130.11	35.62
						50		20.45	−40.49	15.63	135.4	36.38	−23.28	74.53	126.50	33.22

Figure 1. Wave amplitude plotted against ocular perfusion pressure (OPP).

A. A-wave, B. b-wave, C. PhNR, and D. flicker amplitudes plotted against OPP. Systolic and diastolic blood pressures (BP) of the donor at the time of measurement acquisition indicated in the legend. Linear mixed effect model estimates reported for each sublot as insets. Black circles, AOC039 OD; pink squares, AOC040 OD; green triangles, AOC040 OS; dark purple inverted triangles, AOC041 OD; purple diamonds, AOC041 OS.

Table 2.

Modeling results evaluating the association between intraocular pressure (IOP) and a-wave, b-wave, and photopic negative response (PhNR), and flicker amplitudes (mV) and implicit times (ms).

ERG parameter		Estimates	Standard Error	r2	p-value
a-wave	Amplitude (mV)	0.191	0.101	0.04	0.087
	Implicit time (ms)	0.03	0.025	0.065	0.254
b-wave	Amplitude (mV)	−1.33	0.54	0.136	0.032
	Implicit time (ms)	0.09	0.037	0.302	0.029
PhNR	Amplitude (mV)	0.37	0.09	0.284	0.002
	Implicit time (ms)	0.009	0.026	0.0005	0.75
Flicker	Amplitude (mV)	−1.15	0.49	0.097	0.04
	Implicit time (ms)	0.047	0.048	0.039	0.353

Table 3.

Modeling results evaluating the association between ocular perfusion pressure (OPP) and a-wave, b-wave, and photopic negative response (PhNR), and flicker amplitudes (mV) and implicit times (ms).

ERG parameter		Estimates	Standard Error	r2	p-value
a-wave	Amplitude (mV)	−0.187	0.093	0.08	0.068
	Implicit time (ms)	−0.04	−0.04	0.23	0.069
b-wave	Amplitude (mV)	1.414	0.0471	0.36	0.01
	Implicit time (ms)	−0.085	0.029	0.35	0.012
PhNR	Amplitude (mV)	−0.337	0.083	0.28	0.002
	Implicit time (ms)	−0.011	0.024	0	0.673
Flicker	Amplitude (mV)	1.19	0.433	0.24	0.017
	Implicit time (ms)	−0.02	0.04	0.02	0.607

Figure 2. Electroretinography (ERG) traces and ocular coherence tomography angiography (OCT/A) images following elevated intraocular pressure (IOP) in one donor (AOC041) with high (A-B) and low (C-D) blood pressure (BP).

A-B. AOC041 OS, average BP 143/87. A. W/W full field (ffERG), flicker ERG, and R/B ffERG traces immediately following IOP of 10 mmHg (black lines), 30 mmHg (pink lines), and 50 mmHg (green lines), and B. OCTA images following IOP elevation. C-D. AOC041 OD, BP 103/64. C. W/W ffERG, flicker, and R/B ffERG traces immediately following IOP of 10 mmHg (black lines), 30 mmHg (pink lines), and 50 mmHg (green lines), and D. OCTA images following IOP elevation.

Figure 2 shows and illustrative example of W/W ffERG, flicker ERG, and R/B ffERG traces along with OCTA images in two tested eyes in the same donor, AOC041. During imaging and functional testing of the left eye (OS), the average BP was 143/87 (Figure 2A and 2B). However, during the testing interval available for the right eye (OD), the BP had fallen to 103/64 (Figure 2C and 2D). Unfortunately, due to the limited time possible in this setting for testing both eyes of this donor were not able to be tested during this period of low and high pressure. The right eye (OD, lower BP) showed greater reduction in all three ERG traces (Figure 2C) than the left eye (OS, higher BP) (Figure 2A). These functional losses were concomitant with reduction of perfusion apparent on OCTA scans (Figure 2D) that was less pronounced when the BP was higher (Figure 2A).

Figure 3 shows the results of R/B ffERG testing in the donor eye with a prolonged elevation of IOP at 30 mmHg. The a- and b-wave remained unaffected throughout the testing. These data show that while a- and b-waves are preserved at this level of IOP elevation, the PhNR-wave is lost over time, consistent with the origin of the PhNR being within the RGC layer (spiking tertiary retinal neurons) [38] and being selectivity impacted with prolonged IOP elevation (Figure 3).

Figure 3. Photopic electroretinography (ERG) traces show selective loss of photopic negative response (PhNR) wave (arrows) following a prolonged (4 hour) 30 mmHg sub-ischemic intraocular pressure (IOP) elevation in AOC040.

A. R/B ffERG immediately following 10 mmHg IOP elevation (black), B. R/B ffERG immediately following 30 mmHg IOP elevation (pink), and C. R/B ffERG 4 hours after sustained 30 mmHg elevation (pink, dashed).

Discussion

While the approaches employed in the project are novel to studies of the eye, there has been growing research involving research-consented brain-dead subjects since the 1980s [39] [40–43]. While brain death results in a non-functioning brain stem, and thus, requiring mechanical ventilation, individual organs such as the eye can still display normal organ anatomy and physiology. This creates a unique research opportunity to perform invasive testing in the in vivo human eye that heretofore was only possible by employing animal models that incompletely reflect the ocular anatomy and physiology of the human eye. Additionally, the ability to acquire the eyes of these donors as part of organ harvest allows for the unprecedented ability to correlate ex vivo experiments with in vivo evaluation in the living human eye for the first time.

Research within LoH only occurs if not at the expense of organ donation, with IRB approval, and with specific consent. All research consents are obtained from the donor’s family by dedicated staff who are trained to speak with families in a culturally sensitive and compassionate manner. The LoH furnishes a dedicated, comfortable, private, and on-site family room for this purpose. All subjects are taken care of with dignity in ICU level bays with 24-hour critical care nursing. This program was created to address the unique ethical needs of this type of research recognized with several prior research projects involved hematologic [39], cardiovascular, and oncologic [44] diseases [45]. We began the Living Eye Project in 2014 to develop a model to enable the ex vivo evaluation of human eyes that can be tested with invasive in vivo testing to provide a unique translational model. These studies have provided the first histologic validation of OCT images of the optic nerve head [34] and evaluated the ONH biomechanical response to changes in IOP[46] and cerebrospinal fluid pressure [47].

Using the first manometrically-controlled acute ocular hypertension model in the living human eye, the current study demonstrates that inner retinal function is selectively impacted by acutely increased IOP and a resultant decrease in OPP. Additionally, prolonged sub-ischemic IOP elevation causes selectively diminished PhNR amplitude. These results are similar to findings in ocular hypertensive studies in animals which have demonstrated a profound sensitivity of the inner retina to physiologic stress induced by IOP elevation or reduced BP [6–16]. These changes are greatest in the eyes that were tested with lower BP and OPP and illustrate the resilient autoregulation of the human retina.

Few previous studies have directly evaluated the ERG responses in living subjects to isolated changes in IOP in living humans and due to positional changes. While positional changes not only alter IOP but also change choroid thickness and CSF pressure which may confound the direct impact of changes in IOP alone, isolated elevation of IOP has been elevated in living subjects by compressing the anterior globe with ophthaldymomanometry or a suction cup to increase the IOP. These studies also demonstrated similar inner retinal sensitivity to increase IOP. However, the precise IOP is not measured simultaneously to the ERG recording with this approach and sustained IOP elevation cannot be performed.

Prior animal models have explored the impact of acute moderate sub-ischemic pressure elevation and ischemia/reperfusion injury. In these models, ERG components arising from the inner retina can be selectively impacted at lower pressures whereas at higher pressures, there is a more generalized loss of ERG response. This generalized loss of ERG response likely represents more complete retinal ischemia once IOP significantly exceeds the BP perfusing the retina and choroid [16]. Across animal models, this sensitivity to IOP-induced ERG changes appear tightly coupled to BP and thus is thought to be driven by OPP [11, 13, 31]. For this reason, ERG measurements have been proposed to ensure that animal models of glaucoma are sub-ischemic [33].

Short-term sub-ischemic ERG changes include the greatest reductions in the pattern ERG (PERG), PhNR and the positive and negative scotopic threshold response (STR). One study using a suction cup to produce moderately elevated pressure in human patients also showed a reduction in PERG responses [15]. However, because this approach cannot be used to produce a reliable, controlled, graduated IOP elevation and is too uncomfortable to use in most patients, it has never been widely employed. Moreover, PERG depends on intact outer retinal feed-forward signals, hence PERG reduction is not necessarily an indicative or a specific probe of inner retinal function, whereas photopic ffERG provides simultaneous probe of cones (early part of A-wave), cone bipolars (A-wave near its trough and B-wave) and RGCs (PhNR). Collectively, these changes indicate a reduction in RGC function as well as a longer functional recovery period for RGCs compared with the outer retinal layers following acute IOP elevation in the mammalian retina [19].

While this study is the first of its kind in the living human eye, the model has several limitations. First, due to the logistics of organ procurement, we are currently limited to testing photopic responses. Thus, we cannot evaluate the effect of IOP and OPP on scotopic responses as can be performed more readily in small animal models. However, the reliance on scotopic responses in small animal models is because the PhNR is not robust in rodent models as it is in higher primates. While the addition of scotopic ERG testing in this model would be complementary, photopic ERG and PhNR can quite readily distinguish changes of the inner and outer retina and RGCs in resposne to changes in IOP in this living eye model. Also, our study only evaluated normal eyes, and the availability of eyes from donors with glaucoma is limited in this model. This model can only practically evaluate prolonged ischemia to a maximum of 8–10 hours, due to the time restraints of organ procurement, thus, the long-term effects cannot be evaluated in this model. However, the short-term IOP elevation in animal models has provided valuable insight into the physiologic and mechanobiological response to IOP challenge. Moreover, many of the electrophysiologic and cellular responses in these short-term models parallel changes seen with longer term animal studies suggest that the initial insult sets in motion a cascade of damaging events within the inner retina. Additionally, the extrapolation of the absolute level of perfusion pressure to larger populations is limited. Arterial BP was measured with the donor in the supine position, but not completely flat. The body position of the organ donor was never significantly adjusted in the existing protocol. Hence, we did not zero the arterial manometer to eye level. Given they are not completely flat, it is difficult to define which corrections factor is appropriate and if that correction factor is appropriate in the organ donor setting. Future studies directly evaluating central retinal artery pressure would be helpful to refine the experimental approach.

This pioneering study to evaluate the ERG response in the living human eye has demonstrated ERG changes in response to acute and prolonged elevation of IOP which are similar to those observed in animal models of acute ocular hypertension. Future work evaluating the relationship between IOP change at a more granular level and associating regional change in blood flow with retinal function is underway. The ability to deliver a control sustained IOP elevation while preserving retinal perfusion and function in the brain dead organ donor eye will provide a unique model to study the acute impact of IOP on the human optic nerve and retina. This groundbreaking model can provide a transformative tool in the study of the impact of IOP on retinal and optic nerve structure, function, and perfusion, which will have broad applications to studies of glaucoma and ischemia-reperfusion injury.

Supplementary Material

Supplment Fig. 1

Figure S1. Electroretinography (ERG) traces and ocular coherence tomography angiography (OCTA) images following elevated intraocular pressure (IOP) in donor AOC039 right eye (OD). A. W/W ffERG, flicker, and R/B ffERG traces immediately following IOP of 10 mmHg (black lines), 30 mm Hg (pink lines), and 50 mmHg (green lines) and B. OCTA images following IOP of 10, 30, and 50 mmHg as indicated.

Supplement Fig. 2

Figure S2. Electroretinography (ERG) traces and ocular coherence tomography angiography (OCTA) images following elevated intraocular pressure (IOP) in donor AOC040 right eye (OD). A. W/W ffERG, flicker, and R/B ffERG traces immediately following IOP of 10 mmHg (black lines), 30 mm Hg (pink lines), and 50 mmHg (green lines) and B. OCTA images following IOP of 10, 30, and 50 mmHg as indicated.

Supplement Fig. 3

Figure S3. Electroretinography (ERG) traces and ocular coherence tomography angiography (OCTA) images following elevated intraocular pressure (IOP) in donor AOC040 left eye (OS). A. W/W ffERG, flicker, and R/B ffERG traces immediately following IOP of 10 mmHg (black lines), 30 mm Hg (pink lines), and 50 mmHg (green lines) and B. OCTA images following IOP of 10, 30, and 50 mmHg as indicated.

Abbreviations:

IOP

Intraocular Pressure

OPP

Ocular Perfusion Pressure

OCT/A

Optical Coherence Tomography With Angiography

ERG

Electroretinogram

BP

Blood Pressure

PhNR

Photopic Negative Response

RGC

Retinal Ganglion Cell

ONH

Optic Nerve Head

W/W ffERG

White/White Full-Field ERG

R/B ffERG

Red/Blue Full-field ERG