Functional Changes in the Retina during and after Acute Intraocular Pressure Elevation in Mice

Functional Changes in the Retina during and afterAcute Intraocular Pressure Elevation in Mice

Yu X. Kong,1,2 Jonathan G. Crowston,1 Algis J. Vingrys,2 Ian A. Trounce,1,3 andBang V. Bui2

PURPOSE. To examine retinal function using the full-field elec-troretinogram (ERG) during and after acute intraocular pres-sure (IOP) elevation in wild-type mice.

METHODS. IOP was elevated by anterior chamber cannulation inwild-type C57/BL6 mice. The pressure–function relationshipwas determined by IOP elevation in steps from baseline to 80mm Hg. The rate of functional recovery was assessed for 60minutes after an IOP spike of 50 mm Hg for 30 minutes. Duringand immediately after IOP elevation, scotopic ERG signals wererecorded in response to dim and bright flashes (�4.54, �2.23,and 0.34 log cd � s � m�2) and analyzed for photoreceptoral(a-wave), ON-bipolar (b-wave), oscillatory potentials (OPs),and scotopic threshold responses (positive [p]STR/negative [n]STR). A full ERG protocol was collected 2 days before and 7days after the single 50-mm Hg IOP spike.

RESULTS. The pSTR was most sensitive to IOP elevation with50% amplitude loss (�) at 41 mm Hg (�, 95% confidence limits(CL): 37.7, 45.6) followed by nSTR at 45 mm Hg (95% CL: 41.0,49.1). pSTR was significantly more sensitive than the b-wave(95% CL: 41.4, 49.1), a-wave (95% CL: 47.6, 55.3), and OPs(95% CL: 49.6, 59.2). pSTR showed slower recovery immedi-ately after the 50 mm Hg spike compared with the b-wave (P �0.02). One week after the 50-mm Hg spike, pSTR (�30% � 6%,P � 0.001) and OP (�27% � 2%, P � 0.001) amplitudes werereduced, whereas other components were unaffected.

CONCLUSIONS. The STR in mice is more sensitive to acute IOPelevation and recovers slower than other ERG components.Reduction in pSTR and OP amplitude at 1 week suggestspersistent impairment of inner retinal function can occur aftera single IOP spike. (Invest Ophthalmol Vis Sci. 2009;50:5732–5740) DOI:10.1167/iovs.09-3814

Experimental mouse models are increasingly important instudies of neurodegeneration and glaucoma, largely due to

an improved understanding of mouse genetics, the relativeease of genetic modifications and an ever-increasing number ofmurine lines with well-defined genotypes and phenotypes.

Several models of intraocular pressure (IOP) elevation, bothinduced and spontaneously occurring, have been used to studyretinal neuronal injury in mice.1–5 Although retinal functionhas been assessed after IOP elevation in mice, detailed data ofthe retinal response during and after a well-controlled acuteIOP elevation remain sparse.

Investigators have examined the effect of acute IOP eleva-tion on retinal function in humans and in some animal mod-els.6–9 In general, their studies show that an IOP elevation of30 to 35 mm Hg is needed to induce retinal dysfunction. Bui etal.10 showed in rats that the inner retina derived scotopicthreshold response (STR) was more sensitive to acute IOPelevation than ERG components arising from photoreceptors(a-wave) and bipolar cells (b-wave).10

The effect of a single acute IOP elevation on retinal functionand its recovery has been investigated in the DBA/2J mice, astrain predisposed to spontaneous glaucomatous optic neurop-athy. Nagaraju et al.11 showed that a 32% to 38% increase inIOP (�5 mm Hg) could be achieved by a 60° head downposition. Although this IOP change had no effect on the pat-tern ERG in 3-month-old DBA/2J mice, it produced a �65%amplitude reduction in 10-month-old mice. The pressure–func-tion relationship to a moderate IOP challenge has yet to beassessed in wild-type mice.

In this study, we examined in detail the pressure (IOP)–function relationship in adult wild-type mice during a step-wiseincrease in IOP. In particular, we examined whether ERGcomponents arising from the inner, middle, and outer retinashow different sensitivity to IOP elevation. We also consideredwhether there was a difference in the immediate (60 minutes)and medium-term (7 days) recovery of ERG components aftera single IOP spike.

MATERIALS AND METHODS

Animals

All experimental methods and animal care procedures conformed tothe ARVO Statement for the Use of Animals in Ophthalmic and VisionResearch and were approved by our Institutional Animal ExperimentEthics Committee (The University of Melbourne, 07087327.1). Wild-type C57/BL6 mice of 12 months of age (30–35 g, Royal Victorian Eyeand Ear Hospital) were maintained in a 22°C, 12-hour light (�40lux)/12-hour dark environment with normal murine chow (WEHIbreeder mix; Barastoc, VIC, Australia) and water available ad libitum.

Electroretinography

Full-field ERGs were recorded simultaneously from both eyes. In brief,animals were dark-adapted overnight (�12 hours) and prepared forcannulation and ERG recording in the dark with head-mounted night-vision goggles (Scout2; Trivisio Prototyping GmbH, Dreieich, Ger-many). Animals were anesthetized with intraperitoneal injection ofketamine-xylazine (70 mg/kg and 7 mg/kg, respectively; Troy Labora-tories Pty Ltd., Smithfield, NSW, Australia) followed by supplementa-tion with 20% of the initial dose every 30 minutes as previously

From the 1Center for Eye Research Australia, University of Mel-bourne, East Melbourne, Victoria, Australia; the 2Department of Op-tometry and Vision Sciences, University of Melbourne, Parkville, Vic-toria, Australia; and the 3Department of Medicine, St. Vincent’sHospital, University of Melbourne, Fitzroy, Victoria, Australia.

Supported by National Health and Medical Research CouncilGrants 475603 (JGC) and 400127 (BVB); Ophthalmic Research Insti-tute of Australia grants; the Glaucoma Australia Fund; the Henry Green-field Research Fund; and the Cranbourne Trust Fund.

Submitted for publication April 5, 2009; revised June 4 and 27,2009; accepted September 8, 2009.

Disclosure: Y.X. Kong, None; J.G. Crowston, None; A.J. Vin-grys, None; I.A. Trounce, None; B.V. Bui, None

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be marked “advertise-ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Bang V. Bui, Department of Optometryand Vision Sciences, University of Melbourne, Parkville, 3010 Victoria,Australia; [email protected].

Investigative Ophthalmology & Visual Science, December 2009, Vol. 50, No. 125732 Copyright © Association for Research in Vision and Ophthalmology

described.12,13 Mydriasis was achieved with 1 drop of tropicamide(0.5%, Alcon Laboratories, Pty, Ltd., Frenchs Forest, NSW, Australia)and phenylephrine (2.5%, Minims; Chauvin Pharmaceuticals, Surrey,UK). Corneal anesthesia was achieved with a single drop of proxymeta-caine hydrochloride (0.5%; Alcon Laboratories, Inc.). Animals werelightly secured to a platform with wire loops across the upper back andnose. A circulating warm water heating pad was used to maintain bodytemperature (37–38°C).

The pulse was monitored continuously throughout each experi-ment and systolic blood pressure was measured at regular intervals byusing a noninvasive tail-cuff sphygmomanometer (ML125; ADInstru-ments, Bella Vista, NSW, Australia). This method was used to ensureconstant blood pressure throughout the experiment, to avoid con-founding readings induced by changes in ocular perfusion pressure.There was no significant change in blood pressure (average 105 � 5mm Hg) over the experimental period (ANOVA P � 0.63) consistentwith similar observations made in rat.14

Signals were recorded using custom-made silver/silver-chlorideelectrodes (99.99% purity, 0.329 mm 1⁄4 29 G; A&E Metal Merchants,Sydney, NSW, Australia) adapted from those used for rats.14,15 Theactive electrode was centered on the cornea using micromanipulators(KITE; WPI). The inactive electrode comprises a loop (3 mm diameter)that fits around the equator of the eye, to rest behind the limbus. Bothof these are referenced to a stainless steel ground (F-E2–60; GrassTechnologies, West Warwick, RI) inserted into the tail. We found thatthis montage offered the low noise levels needed for scotopic thresh-old response (STR) recordings.16

After the mouse was placed on the heated platform, electrodeplacement and anterior chamber cannulation (described later) wereperformed in darkness with the assistance of monocular night-visionscopes (NVMT1; Yukon Advanced Optics, Mansfield, TX) fitted inplace of the eye pieces of a dissecting microscope (MZ6; Leica, Wet-zlar, Germany). Illumination was provided by an infrared light source(QC3650; Jaycar Electronics, Rydalmere, NSW, Australia). This allowedaccurate cannulation and electrode positioning while maintaining darkadaptation. The eyes were lubricated after electrode placement andperiodically throughout the session with 1.0% carboxymethylcellulosesodium (Celluvisc; Allergan, Irvine, CA). Signals were amplified 1000�and recorded with a band-pass setting of 0.3 to 1000 Hz (�3 dB) (P511AC Preamplifier, Grass Telefactor) and 4 kHz acquisition (Powerlab8SP; ADIntruments).

Light stimuli were brief (1 ms) white flashes (5-W white LEDs,5500°K; Luxeon Calgary, ALB, Canada) delivered via a Ganzfeld inte-grating sphere (Photometric Solutions International, Huntingdale, VIC,Australia). The integrated luminous energy was calibrated with a pho-tometer (IL1700; International Light Research, Peabody, MA) with ascotopic luminosity filter (Z-CIE) in place. The scotopic luminosityfilter was calibrated in terms of rodent photopigment absorbance (�max

� 502 nm).17,18 Based on an average pupil area of 7 mm2, our flash of0 log cd � s � m�2 gave 516 �/rod, consistent with previous calcula-tions.12,19

Before IOP elevation, all animals underwent baseline ERG record-ings at a range of luminous energy. From �5.92 to �4.73 log cd � s �

m�2 20 to 40 responses were averaged, with an interstimulus interval(ISI) of 1.5 to 3 seconds. Between �4.54 and �1.90 log cd � s � m�2 2to 10 responses were averaged with an ISI of 3 to 10 seconds. From1.32 and 2.22 log cd � s � m�2 single responses were recorded with theISI progressively lengthened from 10 to 120 seconds. Acute IOP ele-vation experiments were conducted on the same animal within 2 daysof this baseline session.

Acute IOP Elevation

After inactive electrode placement, the anterior chamber of the left eyewas cannulated with a glass Pasteur pipette (�50 �m, 1B100–6; WPI,Sarasota, FL) connected via polyethylene tubing (0.97 mm inner diam-

eter; Microtube Extrusions, North Rocks, NSW, Australia) to a pressuretransducer (Transpac IV; Abbott Critical Care Systems, Sligo, Ireland),which was in series with a sterile Hanks’ balanced salt solution reser-voir (JRH Biosciences, Lenexa, KS). IOP was controlled by adjustingthe height of the reservoir to precalibrated levels and maintainedwithin 1 mm Hg of the target pressure. IOP calibration was performedby reference to an aneroid sphygmomanometer (Livingstone, Rose-bery, NSW, Australia) as previously described.14 Cannulation was per-formed with the reservoir height set to eye level and the reservoir valveclosed. Variation in needle diameter can lead to differences in flowrate, which may vary the time taken for IOP to stabilize. The anteriorchamber was viewed under infrared illumination during valve opening,to ensure that these differences did not significantly affect IOP equili-bration, and rapid filling was observed in all cases (�5 seconds). Onintraocular pressure equilibration, aqueous flow through normal out-flow channels can result in a small difference between measured IOPand actual IOP due to resistance in the glass pipette. Our calibration offlow rate (0.23 � 0.01 mm Hg per �L/min) through a glass needlefound that within the range of IOP used in our experiments (up to 80mm Hg) the overestimation of IOP due to pipette resistance (�0.2 mmHg) was negligible (see Supplementary Material, online at http://www.iovs.org/cgi/content/full/50/12/5732/DC1). This calculation for miceeyes assumes conventional outflow to be 0.0051 � 0.0006 �L/min permm Hg of IOP and uveoscleral flow to be 0.148 �L/min at a pressureof 80 mm Hg.20 As such, we believe that the resistance through thepipette did not alter the interpretation of our experimental results.

Baseline ERG responses were obtained after 10 minutes of furtherdark adaptation, with IOP maintained manometrically at baseline (12mm Hg). Baseline responses were recorded to luminous energies thatelicited an STR (40 signals averaged, �4.54 log cd � s � m�2), a saturatedrod ON-bipolar cell dominated b-wave (single flash, �2.23 log cd � s �

m�2) and a photoreceptoral a-wave response (single flash, 0.34 log cd �

s � m�2). The choice of luminous energy for the STR is consistent withthat in previous work in mice.1,12 On completion of baseline ERGmeasurements, the saline reservoir was adjusted to give IOP levelsaccording to one of the following protocols.

Protocol 1: IOP-Response Function. IOP was raised in a

step-wise manner starting from 25 up to 50 mm Hg in 5-mm Hgincrements and from there up to 80 in 10-mm Hg increments. At eachstep, IOP was stabilized for 10 minutes before the same ERG record-ings (3 luminous energies) were made (n � 6, animals).

Protocol 2: Time Course of Fixed IOP Spike. IOP was

raised to 50 mm Hg for 30 minutes (spike) and then decreased tobaseline (�12 mm Hg) for recovery. During both the insult andrecovery (60 minutes) phases, ERG recordings were made to dim(�4.54 log cd � s � m�2) and intermediate luminous energies (�2.23 logcd � s � m�2, rod b-wave response) at 10-minute intervals (n � 10,animals).

On completion of the experiment, the cannula was removed, andanimals were allowed to recover on an electric heating pad. Negativecontrols were established with sham experiments in which the eyewas cannulated and IOP remained at baseline (n � 4), to control forthe effect of experimental manipulation. A full scotopic series of ERGrecordings was repeated in the same animals 7 days after IOP chal-lenge.

Data Analysis

Photoreceptor Response. Analysis of rodent electroretino-

gram components in this laboratory have been described in detailelsewhere.10,14–16 Briefly, for the full scotopic ERG protocol the lead-ing edge of the photoreceptor (P3) a-wave, which reflects photorecep-tor activity,21 was quantified by modeling the highest four luminousenergies (1.29, 1.62, 1.92, and 2.22 log cd � s � m�2) as an ensembleusing a delayed Gaussian function.22 This was achieved by (see Wey-mouth and Vingrys15 for details) floating the saturated amplitude

IOVS, December 2009, Vol. 50, No. 12 Acute Intraocular Pressure and Retinal Function in Mice 5733

(Rmp3; �V), sensitivity (S; m2 � cd�1 � s�3), and delay (td; ms) tominimize the sum-of-squares merit function (Excel Solver; Microsoft,Redmond, WA). For the single flash responses measured during or afterIOP challenge, P3 modeling is not viable and a-wave amplitude wasquantified at a fixed time of 6 and 8 ms after stimulus onset, tominimize contamination from nonphotoreceptoral contributions.23,24

The data presented herein are for 8 ms, to improve signal-to-noise;however, the 6-ms analysis gave the same outcome.

Bipolar Cell Response. The postreceptoral P2 waveform re-

flects the activity of bipolar cell responses.22,25–27 The P3 derived fromthe delayed Gaussian model was digitally subtracted from the raw ERGto expose the P2–OP (oscillatory potential) complex, which was thendigitally low-pass–filtered (47 Hz, �3 dB) to return the P2.15 P2 am-plitude was measured from the filtered waveform at a fixed time of 110ms from stimulus onset, which corresponds to the average peak ofcontrol eyes (n � 10) at �2.23 log cd � s � m�2.

Scotopic Threshold Response. The STR has been shown in

mice12,28 and rats16 to contain ganglion cell and some amacrine cellcontributions. Response amplitudes of the positive (p)STR and nega-tive (n)STR components were measured at fixed times of 120 and 220ms after the stimulus flash, respectively. These fixed times were cho-sen to correspond to the STR peak and trough of control eyes (n � 10).

Oscillatory Potentials (OPs). OPs were isolated from the

flash response to 0.34 log cd � s � m�2. The effect of the a-wave on theOPs was reduced by removing the raw waveform up to the minimumof the first electronegative deflection.29 The resulting waveform wasthen digitally band-pass–filtered (fifth-order Butterworth, �3 dB at 55and 210 Hz) to isolate the scotopic OPs. The OPs were then modeledusing a Gabor function, as described previously.29 A Gabor comprisesa Gaussian envelope that locates the oscillation as a function of time(m; milliseconds) with maximum amplitude (a; microvolt) and spread(s; milliseconds), and a resonating element that specifies the frequency(h; hertz) and phase (p; radians) of the oscillation.29 The parameters ofthe Gabor were optimized by minimizing the sum-of-squares meritfunction (Excel Solver; Microsoft).

Retinal Function versus IOP. IOP effects were expressed as

relative amplitudes (treated/baseline, %) for the various components ateach IOP step. Group data were shown as mean (�SEM) and plotted asa function of IOP for each ERG parameter. The IOP pressure-functionrelationship was modeled using an inverse cumulative normal.10 Themodel returns the mean (�), which indicates the IOP resulting in 50%amplitude reduction; and the SD (�). A smaller SD equates to a steeperslope.30 The model was fitted to the relative amplitude at all pressurelevels, with the maximum fixed to 100%.

Sensitivity of various ERG components to acute IOP elevation wascompared by contrasting parameters in terms of their 95% bootstrapconfidence limits, with an effective nonparametric � of 0.05. Thebootstrap method has been described elsewhere.10,14,29,31 In short,several synthetic populations of size n (bootstrap samples) were gen-erated by random selection (with replacement) from the available datasets across IOP.32 Each synthetic sample was parameterized with theinverse cumulative normal function to give an estimate of � and � forthat particular bootstrap sample. After 1000 repetitions, the nonpara-metric 2.5% and 97.5% confidence limits for � and � could be derived.

Statistical Methods. One-way, repeated-measures analysis of

variance (ANOVA; SPSS ver. 15.00; SPSS Inc., Chicago, IL) was appliedto test the null hypothesis that there was no effect of IOP elevation onERG component (� � 0.05). Two-way ANOVA was used to test thestatistical difference between groups across time or luminous energy,and Tukey’s test was used for post hoc analysis of subcategories.

RESULTS

Differential Sensitivity of ERG Components toIOP Challenge

Figure 1 shows the effect of step-wise IOP elevation (protocol1) on the retinal function of a representative animal. Thewaveform elicited with the dim flash demonstrates the charac-

FIGURE 1. Representative ERG wave-forms measured at dim (A, �4.54 logcd � s � m�2), medium (B, �2.23 logcd � s � m�2) and bright (C, 0.34log cd � s � m�2) luminous energies.Responses from the IOP challengedeye (thick traces) at progressively in-creasing IOPs are compared with thewaveform recorded at baseline (thintraces). Dotted vertical lines: repre-sent the fixed times at which the am-plitudes of pSTR (120 ms), nSTR (220ms), b-wave (110 ms) and a-wave(8 ms) were measured.

5734 Kong et al. IOVS, December 2009, Vol. 50, No. 12

teristic STR of mice with a positivity at approximately 120 msand a negativity at 220 ms.12 The response to the brightest flash(0.34 log cd � s � m�2) showed an a-wave trough followed by adominant corneal positive b-wave. Oscillatory potentials canbe seen superimposed on the b-wave.

Consistent with a study in rats,10 increased IOP attenuatedthe ERG in a dose-dependent manner. The STR was affected atIOP levels that were lower than those needed to reduce otherscotopic ERG components. In particular, the pSTR was re-duced at 30 mm Hg, whereas the a- and b-wave remainedlargely unaffected. By 80 mm Hg, only a small slow negativea-wave remained with all other ERG components abolished.

In addition to the reduction in amplitude, ERG componentsshowed an increasing delay with higher IOPs. Delay in peaktimes of up to 13 � 4 ms for pSTR, 38 � 11 ms for nSTR, and9 � 4 ms for b-wave were found when IOP was elevated frombaseline to 45 mm Hg (one-way ANOVA, P � 0.001, P � 0.04,P � 0.03, respectively). As such, ERG component amplitudeswere analyzed at fixed times to better capture the combinedamplitude and timing changes.

To consider the relative sensitivity of ERG components toIOP elevation, data have been expressed relative to baseline.Figure 2A shows that IOP elevation had similar effects on bothpSTR and nSTR (two-way ANOVA, P � 0.46).

Figure 2B shows that the pSTR pressure–function relation-ship was leftward of the b- and a-wave and had a steeper slope,

suggesting greater pSTR sensitivity to IOP manipulation. Thiswas confirmed with the pSTR pressure–function relationshipreturning a mean (�) significantly below the lower confidencelimit for the b- and a-wave (Table 1). In addition, the cumula-tive normal function fitted to the pSTR returned an SD (�) thatwas significantly smaller than the lower limit for the a-wave �,indicating a steeper slope. The nSTR was not significantly moresensitive than the b-wave (Table 1). The b-wave pressure–function relationship also had a significantly lower mean andsteeper slope than did the a-wave (Table 1).

Figure 2A shows that scotopic OP amplitudes were signifi-cantly less sensitive to IOP elevation compared to the pSTR(two-way ANOVA, P � 0.001) and nSTR (P � 0.001). Furtheranalysis of IOP effects on OP characteristics was achieved byfitting the filtered OP waveforms with the Gabor model. Figure3 shows that IOP-related reduction in OP amplitude was ac-companied by a reduction of OP frequency (by �19 � 4 Hz,ANOVA, P � 0.03), delay in OP peak time (from 34 � 1 to 55 �11 ms, P � 0.05) and increased OP waveform spread (from 4 �3 to 16 � 4 ms, P � 0.007). There was also a delay in phase forIOP levels of 35 mm Hg (phase change � �1.94 � 0.55, t-test,P � 0.02) and 40 mm Hg (phase change � �1.63 � 0.60, P �0.04). IOPs of 70 and 80 mm Hg resulted in OP amplitudes thatwere within the noise of the system (�10 �V), thereforereturning unreliable Gabor parameters; these recordings havebeen omitted from the figure.

Rate of Functional Impairment and Recoveryfrom an IOP Spike

To fully characterize the response of ERG components duringand after an acute IOP spike, we subjected treated eyes to afixed IOP of 50 mm Hg for 30 minutes followed by return tobaseline for 60 minutes (protocol 2). Figure 4 shows the STRand scotopic bright-flash waveforms for a representative ani-mal at baseline, during IOP challenge and at various timesduring recovery. Attenuation in the STR and b-wave amplitudewas evident at 30 minutes after IOP spike onset. Functionalrecovery could be seen after IOP returned to baseline.

Figure 5 shows group data (n � 10) for relative pSTR, nSTR,and b-wave amplitude during and subsequent to the IOP spike.During the IOP spike, all ERG components declined gradually.At 30 minutes, the pSTR, nSTR, and b-wave amplitudes werereduced by 56% � 8%, 49% � 13%, and 43% � 6%, respec-tively, relative to baseline. Significant delays in peak time forpSTR and nSTR components were also observed during IOPelevation (Figs. 5B, 5C). When compared to the gradual step-wise IOP elevation to 50 mm Hg (protocol 1), the 50 mm HgIOP spike (protocol 2) showed a similar degree of pSTR, nSTR,and b-wave amplitude loss (t-test; P � 0.32, P � 0.56, P � 0.22,respectively).

After IOP restoration to baseline, pSTR amplitude recoverywas significantly slower compared with b-wave recovery dur-

TABLE 1. Parameters and 95% Bootstrap Confidence Limits for theCumulative Normal Function Used to Describe the Amplitude-Pressure Relationship

ERGComponent

Mean(mm Hg) � 95% CL SD (�) 95% CL

pSTR 41.2 37.7, 45.6 10.8 4.6, 16.1nSTR 45.0 41.0, 49.1 12.2 7.0, 15.7b-Wave 45.1 41.4, 49.1 12.2 6.5, 15.9a-Wave 51.0 47.6, 55.3 19.1 14.5, 23.0OP 55.1 49.6, 59.2 7.2 1.6, 11.0

FIGURE 2. Relative change in ERG amplitude at various IOP levels(mean � SEM, n � 6). (A) Inner retinal components of the ERG (pSTR,nSTR, and OP amplitudes) in response to IOP. Pressure–function rela-tionships for pSTR (thin solid line) and OP (thick solid line) areshown. (B) Comparison of pSTR, b- and a-wave responses to IOP.Pressure–function relationships for pSTR (thin solid line), b-wave(dashed line), and a-wave (thick solid line) were described by using acumulative normal function.

IOVS, December 2009, Vol. 50, No. 12 Acute Intraocular Pressure and Retinal Function in Mice 5735

ing the 60-minute period (two-way ANOVA, P � 0.02). Therewas no statistical significance between nSTR and pSTR recov-ery (two-way ANOVA, P � 0.13). Although nSTR amplitudewas within normal limits by 60 minutes of recovery, thereremained a significant nSTR peak time delay of 19 � 5 ms (P �0.01) (Fig. 5C). There was also significant pSTR delay of 7 � 2ms (P � 0.02) at 50 minutes recovery (Fig. 5B).

Retinal Function 1 Week after IOP Spike

To examine the longer term effects of a single acute IOP spikeon ERG components, we reassessed full scotopic ERG record-ings 7 days after the single IOP spike used in protocol 2. Therewas no significant difference in IOP measured before or 7 daysafter the acute spike (13 � 1 mm Hg vs. 14 � 1 mm Hg; t-test,P � 0.54). These value represent the average of three repeatedreadings on lightly anesthetized animals under room lighting(�100 lux) using a noninvasive rebound tonometer (iCareFinland, Espoo, Finland). Figure 6 shows that there was nosignificant change in b-wave (two-way ANOVA, P � 0.46),

a-wave (two-way ANOVA, P � 0.09), or nSTR amplitude (two-way ANOVA, P � 0.15) at 7 days after IOP treatment comparedwith baseline. Phototransduction amplitude (RmP3) and sensi-tivity (S) were similar at baseline and 7 days after the IOP spike(RmP3: 355 � 28 �V vs. 340 � 36 �V, P � 0.75; S: 1429 � 117vs. 1390 � 91 m2 � cd�1 � s�3, P � 0.80, data not shown). Incontrast, the pSTR was significantly reduced by an average of30% � 6% (ANOVA; P � 0.001) compared to baseline (acrossluminous energies from �5.92 to �4.73 log cd � s � m�2).Likewise, OP amplitude was significantly reduced by an aver-age of 27% � 2% (ANOVA; P � 0.001 across luminous energiesfrom �1.9 to 2.22 log cd � s � m�2).

DISCUSSION

To our knowledge, this is the first study to describe in detailthe pressure–function relationship of wild-type mouse ERGcomponents, during and after acute IOP elevation. We show

FIGURE 3. Changes in scotopic OPsat various IOP levels (mean � SEM,n � 6). Parameters are derived byfitting a Gabor model to the ex-tracted OP waveform recorded to0.34 log cd � s � m�2. (A) Gabor func-tion is composed of a Gaussian enve-lope and sine wave carrier. (B) Rep-resentative extracted OP waveformsobtained at baseline IOP, 35, 50, and80 mm Hg. (C) Relative OP ampli-tude; (D) OP frequency h; (E) OPpeak time m; (F) overall spread s ofthe oscillations in response to in-creasing IOP. The data for parame-ters m, h, s at IOP of 70 and 80 mmHg have been omitted, as the smallamplitudes of OPs at these IOP levels(see B) preclude a sensible fit of theGabor model.

5736 Kong et al. IOVS, December 2009, Vol. 50, No. 12

that the pSTR in mice was more sensitive to acute IOP eleva-tion than those components arising from the middle (the b-wave) and more distal retinal layers (the a-wave, Fig. 2). Re-covery of the pSTR after a single IOP spike was also slowercompared with that of the b-wave (Fig. 5). In addition, thepSTR and the OPs showed persistent impairment 1 week aftera single IOP spike (Fig. 6).

Pressure–Function Relationship

The pSTR has been shown to have a significant ganglion cellcontribution,12,16,33–35 and is the full-field ERG componentmost sensitive to chronic IOP elevation in both rats andmice.1,36 The higher sensitivity of the STR compared withother ERG components during acute IOP elevation is consis-tent with a rat model of acute IOP challenge.14 The pSTRamplitude attenuation was detectable at IOP levels as low as 30mm Hg. This is similar to results in rats, where STR timingchanges were noted at a similar pressure10 and in mice sub-jected to chronic IOP elevation of 35 to 38 mm Hg.1 Given thatour mice had systolic blood pressures of 105 � 5 mm Hg, ourdata indicate that the pSTR becomes affected at an ocularperfusion pressure (blood pressure – IOP)37 of �75 � 5 mmHg. Previous studies have found that b-wave changes are seenwhen ocular perfusion pressure is reduced to 30 to 45 mmHg.38,39 This is consistent with other reports that ocular per-fusion pressures as low as �35 mm Hg are needed before opticnerve oxygen tension in porcine40 and cat eyes41 and cyto-chrome activity in cat optic nerve becomes significantly re-duced.42 It is not clear whether ischemia is the sole mechanismunderlying the functional changes we observed at an ocularperfusion pressure of 75 mm Hg.

Of interest, the reduction in OP amplitudes occurred atIOPs that were higher than that required for nSTR and pSTR,

even though OPs are also believed to be generated in the innerretina.43–45 During acute IOP elevation, OP amplitude wasrelatively unaffected at 50 mm Hg yet showed reduced fre-quency, increased waveform spread, and delayed peak time.We speculate that the lack of OP amplitude loss during mildacute IOP elevation may be related to altered inhibitory feed-back circuits, which might enhance OP amplitude. For exam-ple, larger OPs are found after pharmacologic inhibition ofGABAc receptors46 and in mice lacking inhibitory GABAc re-ceptors.47

It is worth noting that light-activated responses from innerretinal neurons depend on the integrity of distal retinal ele-ments. As such, IOP-induced deficits in the a-wave, affect theb-wave, which in turn should affect the STR. The degree towhich these downstream effects manifest depends on the gainrelationships between the various cellular layers. It is knownthat a reduction in the ERG a-wave can result in a proportionalb-wave attenuation.48 Our results show that the b-wave IOP-response function had a significantly lower mean and steeperslope than the a-wave, suggesting that bipolar cells are moresensitive to IOP challenge than photoreceptors. The gain rela-

FIGURE 5. Functional changes during and immediately after a singleIOP spike of 50 mm Hg for 30 minutes (mean � SEM, n � 10). Hatchedarea in the box below each graph indicates the duration of IOPelevation. Open area in the box indicates 60 minutes of recovery, withIOP returned to baseline (�12 mm Hg). (A) Relative amplitudes (treat-ment/baseline, %) for pSTR, nSTR, and b-wave during IOP insult andrecovery. The x-axis for the nSTR and b-wave were jittered by �1 and1 minute, respectively, to improve clarity. Relative b-wave amplitudefor sham (n � 4) experiments is shown, with the shaded area indicat-ing the 95% CL across the 90 minutes of experimentation. (B) Peaktime delay for pSTR, (C) nSTR, and (D) b-wave. *Statistically significantdelays (post hoc; P � 0.05).

FIGURE 4. ERG waveforms during and immediately after a single IOPchallenge. Representative dim flash (A, �4.54 log cd � s � m�2) and rodb-wave flash (B, �2.23 log cd s m�2) waveforms at baseline (thintrace), 30 minutes after 50 mm Hg IOP elevation (maximal dysfunc-tion), and at 10, 30 and 60 minutes during recovery (thick traces).Vertical reference lines: fixed times used to quantify the amplitudechange for pSTR (120 ms), nSTR (220 ms), and b-wave (110 ms).

IOVS, December 2009, Vol. 50, No. 12 Acute Intraocular Pressure and Retinal Function in Mice 5737

tionship between the outer retinal ERG components and theSTR is less well characterized. Should outer retinal deficits beamplified at the ganglion cell level under these circumstances,a more sensitive pSTR pressure–response function could beanticipated. However, our finding that there was selective,persistent pSTR impairment 7 days after IOP challenge in thepresence of normal b-waves provides evidence that inner ret-inal function involving ganglion cells is more susceptible toIOP challenge.

The mechanism of increased inner retinal sensitivity to IOPelevation is likely to involve many factors, including mechani-cal, vascular, and oxidative stress. Ganglion cells may be par-ticularly susceptible as they receive insults both at their axonsat the level of the optic nerve head49–51 and at their cellbodies2,52 during IOP elevation. Therefore, additional mechan-ical and vascular stress at the optic nerve head could over-whelm the bioenergetics required for normal ganglion cellfunction.53

Recovery from IOP Spike

The time course of STR recovery in our study is consistent withprevious studies in rats.14,54 This was much slower (60 min-utes) than could be accounted for by the return of retinalreperfusion, as demonstrated in the porcine retina.40 The pro-cesses underlying functional loss and recovery are not wellunderstood, and further work is needed to characterize thefactors that influence the rate of this recovery. Of interest,although STR amplitude showed recovery during the 60 min-utes after IOP challenge, the delay in peak time remained. Adelay in timing of ERG components might reflect either areduced efficacy in the transduction cascades mediatingchanges in membrane potential, or reduced efficacy of neuro-transmission from distal neuronal components.48 These possi-bilities require further investigation.

The attenuation of the pSTR and OPs at 7 days after an IOPspike shows that a single episode of stress can produce aselective inner retinal deficit in mice. As we have yet to assessganglion cell anatomy at 7 days after the IOP challenge, it is notclear whether the functional reduction reflects cell loss orthere was dysfunction in a normal cohort of inner retinal cells.

Limitations

The approach used in this study does not attempt to model thechronic IOP elevation observed in glaucoma; however, it doeshighlight the impact of acute IOP-induced stress on retinalfunction. In rat and mouse models of chronic IOP elevation,IOP can regularly spike up to and above 30 mm Hg.1,5,36,55

Thus, in these chronic rodent models there are likely to beperiods of acute ganglion cell dysfunction. It is clear thatrodent eyes have different anatomic and biomechanical prop-erties; thus, extrapolation of our outcomes to other speciesshould be made cautiously.

The full-field ERG is unable to identify region-specific sen-sitivity to IOP challenge. This would be particularly importantin humans and animal models that have a fovea or area cen-tralis.41 A functional modality such as the multifocal ERG maybe more useful as a function marker of regional stress.

SUMMARY

We showed that in mice the ganglion cell–dominated pSTR ismore sensitive to acute IOP spikes than are ERG componentsarising from more distal retinal cell classes. This increasedsensitivity manifests as leftward shifts as well as a steeper slopeof the pSTR IOP-response function; a slower recovery imme-diately after a constant IOP challenge; and a selective pSTR andOP loss 7 days after a single 50-mm Hg IOP spike. These dataalso suggest that chronic IOP may not be needed to generatepermanent dysfunction. The ability to simultaneously assessretinal function during and after acute IOP challenge providesa means to quantify the susceptibility of retinal neurons to IOPstress.

Acknowledgments

The authors thank Karl A. J. Bromelow, Zheng He, and Vickie Wong forassistance in the daily care and monitoring of the animals.

FIGURE 6. ERG amplitudes (mean � SEM, n � 7) versus luminousenergy at baseline and 7 days after a single IOP spike for (A) peakamplitudes, (B) OP peak amplitudes, (C) nSTR amplitudes (at 220 ms);and (D) a-wave amplitude (at 8 ms). Relative amplitudes (treated/baseline %) are shown in the lower box of each panel.

5738 Kong et al. IOVS, December 2009, Vol. 50, No. 12

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