Free
Retina  |   April 2014
Remote Ischemia Influences the Responsiveness of the Retina: Observations in the Rat
Author Notes
  • Discipline of Physiology and Bosch Institute, School of Medical Sciences, University of Sydney, Sydney, Australia 
  • Correspondence: Alice Brandli, Department of Physiology, University of Sydney, F13 NSW 2006, Australia; abra4641@uni.sydney.edu.au
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2088-2096. doi:10.1167/iovs.13-13525
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Alice Brandli, Jonathan Stone; Remote Ischemia Influences the Responsiveness of the Retina: Observations in the Rat. Invest. Ophthalmol. Vis. Sci. 2014;55(4):2088-2096. doi: 10.1167/iovs.13-13525.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: Remote ischemic preconditioning (RIP) has been found to be protective of heart and brain against ischemic injury. We have tested the effects of RIP on retinal function using the electroretinogram.

Methods.: Ischemia remote from the retina was induced in one hindlimb, using a pressure cuff applied for between 5 and 10 minutes. A temperature probe on the footpad confirmed blockage of the circulation. To test the impact of RIP on retinal function, we recorded the dark-adapted flash electroretinogram (ERG) in four groups (n = 5 per group) of Sprague-Dawley rats (sham, 5-minute, 10-minute, and 2 × 5-minute ischemia). Heart rate, breath rate, and peripheral oxygen saturation were monitored using infrared pulse oximetry.

Results.: RIP increased both the a- and b-waves by up to 14%, more markedly after the longer periods (10 minutes or 2 × 5 minutes) of ischemia. The effect was tested up to 30 minutes after ischemia and retested at 1 week and 1 month. RIP did not appear to accelerate the initial stages of recovery from photopigment bleach. Systemic oxygen saturation, heart rate, and respiration did not vary consistently during or after remote ischemia.

Conclusions.: The effect of RIP on the ERG is a novel finding. Possible mechanisms of this effect are discussed and related to the idea of neuroprotection and to fundamentals of the electroretinogram.

Introduction
Ischemic preconditioning (IP) refers to the remarkable ability of nonlethal ischemia to upregulate mechanisms in the affected tissue, which make it resistant to subsequent ischemia. 1,2 Ischemic preconditioning was first demonstrated in cardiac muscle 1 by occluding a single coronary artery to make a patch of cardiac muscle ischemic. The protective effect of IP was confirmed in other tissues, including lung, 3 brain, 4 and retina. 5,6 The effectiveness of IP in such diverse tissues suggests that an endogenous mechanism of self-protection, common to many tissues, is upregulated by ischemia. One candidate mechanism is the regulation of adenosine A1 receptors and adenosine triphosphate (ATP)-sensitive K+ channels. 7  
Subsequent investigators noted that the area of heart made resistant to subsequent ischemia by the occlusion a coronary artery was much larger than the area of heart muscle supplied by that artery. 8 This led to the idea of remote ischemic preconditioning (RIP)—that a vital organ can be protected by a period of ischemia in remote body tissues. For example, in rats, limb ischemia protected the brain from a subsequent episode of focal ischemia 9 ; and in humans, ischemia of the arm has been reported to protect the heart in patients undergoing angioplasty. 10 A study of healthy human volunteers provided evidence that remote ischemic protection of cardiac muscle involves the opening of ATP-sensitive K+ channels, 11 consistent with mechanisms found in direct ischemic conditioning studies. 7  
The function of the retina, an easy-to-access extension of the brain, 12 can be assessed using the electroretinogram (ERG). The ERG allows identification of the activities of photoreceptors and inner retinal neurons and is a sensitive measure of panretinal changes in retinal function, which has been used extensively in clinical diagnosis and monitoring of disease progression. 13  
This study assesses the impact of remote ischemia on the function of retina, using the rat as a model. The impact of remote ischemia on the function of otherwise nondegenerative organs has not been tested previously. A test is possible in the retina because of the sensitivity of the ERG as a measure of retinal function. Our working hypotheses were two—that remote ischemia would influence membrane currents responsible for the a- and b-waves and that, because the influence would likely affect the entire retina, it would be detectable in the flash ERG. 
Methods
All experimental methods and animal care procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by University of Sydney Animal Ethics Committee (K22/6-2011/1/5563). 
Rearing
Sprague-Dawley rats were sourced from Animal Resource Center (Perth, WA, Australia). Animals were raised from birth in controlled scotopic conditions (22°C; 12 hours at 5–8 lux, 12 hours dark). Normal chow (WEHI; Barastoc, VIC, Australia) and water were available ad libitum. 
Preparation for ERG
Animals were dark adapted for 12 to 15 hours, first overnight and then, on the morning of the recording, until they were prepared for the recording session. Animals were anesthetized by intraperitoneal injection of ketamine and xylazine (60 and 7 mg/kg, respectively; Parnell Manufacturing Pty. Ltd., Alexandria, NSW, Australia) and then prepared for recording under dim red illumination. Mydriasis was achieved with topical application of atropine sulphate 1.0% (Bausch & Lomb Australia Pty. Ltd., Macquarie Park, NSW, Australia). Proxymetacaine (0.5%; Alcon Laboratories Pty. Ltd., French Forrest, NSW, Australia) was applied topically for corneal anesthesia. The eyeball was drawn forward with a loosely tied thread behind its equator to minimize contamination of recordings by lid activity. Corneal hydration was maintained during recordings with Carbomer (polyacrylic acid) (2 mg/g; Novartis Pharmaceuticals, North Ryde, NSW, Australia). The animal's body temperature was reliably maintained at 37°C to 37.5°C as monitored with a rectal temperature probe (Harvard Apparatus, Holliston, MA, USA). 
Limb Ischemia
Maintaining the dim red illumination, and with the animal under anesthesia (as specified above), one hindlimb was made ischemic with a neonatal blood pressure cuff (Cas Medical Systems, Branford, CT, USA). The cuff was applied on either the right or left hindlimb < 1 cm above the “knee.” It was inflated manually with an aneroid sphygmomanometer (DS56; Welch Allyn, Inc., Skaneateles Falls, NY, USA) to above systolic blood pressure (160 mm Hg), and then eased and maintained at 160 mm Hg. The effect of ischemia was tracked by a decline in footpad temperature (Thermistor Pod; AD Instruments Pty. Ltd., Bella Vista, NSW, Australia). This reliably fell by ∼2°C over 5 minutes (Fig. 1), and recovered steadily after the pressure was released. 
Figure 1
 
Footpad temperature fell during ischemia and rose during reperfusion. The three traces show temperature changes induced by 5-minute ischemia (blue), 10-minute ischemia (red), and two 5-minute periods of ischemia separated by 5-minute reperfusion (green).
Figure 1
 
Footpad temperature fell during ischemia and rose during reperfusion. The three traces show temperature changes induced by 5-minute ischemia (blue), 10-minute ischemia (red), and two 5-minute periods of ischemia separated by 5-minute reperfusion (green).
Full-Field ERG Recordings
The right eye was exposed to a Ganzfeld integrating sphere (Photometric Solutions International, Huntingdale, VIC, Australia), in which flashes of programmable intensity and duration could be generated from white light emitting diodes (5500°K; Luxeon, Calgary, AB, Canada). Once the setup was complete and the red setup light removed, 10 minutes of dark adaptation was allowed before commencement of recording. 
The brightness of each flash intensity was calibrated with a photometer (International Light Research, Peabody, MA, USA) using a scotopic rod filter (ZCIE/R) supplied by the manufacturer. This filter replicates human rod absorption of light under scotopic conditions. The measure of intensity obtained, as log cd.s.m−2, was then adjusted to rat rod maximum wavelength sensitivity (500 nm), with a conversion factor of −0.009 log units, to give light stimulus recordings in scot cd.s.m−2. The conversion factors followed Weymouth and Vingrys. 14  
The electroretinogram was recorded between a custom-made 4-mm platinum positive electrode lightly touching the cornea and a 2-mm-diameter Ag/AgCl pellet electrode (No. E206; SDR Clinical Technology, Middle Cove, NSW, Australia) in the mouth. Both were referenced to a stainless steel ground (23 g × 1.25 inch; Terumo Medical, Somerset, NJ, USA) inserted into the skin of the rump. Signals were recorded with band-pass setting of 0.3 to 1000 Hz (−3 dB), with a 2-kHz acquisition rate (PowerLab 4SP system; AD Instruments Pty. Ltd.). 
Remote Ischemia Protocols
In these protocols, the light stimulus used to elicit the ERG was a brief white flash. The duration of the flash was programmable (we used flashes 1–2 ms in duration), as was its intensity (−4.4 to 2.0 log scot cd.s.m−2). Luminous energy was calibrated (IL1700; International Light Research, Peabody, MA, USA) to give rodent (λmax = 502 nm) scotopic (Z-CIE luminosity filter) luminous measures (log cd.s.m−2). Two patterns of stimulation were used. 
Pre- Versus Postischemia Comparisons.
These comparisons were made to establish the effect of RIP on the ERG. Before and after ischemia, we recorded responses to a series of flashes, whose intensity ranged in 11 steps over the range stated above (example of series responses, Fig. 2C). At lower flash intensities (−4.4 to −0.3 log scot cd.s.m−2), responses were averaged from four flashes delivered at 0.2 Hz. At higher intensities, fewer responses were averaged and interstimulus intervals were increased, up to 120 seconds. 
Figure 2
 
Experimental groups and ERG measurements. (A) Four experimental groups were used to demonstrate the impact of RIP on retinal function. In one group, ischemia was generated in one hindlimb for 5 minutes; in a second group the ischemia lasted 10 minutes; in a third group, the limb was made ischemic for two periods of 5 minutes, separated by a 5-minute period of reperfusion. In a fourth group (sham), the pressure cuff was wrapped around the limb but not inflated. The ERG was recorded at the time points shown by the red arrows. (B) The amplitude of the a-wave was measured from the baseline to the first negative peak (left arrow). The amplitude of the b-wave was measured as shown by the right arrow. (C) A representative set of responses to flashes of 11 intensities (−4.4 to 2.0 log scot cd.s.m−2). (D) For the bleach-recovery experiment, the retina was conditioned by the 2 × 5-minute pattern of limb ischemia followed immediately by a 60-second period of light exposure (14.2 cd/m−2). The response of the retina to tracking flash (a single flash at 1.4 log scot cd.s.m−2) was used to trace the recovery of responsiveness after the bleach at 5-minute intervals after the flash.
Figure 2
 
Experimental groups and ERG measurements. (A) Four experimental groups were used to demonstrate the impact of RIP on retinal function. In one group, ischemia was generated in one hindlimb for 5 minutes; in a second group the ischemia lasted 10 minutes; in a third group, the limb was made ischemic for two periods of 5 minutes, separated by a 5-minute period of reperfusion. In a fourth group (sham), the pressure cuff was wrapped around the limb but not inflated. The ERG was recorded at the time points shown by the red arrows. (B) The amplitude of the a-wave was measured from the baseline to the first negative peak (left arrow). The amplitude of the b-wave was measured as shown by the right arrow. (C) A representative set of responses to flashes of 11 intensities (−4.4 to 2.0 log scot cd.s.m−2). (D) For the bleach-recovery experiment, the retina was conditioned by the 2 × 5-minute pattern of limb ischemia followed immediately by a 60-second period of light exposure (14.2 cd/m−2). The response of the retina to tracking flash (a single flash at 1.4 log scot cd.s.m−2) was used to trace the recovery of responsiveness after the bleach at 5-minute intervals after the flash.
Male and female rats aged 3 to 4 months were randomly assigned to four groups (Fig. 2A): 
  1.  
    Leg made ischemic for 5 minutes (“5 minutes,” n = 5)
  2.  
    Leg made ischemic for 10 minutes (“10 minutes,” n = 6) or
  3.  
    Leg made ischemic for two 5-minute periods, with a 5-minute reperfusion time between (“2 × 5 minutes,” n = 6)
  4.  
    Cuff around the limb but not inflated (“sham,” n = 3)
The patterns of ischemia used in groups 1, 2, and 3 were designed following previous studies with remote ischemia. 15 Intensity–response series were collected at the time points shown in Figure 2A: once before ischemia, once 10 minutes after the end of ischemia, and, in the 2 × 5-minute group, at 35 and 45 minutes after the end of ischemia. 
To test repeatability, the 2 × 5-minute group was restudied at 1 week and 1 month after the initial test. 
Bleach Protocol.
To track the recovery of the ERG from bleaching, RIP was applied in the 2 × 5-minute pattern (Fig. 2D). This period of ischemia was followed immediately by an exposure of the animal to a white Ganzfeld (14.2 cd/m2 for 60 seconds), resulting in a bleach of 1% of total rhodopsin content. 14 The tracking stimulus (a single flash at 1.4 log cd.s.cm−2, every 3 minutes) was then used to trace the recovery of responsiveness from the end of the bleaching period, for up to 60 minutes. 
Data Analysis
We measured the amplitudes of the a- and b-waves of the ERG as shown in Figure 2B. We also measured the latencies of these waves, from the stimulus artifact to the negative peak of the a-wave and to the positive peak of the b-wave. A one-way ANOVA test (GraphPad v5.01, La Jolla, CA, USA) was used to assess the statistical significance of any differences in latencies and amplitudes between experimental groups, together with Bonferroni multiple comparison post hoc analysis. A Student's t-test was used to assess variations in heart rate, breath rate, and oxygen saturation of the blood. 
Results
Confirmation of Ischemia
Ischemia in the cuffed limb was confirmed by a fall in footpad temperature as seen in Figure 1. The preischemia temperature of the footpad varied, but ischemia reliably produced a fall in temperature, and the end of ischemia induced a consistent rise. Confirming this, the color of the affected footpad skin turned from pink-red to a blue-purple color, characteristic of venous blood (data not shown). 
Remote Ischemia Increased a-Wave and b-Wave Amplitudes
Remote ischemia increased the amplitude of the a- and b-waves (Fig. 3). Each pair of traces in Figure 3 is an overlay of the ERG recorded in response to a flash presented before ischemia, and to a second flash presented 10 minutes after the end of ischemia. In the sham group (no pressure applied to the cuff), the response recorded at the two time points did not differ in amplitude or latency at any of the four intensities of flash used (left column in Fig. 3). When ischemia was applied for 5, for 10, and for 2 × 5 minutes (second, third, and fourth columns in Fig. 3), differences in amplitude between before and after traces were apparent. The postischemia ERG was consistently the bigger, more clearly for the b-wave. 
Figure 3
 
Representative ERG recordings before (thinner trace) and after (thicker trace) remote ischemia for the four experimental groups in Figure 1 and for four intensities of flash stimulus. Left column: Essentially identical responses were obtained 10 minutes apart for the sham group. Second column: A 5-minute period of ischemia induced a small increase in amplitude, most noticeably in the b-wave. Third column: A 10-minute period of ischemia induced a greater increase in amplitude. Fourth column: Two 5-minute periods of ischemia separated by 5-minute reperfusion also induced a larger increase in amplitude, apparent also in the a-wave at brighter intensities.
Figure 3
 
Representative ERG recordings before (thinner trace) and after (thicker trace) remote ischemia for the four experimental groups in Figure 1 and for four intensities of flash stimulus. Left column: Essentially identical responses were obtained 10 minutes apart for the sham group. Second column: A 5-minute period of ischemia induced a small increase in amplitude, most noticeably in the b-wave. Third column: A 10-minute period of ischemia induced a greater increase in amplitude. Fourth column: Two 5-minute periods of ischemia separated by 5-minute reperfusion also induced a larger increase in amplitude, apparent also in the a-wave at brighter intensities.
The results obtained for each group of animals are shown quantitatively in Figure 4 for the a-wave and b-waves and for two stimulus intensities (Figs. 4A, 4B). The a-wave showed an increase in amplitude of up to 9%, at both intensities and with all three ischemia protocols. The increase was statistically significant for the 2 × 5-minute group at both intensities (P < 0.05). The b-wave also increased in amplitude after RIP, by up to 14%, and was significant for both the 10- and the 2 × 5-minute groups at the higher intensity (2.0 log scot cd.s.m−2), as well as for all three ischemia groups at the lower intensity (0.4 log scot cd.s.m−2). 
Figure 4
 
RIP-induced changes in the amplitudes of a- and b-wave; normalized data for two flash intensities. For comparisons of remote ischemia–conditioned responses to unconditioned, *P < 0.05, **P < 0.01, ***P < 0.0001. Mean a-wave responses shown in white bars, b-wave mean responses shown in gray bars, n = 3 to 6 ± SEM. (A) Using a flash of 0.4 log cd.s.m−2, the normalized values showed a significant (asterisk) increase in the 2 × 5-minute group for the a-wave and in the 10- and the 2 × 5-minute groups for the b-wave. (B) Using a brighter flash (2.0 log cd.s.m−2), the normalized values showed a significant increase in the 2 × 5-minute group for the a-wave and in all three ischemia groups for the b-wave.
Figure 4
 
RIP-induced changes in the amplitudes of a- and b-wave; normalized data for two flash intensities. For comparisons of remote ischemia–conditioned responses to unconditioned, *P < 0.05, **P < 0.01, ***P < 0.0001. Mean a-wave responses shown in white bars, b-wave mean responses shown in gray bars, n = 3 to 6 ± SEM. (A) Using a flash of 0.4 log cd.s.m−2, the normalized values showed a significant (asterisk) increase in the 2 × 5-minute group for the a-wave and in the 10- and the 2 × 5-minute groups for the b-wave. (B) Using a brighter flash (2.0 log cd.s.m−2), the normalized values showed a significant increase in the 2 × 5-minute group for the a-wave and in all three ischemia groups for the b-wave.
Figure 5 shows the increases in a- and b-wave amplitudes in the 2 × 5-minute ischemia group as a function of flash intensity. The difference in amplitude appears at the higher intensities for both a- and b-waves. Analysis of variance indicated that the unconditioned and conditioned a-wave functions are significantly different (P = 0.006); but post hoc analyses of individual stimulus intensities did not reach significance. Analysis of variance also showed that the unconditioned and conditioned b-wave functions are significantly different (P < 0.0001), and post hoc analyses showed that the b-wave is significantly larger in preconditioned animals at the two highest intensities used (P < 0.05, 1.4 and 2.0 log scot cd.s.m−2). 
Figure 5
 
Intensity–response curves for conditioned (red) 2 × 5-minute group and unconditioned eyes (blue). Group average responses, n = 6 ± SEM. (A) The conditioned a-wave was consistently larger at higher flash intensities. On a two-way ANOVA test, the two curves are significantly different (P < 0.01). (B) The conditioned b-wave was also consistently larger at higher intensities. On a two-way ANOVA test, the two curves are significantly different (P < 0.0001). Post hoc analysis showed significant (*P < 0.05) differences at each of the two top intensities.
Figure 5
 
Intensity–response curves for conditioned (red) 2 × 5-minute group and unconditioned eyes (blue). Group average responses, n = 6 ± SEM. (A) The conditioned a-wave was consistently larger at higher flash intensities. On a two-way ANOVA test, the two curves are significantly different (P < 0.01). (B) The conditioned b-wave was also consistently larger at higher intensities. On a two-way ANOVA test, the two curves are significantly different (P < 0.0001). Post hoc analysis showed significant (*P < 0.05) differences at each of the two top intensities.
Synaptic Responsiveness (b- to a-Wave Ratio)
To assess whether RIP affects synaptic responsiveness in the retina, we examined the ratio of the amplitudes of the a- and b-wave for all four experimental groups (Fig. 3) and at two intensities. At low intensities (e.g., −2.45 log scot cd.s.m−2), the a-wave was not apparent (bottom traces in Fig. 3), and a ratio could not be calculated. At midrange intensities, where a- and b-wave amplitudes were measurable but submaximal (e.g., middle traces in Fig. 3), there was no difference in the b- to a-wave ratio between RIP groups and the sham-conditioned group. Values were between 2.2 and 3.2 (sham: 3.2 ± 0.3; 5 minutes: 2.8 ± 0.7; 10 minutes: 3.0 ± 0.7; 2 × 5 minutes: 2.2 ± 0.2). Similarly, the b- to a-wave ratio for the maximal intensity (2.0 log cd.s.m2) showed no significant difference between the RIP groups and the sham-conditioned group; values ranged from 1.6 to 1.9 (sham: 1.8 ± 0.3; 5 minutes: 1.7 ± 0.2; 10 minutes: 1.9 ± 0.1; 2 × 5 minutes: 1.6 ± 0.2). Our data therefore provided no evidence that RIP amplified synaptic responsiveness from photoreceptors to inner retina. 
Latencies of a- and b-Wave
Latencies were measured from the stimulus artifact to the maximum negativity of the a-wave or the maximum positive of the b-wave at the mid to maximum stimulus intensity (−2.45 to 2.0 log cd.s.m2). We could not identify any significant variation in latency between sham-conditioned and RIP groups (Fig. 6). 
Figure 6
 
Latency of the a- and b-waves in the four groups shown in Figures 2 and 3. A-wave mean timing shown in white bars and b-wave mean timing in gray bars, n = 6 ± SEM. Latency of the a-wave was measured from the stimulus artifact to the first negative peak, latency of the b-wave to the first positive peak. Latencies appeared unaffected by RIP (a-wave P = 0.4 and b-wave P = 0.6, one-way ANOVA).
Figure 6
 
Latency of the a- and b-waves in the four groups shown in Figures 2 and 3. A-wave mean timing shown in white bars and b-wave mean timing in gray bars, n = 6 ± SEM. Latency of the a-wave was measured from the stimulus artifact to the first negative peak, latency of the b-wave to the first positive peak. Latencies appeared unaffected by RIP (a-wave P = 0.4 and b-wave P = 0.6, one-way ANOVA).
Duration and Repeatability of the Response
Using the 2 × 5-minute pattern of ischemia, we applied the intensity series 25, 35, and 45 minutes after the end of ischemia (Fig. 7A). For both a- and b-wave, the increase of amplitude was consistent at all three times. At the same three time points, in the sham-treated group (Fig. 7B), a- and b-waves showed no significant increase. 
Figure 7
 
Stability and repeatability of RIP-induced increase of a- and b-waves. (A) The a- (white) and b-waves (gray) ± SEM were measured 25, 35, and 45 minutes after the commencement of 2 × 5-minute RIP; the increase of their amplitudes persisted (P < 0.0001, n = 4 for differences between sham and RIP treatment). Post-RIP differences were stable at 25, 35, and 45 minutes (n = 4; a-wave P = 0.8 and b-wave P = 0.9, one-way ANOVA). (B) In sham-treated animals, the a- and b-waves were stable over the same testing times (C) in each of a group of six animals. The effect of RIP on the amplitudes of the a- and b-wave was assessed 25 minutes after 2 × 5-minute RIP on three occasions—an initial trial, and then 1 week and 1 month later. The effect recurred reliably at each time point (n = 4; a-wave P = 0.8, b-wave P = 0.7, one-way ANOVA).
Figure 7
 
Stability and repeatability of RIP-induced increase of a- and b-waves. (A) The a- (white) and b-waves (gray) ± SEM were measured 25, 35, and 45 minutes after the commencement of 2 × 5-minute RIP; the increase of their amplitudes persisted (P < 0.0001, n = 4 for differences between sham and RIP treatment). Post-RIP differences were stable at 25, 35, and 45 minutes (n = 4; a-wave P = 0.8 and b-wave P = 0.9, one-way ANOVA). (B) In sham-treated animals, the a- and b-waves were stable over the same testing times (C) in each of a group of six animals. The effect of RIP on the amplitudes of the a- and b-wave was assessed 25 minutes after 2 × 5-minute RIP on three occasions—an initial trial, and then 1 week and 1 month later. The effect recurred reliably at each time point (n = 4; a-wave P = 0.8, b-wave P = 0.7, one-way ANOVA).
Again using the 2 × 5-minute pattern of ischemia, we assessed a- and b-wave amplitudes 10 minutes after the end of ischemia three times in each of a group of animals (n = 6), first in an initial test, then 1 week and 1 month later. In these experiments, the ischemia was repeated on each occasion. The increases of a- and b-wave amplitudes were consistent in these successive tests (Fig. 7C). 
Remote Ischemia Increases Recovery From Bleach
The recovery of retinal responsiveness following photobleaching was tracked using a 1.4 log scot cd.s.m−2 flash delivered at 5-minute intervals for up to 60 minutes. The amplitude of b-wave recovered monotonically, suggesting that the bleach was limited to rods (Figs. 8A, 8B). The amplitude of the b-wave following RIP followed a similar time course up to ∼50% recovery, at ∼15 minutes; thereafter the b-wave was consistently larger than in the sham-conditioned control. A two-way ANOVA test indicated that the difference between the curves was significant (P = 0.003). For the a-wave, the RIP- and sham-conditioned recovery curves did not separate consistently (P = 0.06 on a two-way ANOVA test, Fig. 8C). 
Figure 8
 
(A) Representative ERG responses at 0 to 60 minutes after a 60-second bleach (∼1% bleach of rhodopsin). (B, C) Amplitude recovery with and without RIP for the a- and b-wave, unconditioned eyes (blue) compared to conditioned 2 × 5 minutes (red) ± SEM. For the b-wave the curves were significantly different (n = 4; P = 0.03, two-way ANOVA) (B). For the a-wave the difference between the two curves did not reach significance (n = 4, P > 0.05, two-way ANOVA) (C).
Figure 8
 
(A) Representative ERG responses at 0 to 60 minutes after a 60-second bleach (∼1% bleach of rhodopsin). (B, C) Amplitude recovery with and without RIP for the a- and b-wave, unconditioned eyes (blue) compared to conditioned 2 × 5 minutes (red) ± SEM. For the b-wave the curves were significantly different (n = 4; P = 0.03, two-way ANOVA) (B). For the a-wave the difference between the two curves did not reach significance (n = 4, P > 0.05, two-way ANOVA) (C).
Oxygen Saturation, Pulse Rate, Respiration Rate: No Apparent Relation to RIP
In six animals, we used pulse oxymetry to record blood saturation, pulse rate, and respiration rate during and after 2 × 5-minute remote ischemia to test whether RIP influenced any of these parameters, which might then have influenced the ERG. We could not, however, detect any consistent relationship between any of the three parameters and the onset, maintenance, or termination of remote ischemia (data not shown). Oxygen saturation in some animals was relatively low (90%) by the time the oximeter was applied (after anesthesia and preparation for ERG recording); if initially low, saturation then rose steadily toward values > 95%, without correlation to periods of ischemia. Pulse and breath rates also showed variability during the period of study but no correlation with remote ischemia (see Supplementary Figs.). 
Discussion
Summary of Findings
Remote ischemia of the hindlimb was found to increase the amplitudes of two major components of the dark-adapted flash ERG, the a- and b-waves, by up to 14%. Initial experiments showed that the increase was detectable when the ischemia lasted 5 minutes and was consistently greater when the ischemia lasted 10 minutes. Ischemia for two periods of 5 minutes, separated by a 5-minute period of reperfusion, was used for further experiments. The increase was detectable over a wide range of flash intensities; the increase was stable for up to 30 minutes after the ischemia (the longest period tested) and was repeatable in the same animal at 1-week and 1-month intervals. RIP did not affect the latency of either the a- or b-wave. Ischemia of the hindlimb was not associated with consistent changes in blood oxygen saturation or pulse or respiration rate. 
As far as we are aware, this is the first report of remote ischemia affecting the function of central nervous tissue. Although the mechanism of the effect remains unknown, we suggest that the effect is unlikely to be specific to the retina; we have been able to detect it in the retina because the ERG is a sensitive, high-amplitude signal generated by a highly ordered array of neurons in the retina when they are activated synchronously. This evidence that remote ischemia regulates retinal function raises the question, which we are currently exploring, whether it will condition the retina against damage. 
Other Instances of Supernormal ERG
Supernormality of the ERG has been reported in a rat model of experimental uveitis. 16 Clinically, supernormality of the ERG, with no change in the b- to a-wave ratio, has been reported in cases of inflammatory disease of the eye. 17 Supernormality of b-wave, without modification of a-wave amplitude, can be induced experimentally by suppressing retinal autoregulation 18 and by antagonists of amacrine cell inhibition, such as bicucilline or strychnine. 19 Mutations in ATP-sensitive potassium channels, which cause loss of cone function, are diagnosed by an increase (supernormality) of the rod b-wave, coupled with a suppression of the a-wave. 20  
Possible Mechanisms
The amplitudes of components of the ERG, such as the a- and b-waves, are determined by the currents generated by the light stimulus, initially along the length of the photoreceptors and then by the synaptic action of photoreceptors on inner retinal neurons. Previous work has shown that determinants of the amplitude of ERG components include the integrity of retinal structure and physiological parameters, including the state of adaptation of photoreceptors, blood supply, metabolite availability, and ionic conditions. 14,21 The present results provide evidence that ERG amplitude is also regulated by a previously unsuspected factor—ischemia in remote tissues. 
Blood/Oxygen Supply.
Overall, the ERG is quite robust in the face of variations in blood supply or oxygen saturation. Severe ischemia, induced by carotid occlusion, was shown by Granit 22 to reduce the amplitude of the b-wave, while the amplitude of the a-wave was maintained. Subsequent research has confirmed this observation using several techniques of reducing blood flow to the eye, including clamping the retrobulbar vascular supply, 23 elevating intraocular pressure, 24,25 and varying the combination of blood pressure and intraocular pressure, known as ocular perfusion pressure. 26 The ERG is also resistant to moderate hypoxemia. 27 Conversely, an increase in blood oxygen saturation, achieved by breathing 100% O2, did not alter the amplitude of either the a- or b-wave. 28,29 In the present experiments, oxygen saturation, pulse rate, and respiration rate did not vary consistently with remote ischemia and seem unlikely to be the cause of the amplitude increase observed. 
Adenosine, Glucose.
Adenosine can act as a neuromodulator, as a modulator of vasomotor tone, notably of the autoregulation of retinal arterioles, and as an activator of ionic channels in the membrane of mitochondria. 3032 Further, A1 receptor activation has been considered important in RIP-induced protection of the myocardium 33 ; adenosine turnover has been reported to be upregulated in myocardium by direct ischemia, 34 and adenosine infusion in perfused cat eyes resulted in supernormal b-waves and vasodilation. 18,35 Adenosine is then a candidate mediator for the effect reported here, the RIP-induced supernormality of the a- and b-waves; to test this, further investigation is required. 
We also considered whether remote ischemia might release a major metabolite, such as glucose, into the bloodstream in quantities sufficient to induce supernormality of the ERG. The ERG is remarkably stable, however, in the face of moderate hypoglycemia 36 and hyperglycemia. 37 Sun and colleagues 38 reported that limb ischemia did not alter blood glucose levels. 
Ionic Environment.
Regulation of ATP-sensitive K+ channels has been considered important in the protection of brain tissue induced by remote and direct IP. 39,40 This point has not yet been tested in the retina; but the b-wave of the ERG, which originates from cells postsynaptic to the photoreceptor, is modulated by a number of potassium currents between distal and proximal retina. 41 Müller cell–generated K+ currents are believed to contribute to the b-wave 42 and to slower components, including the slow PIII (transretinal spatial buffering) current, 43 the M-wave, 44 and the photopic negative response. 45  
More generally, the Müller cell provides spatial buffering of extracellular K+ via inward-rectifying K+ channels, notably the Kir 4.1 (ATP-sensitive inward-rectifying K+ channel) and Kir 2.1 (inward-rectifying K+ channel). 43,46  
In summary, K+ channels in the membranes of mitochondria and Müller cells are potent determinants of the current flows detected by electroretinography and may therefore mediate RIP-induced supernormality of ERG components. Again, further investigation is needed to link limb ischemia to regulation of K+ channels. 
Overall, the mechanism of RIP-induced increase of the ERG remains difficult to define, and further experimentation is needed. 
Implications
The present results have implications for both understanding of the regulation of retinal function and the concept of “protective conditioning” of the retina. Although remote ischemia has been studied in some detail for its protective conditioning of vital organs, the present evidence—that remote ischemia influences the function in the retina—is the first evidence that the function of a vital organ is regulated by the state of peripheral body tissues. The implications of this finding are hard to foresee; but the state of body tissues may, like environmental enrichment, prove to be a significant (and previously unrecognized) factor in regulating aspects of the function of the central nervous system. It should be noted, however, that it is a damaging state of remote tissue (i.e., ischemia) that, in the present experiments, appears to increase retinal responsiveness. 
The idea of protective conditioning of the retina can be traced back to Penn and Anderson's 47 1987 work on the impact of environmental light conditions on retinal stability. They reported that the retina of rats reared in scotopic (dim) illumination showed less cell death and less membrane damage than that in rats reared in photopic (brighter) illumination. Nevertheless, when the retina was challenged with long exposure to very bright, potentially damaging light, the light-naïve but structurally more intact retinas were extremely vulnerable, and their photoreceptor population was almost totally destroyed. The light-conditioned but more damaged retinas were, by contrast, much more resistant to the damaging light, surviving intact and functional. 
The idea that ambient light may be measurably stressful to the retina, but upregulates endogenous protective mechanisms that make the retina resistant to acute stress, has been expanded in various ways. Evidence is now available that the retina can be protectively conditioned by light, 48 by hypoxia and hyperoxia, 49 by low doses of gamma rays, 50 by irradiation with red-near infrared light, 51,52 and by dietary saffron. 52,53 We are currently testing whether remote ischemia also conditions the retina protectively as it does the heart, brain, and other organs. 54,55  
Both issues—the impact of remote tissue on retinal responsiveness and protective conditioning—seem of fundamental importance, and it will be of interest to elucidate whether the same molecular mechanisms are involved. 
Supplementary Materials
Acknowledgments
Supported by funding from the Australian Research Council Centre of Excellence in Vision Science and from the Sir Zelman Cowen Universities Fund. 
Disclosure: A. Brandli, None; J. Stone, CSCM Pty Ltd. (E) 
References
Murry CE Jennings RB Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation . 1986; 74: 1124–1136. [CrossRef] [PubMed]
Zhao ZQ Corvera JS Halkos ME Kerendi F Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol . 2003; 285: H579–H588. [CrossRef] [PubMed]
Harkin DW D'Sa A McCallion K Hoper M Campbell FC. Ischemic preconditioning before lower limb ischemia-reperfusion protects against acute lung injury. J Vasc Surg . 2002; 35: 1264–1273. [CrossRef] [PubMed]
Barone FC White RF Spera PA Ischemic preconditioning and brain tolerance - temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke . 1998; 29: 1937–1950. [CrossRef] [PubMed]
Casson RJ Wood JPM Melena J Chidlow G Osborne NN. The effect of ischemic preconditioning on light-induced photoreceptor injury. Invest Ophthalmol Vis Sci . 2003; 44: 1348–1354. [CrossRef] [PubMed]
Roth S Li B Rosenbaum PS Preconditioning provides complete protection against retinal ischemic injury in rats. Invest Ophthalmol Vis Sci . 1998; 39: 777–785. [PubMed]
Heurteaux C Lauritzen I Widmann C Lazdunski M. Essential role of adenosine, adenosine-A1-receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning. Proc Natl Acad Sci U S A . 1995; 92: 4666–4670. [CrossRef] [PubMed]
Przyklenk K Bauer B Ovize M Kloner RA Whittaker P. Regional ischemic preconditioning protects remote virgin myocardium from subsequent sustained coronary-occlusion. Circulation . 1993; 87: 893–899. [CrossRef] [PubMed]
Ren C Gao X Steinberg GK Zhao H. Limb remote-preconditioning protects against focal ischemia in rats and contradicts the dogma of therapeutic time windows for preconditioning. Neuroscience . 2008; 151: 1099–1103. [CrossRef] [PubMed]
Botker HE Kharbanda R Schmidt MR Remote ischaemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomised trial. Lancet . 2010; 375: 727–734. [CrossRef] [PubMed]
Moses MA Addison PD Neligan PC Mitochondrial K-ATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction. Am J Physiol Heart Circ Physiol . 2005; 288: H559–H567. [CrossRef] [PubMed]
Dowling J. The Retina: An Approachable Part of the Brain . Cambridge, MA: Harvard University Press; 2012.
Marmor MF ISCEV standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol . 2009; 118: 69–77. [CrossRef] [PubMed]
Weymouth AE Vingrys AJ. Rodent electroretinography: methods for extraction and interpretation of rod and cone responses. Prog Retin Eye Res . 2008; 27: 1–44. [CrossRef] [PubMed]
Kanoria S Jalan R Seifalian AM Williams R Davidson BR. Protocols and mechanisms for remote ischemic preconditioning: a novel method for reducing ischemia reperfusion injury. Transplantation . 2007; 84: 445–458. [CrossRef] [PubMed]
Stanford MR Robbins J. Experimental posterior uveitis and interpretation of rod and cone responses. Br J Ophthalmol . 1988; 72: 88–96. [CrossRef] [PubMed]
Ikeda H Franchi A Turner G Shilling J Electroretinography Graham E. and electroculography to localize abnormalities in early-stage inflammatory eye disease. Doc Ophthalmol . 1989; 74: 387–394. [CrossRef]
Frueh B Niemeyer G Onoe S. Adenosine enhances the ERG b-wave and depresses the light peak in perfused cat eyes. Invest Ophthalmol Vis Sci . 1989; 30: 124.
Frumkes TE Nelson R Pflug R. Functional role of GABA in cat retina: 2. Effects of GABA(A) antagonists. Vis Neurosci . 1995; 12: 651–661. [CrossRef] [PubMed]
Wissinger B Dangel S Jagle H Cone dystrophy with supernormal rod response is strictly associated with mutations in KCNV2. Invest Ophthalmol Vis Sci . 2008; 49: 751–757. [CrossRef] [PubMed]
Heckenlively JA Arden GB eds. Principles and Practice of Clinical Electrophysiology of Vision . Cambridge, MA: MIT Press; 2006.
Granit R. Sensory Mechanisms of the Retina, with an Appendix on Electroretinography . London: Oxford University Press; 1947.
Brunette JR Lafond G. Synchronization of ERG signs of retinal ischemia. Doc Ophthalmol . 1983; 37: 309–315.
Block F Schwarz M. The b-wave of the electroretinogram as an index of retinal ischemia. Gen Pharmacol . 1998; 30: 281–287. [CrossRef] [PubMed]
Bui BV He Z Vingrys V Nyguyen CTO Wong VHY Fortune B. Using the electroretinogram to understand how intraocular pressure elevation affects the rat retina. J Ophthalmol . 2013; 2013: 262467. [PubMed]
He Z Nguyen CTO Armitage JA Vingrys AJ Bui BV. Blood pressure modifies retinal susceptibility to intraocular pressure elevation. PLoS One . 2012; 7: e31104. [CrossRef] [PubMed]
Derwent JK Linsenmeier RA. Effects of hypoxemia on the a- and b-waves of the electroretinogram in the cat retina. Invest Ophthalmol Vis Sci . 2000; 41: 3634–3642. [PubMed]
Niemeyer G Nagahara K Demant E. Effects of changes in arterial PO2 and PCO2 on the electroretinogram in the cat. Invest Ophthalmol Vis Sci . 1982; 23: 678–683. [PubMed]
Kergoat H Tinjust D. Neuroretinal function during systemic hyperoxia and hypercapnia in humans. Optom Vis Sci . 2004; 81: 214–220. [CrossRef] [PubMed]
Dunwiddie TV Masino SA. The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci . 2001; 24: 31–55. [CrossRef] [PubMed]
Mubagwa K Flameng W. Adenosine, adenosine receptors and myocardial protection: an updated overview. Cardiovasc Res . 2001; 52: 25–39. [CrossRef] [PubMed]
Gidday JM Park TS. Adenosine-mediated autoregulation of retinal arteriolar tone in the piglet. Invest Ophthalmol Vis Sci . 1993; 34: 2713–2719. [PubMed]
Hu S Dong HL Zhang HP Noninvasive limb remote ischemic preconditioning contributes neuroprotective effects via activation of adenosine A1 receptor and redox status after transient focal cerebral ischemia in rats. Brain Res . 2012; 1459: 81–90. [CrossRef] [PubMed]
Aberg AM Ahlstrom K Abrahamsson P Ischaemic pre-conditioning means an increased adenosine metabolism with decreased glycolytic flow in ischaemic pig myocardium. Acta Anaesthesiol Scand . 2010; 54: 1257–1264. [CrossRef] [PubMed]
Blazynski C Cohen AI Fruh B Niemeyer G. Adenosine: autoradiographic localization and electrophysiologic effects in the cat retina. Invest Ophthalmol Vis Sci . 1989; 30: 2533–2536. [PubMed]
Derwent JJK Linsenmeier RA. Hypoglycemia increases the sensitivity of the cat electroretinogram to hypoxemia. Vis Neurosci . 2001; 18: 983–993. [CrossRef] [PubMed]
Johnson LE Larsen M Perez MT. Retinal adaptation to changing glycemic levels in a rat model of type 2 diabetes. PLoS One . 2013; 8: e55456. [CrossRef] [PubMed]
Sun J Li T Luan Q Protective effect of delayed remote limb ischemic postconditioning: role of mitochondrial K-ATP channels in a rat model of focal cerebral ischemic reperfusion injury. J Cereb Blood Flow Metab . 2012; 32: 851–859. [CrossRef] [PubMed]
Teshima Y Akao M Li RA Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress. Stroke . 2003; 34: 1796–1802. [CrossRef] [PubMed]
Ettaiche M Heurteaux C Blondeau N ATP-sensitive potassium channels (K-ATP) in retina: a key role for delayed ischemic tolerance. Brain Res . 2001; 890: 118–129. [CrossRef] [PubMed]
Lei B Perlman I. The contributions of voltage- and time-dependent potassium conductances to the electroretinogram in rabbits. Vis Neurosci . 1999; 16: 743–754. [CrossRef] [PubMed]
Robson JG Frishman LJ. Dissecting the dark-adapted electroretinogram. Doc Ophthalmol . 1998; 95: 187–215. [CrossRef] [PubMed]
Kofuji P Ceelen P Zahs KR Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: phenotypic impact in retina. J Neurosci . 2000; 20: 5733–5740. [PubMed]
Karwoski C. Spatial buffering of light-evoked potassium increase by retinal muller (glia) cells. Science . 1989; 244: 578–580. [CrossRef] [PubMed]
Viswanathan S Frishman LJ Robson JG Harwerth RS Smith EL. The photopic negative response of the macaque electroretinogram: reduction by experimental glaucoma. Invest Ophthalmol Vis Sci . 1999; 40: 1124–1136. [PubMed]
Raz-Prag D Grimes WN Fariss RN Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration. Proc Natl Acad Sci U S A . 2010; 107: 12710–12715. [CrossRef] [PubMed]
Penn JS Anderson RE. Effect of light history on rod outer-segment membrane composition in the rat. Exp Eye Res . 1987; 44: 767–778. [CrossRef] [PubMed]
Liu C Peng M Wen R. Pre-exposure to constant light protects photoreceptor from subsequent light damage in albino rats. Invest Ophthalmol Vis Sci . 1997; 38: S718.
Bowers F Valter K Chan SW Walsh N Maslim J Stone J Effects of oxygen and bFGF on the vulnerability of photoreceptors to light damage. Invest Ophthalmol Vis Sci . 2001; 42: 804–815. [PubMed]
Otani A Kojima H Guo C Oishi A Yoshimura N. Low-dose-rate, low-dose irradiation delays neurodegeneration in a model of retinitis pigmentosa. Am J Pathol . 2012; 180: 328–336. [CrossRef] [PubMed]
Eells J Henry MM Summerfelt P Therapeutic photobiomodulation for methanol-induced retinal toxicity. Proc Natl Acad Sci U S A . 2003; 100: 3439–3444. [CrossRef] [PubMed]
Natoli R Zhu Y Valter K Bisti S Eells J Stone J Gene and noncoding RNA regulation underlying photoreceptor protection: microarray study of dietary antioxidant saffron and photobiomodulation in rat retina. Mol Vis . 2010; 16: 1801–1822. [PubMed]
Maccarone R Di Marco S Bisti S. Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina. Invest Ophthalmol Vis Sci . 2008; 49: 1254–1261. [CrossRef] [PubMed]
Jensen HA Loukogeorgakis S Yannopoulos F Remote ischemic preconditioning protects the brain against injury after hypothermic circulatory arrest. Circulation . 2011; 123: 714–721. [CrossRef] [PubMed]
Walsh SP Tang TY Sadat U Gaunt ME. Remote ischemic preconditioning in major vascular surgery. J Vasc Surg . 2009; 49: 240–243. [CrossRef] [PubMed]
Figure 1
 
Footpad temperature fell during ischemia and rose during reperfusion. The three traces show temperature changes induced by 5-minute ischemia (blue), 10-minute ischemia (red), and two 5-minute periods of ischemia separated by 5-minute reperfusion (green).
Figure 1
 
Footpad temperature fell during ischemia and rose during reperfusion. The three traces show temperature changes induced by 5-minute ischemia (blue), 10-minute ischemia (red), and two 5-minute periods of ischemia separated by 5-minute reperfusion (green).
Figure 2
 
Experimental groups and ERG measurements. (A) Four experimental groups were used to demonstrate the impact of RIP on retinal function. In one group, ischemia was generated in one hindlimb for 5 minutes; in a second group the ischemia lasted 10 minutes; in a third group, the limb was made ischemic for two periods of 5 minutes, separated by a 5-minute period of reperfusion. In a fourth group (sham), the pressure cuff was wrapped around the limb but not inflated. The ERG was recorded at the time points shown by the red arrows. (B) The amplitude of the a-wave was measured from the baseline to the first negative peak (left arrow). The amplitude of the b-wave was measured as shown by the right arrow. (C) A representative set of responses to flashes of 11 intensities (−4.4 to 2.0 log scot cd.s.m−2). (D) For the bleach-recovery experiment, the retina was conditioned by the 2 × 5-minute pattern of limb ischemia followed immediately by a 60-second period of light exposure (14.2 cd/m−2). The response of the retina to tracking flash (a single flash at 1.4 log scot cd.s.m−2) was used to trace the recovery of responsiveness after the bleach at 5-minute intervals after the flash.
Figure 2
 
Experimental groups and ERG measurements. (A) Four experimental groups were used to demonstrate the impact of RIP on retinal function. In one group, ischemia was generated in one hindlimb for 5 minutes; in a second group the ischemia lasted 10 minutes; in a third group, the limb was made ischemic for two periods of 5 minutes, separated by a 5-minute period of reperfusion. In a fourth group (sham), the pressure cuff was wrapped around the limb but not inflated. The ERG was recorded at the time points shown by the red arrows. (B) The amplitude of the a-wave was measured from the baseline to the first negative peak (left arrow). The amplitude of the b-wave was measured as shown by the right arrow. (C) A representative set of responses to flashes of 11 intensities (−4.4 to 2.0 log scot cd.s.m−2). (D) For the bleach-recovery experiment, the retina was conditioned by the 2 × 5-minute pattern of limb ischemia followed immediately by a 60-second period of light exposure (14.2 cd/m−2). The response of the retina to tracking flash (a single flash at 1.4 log scot cd.s.m−2) was used to trace the recovery of responsiveness after the bleach at 5-minute intervals after the flash.
Figure 3
 
Representative ERG recordings before (thinner trace) and after (thicker trace) remote ischemia for the four experimental groups in Figure 1 and for four intensities of flash stimulus. Left column: Essentially identical responses were obtained 10 minutes apart for the sham group. Second column: A 5-minute period of ischemia induced a small increase in amplitude, most noticeably in the b-wave. Third column: A 10-minute period of ischemia induced a greater increase in amplitude. Fourth column: Two 5-minute periods of ischemia separated by 5-minute reperfusion also induced a larger increase in amplitude, apparent also in the a-wave at brighter intensities.
Figure 3
 
Representative ERG recordings before (thinner trace) and after (thicker trace) remote ischemia for the four experimental groups in Figure 1 and for four intensities of flash stimulus. Left column: Essentially identical responses were obtained 10 minutes apart for the sham group. Second column: A 5-minute period of ischemia induced a small increase in amplitude, most noticeably in the b-wave. Third column: A 10-minute period of ischemia induced a greater increase in amplitude. Fourth column: Two 5-minute periods of ischemia separated by 5-minute reperfusion also induced a larger increase in amplitude, apparent also in the a-wave at brighter intensities.
Figure 4
 
RIP-induced changes in the amplitudes of a- and b-wave; normalized data for two flash intensities. For comparisons of remote ischemia–conditioned responses to unconditioned, *P < 0.05, **P < 0.01, ***P < 0.0001. Mean a-wave responses shown in white bars, b-wave mean responses shown in gray bars, n = 3 to 6 ± SEM. (A) Using a flash of 0.4 log cd.s.m−2, the normalized values showed a significant (asterisk) increase in the 2 × 5-minute group for the a-wave and in the 10- and the 2 × 5-minute groups for the b-wave. (B) Using a brighter flash (2.0 log cd.s.m−2), the normalized values showed a significant increase in the 2 × 5-minute group for the a-wave and in all three ischemia groups for the b-wave.
Figure 4
 
RIP-induced changes in the amplitudes of a- and b-wave; normalized data for two flash intensities. For comparisons of remote ischemia–conditioned responses to unconditioned, *P < 0.05, **P < 0.01, ***P < 0.0001. Mean a-wave responses shown in white bars, b-wave mean responses shown in gray bars, n = 3 to 6 ± SEM. (A) Using a flash of 0.4 log cd.s.m−2, the normalized values showed a significant (asterisk) increase in the 2 × 5-minute group for the a-wave and in the 10- and the 2 × 5-minute groups for the b-wave. (B) Using a brighter flash (2.0 log cd.s.m−2), the normalized values showed a significant increase in the 2 × 5-minute group for the a-wave and in all three ischemia groups for the b-wave.
Figure 5
 
Intensity–response curves for conditioned (red) 2 × 5-minute group and unconditioned eyes (blue). Group average responses, n = 6 ± SEM. (A) The conditioned a-wave was consistently larger at higher flash intensities. On a two-way ANOVA test, the two curves are significantly different (P < 0.01). (B) The conditioned b-wave was also consistently larger at higher intensities. On a two-way ANOVA test, the two curves are significantly different (P < 0.0001). Post hoc analysis showed significant (*P < 0.05) differences at each of the two top intensities.
Figure 5
 
Intensity–response curves for conditioned (red) 2 × 5-minute group and unconditioned eyes (blue). Group average responses, n = 6 ± SEM. (A) The conditioned a-wave was consistently larger at higher flash intensities. On a two-way ANOVA test, the two curves are significantly different (P < 0.01). (B) The conditioned b-wave was also consistently larger at higher intensities. On a two-way ANOVA test, the two curves are significantly different (P < 0.0001). Post hoc analysis showed significant (*P < 0.05) differences at each of the two top intensities.
Figure 6
 
Latency of the a- and b-waves in the four groups shown in Figures 2 and 3. A-wave mean timing shown in white bars and b-wave mean timing in gray bars, n = 6 ± SEM. Latency of the a-wave was measured from the stimulus artifact to the first negative peak, latency of the b-wave to the first positive peak. Latencies appeared unaffected by RIP (a-wave P = 0.4 and b-wave P = 0.6, one-way ANOVA).
Figure 6
 
Latency of the a- and b-waves in the four groups shown in Figures 2 and 3. A-wave mean timing shown in white bars and b-wave mean timing in gray bars, n = 6 ± SEM. Latency of the a-wave was measured from the stimulus artifact to the first negative peak, latency of the b-wave to the first positive peak. Latencies appeared unaffected by RIP (a-wave P = 0.4 and b-wave P = 0.6, one-way ANOVA).
Figure 7
 
Stability and repeatability of RIP-induced increase of a- and b-waves. (A) The a- (white) and b-waves (gray) ± SEM were measured 25, 35, and 45 minutes after the commencement of 2 × 5-minute RIP; the increase of their amplitudes persisted (P < 0.0001, n = 4 for differences between sham and RIP treatment). Post-RIP differences were stable at 25, 35, and 45 minutes (n = 4; a-wave P = 0.8 and b-wave P = 0.9, one-way ANOVA). (B) In sham-treated animals, the a- and b-waves were stable over the same testing times (C) in each of a group of six animals. The effect of RIP on the amplitudes of the a- and b-wave was assessed 25 minutes after 2 × 5-minute RIP on three occasions—an initial trial, and then 1 week and 1 month later. The effect recurred reliably at each time point (n = 4; a-wave P = 0.8, b-wave P = 0.7, one-way ANOVA).
Figure 7
 
Stability and repeatability of RIP-induced increase of a- and b-waves. (A) The a- (white) and b-waves (gray) ± SEM were measured 25, 35, and 45 minutes after the commencement of 2 × 5-minute RIP; the increase of their amplitudes persisted (P < 0.0001, n = 4 for differences between sham and RIP treatment). Post-RIP differences were stable at 25, 35, and 45 minutes (n = 4; a-wave P = 0.8 and b-wave P = 0.9, one-way ANOVA). (B) In sham-treated animals, the a- and b-waves were stable over the same testing times (C) in each of a group of six animals. The effect of RIP on the amplitudes of the a- and b-wave was assessed 25 minutes after 2 × 5-minute RIP on three occasions—an initial trial, and then 1 week and 1 month later. The effect recurred reliably at each time point (n = 4; a-wave P = 0.8, b-wave P = 0.7, one-way ANOVA).
Figure 8
 
(A) Representative ERG responses at 0 to 60 minutes after a 60-second bleach (∼1% bleach of rhodopsin). (B, C) Amplitude recovery with and without RIP for the a- and b-wave, unconditioned eyes (blue) compared to conditioned 2 × 5 minutes (red) ± SEM. For the b-wave the curves were significantly different (n = 4; P = 0.03, two-way ANOVA) (B). For the a-wave the difference between the two curves did not reach significance (n = 4, P > 0.05, two-way ANOVA) (C).
Figure 8
 
(A) Representative ERG responses at 0 to 60 minutes after a 60-second bleach (∼1% bleach of rhodopsin). (B, C) Amplitude recovery with and without RIP for the a- and b-wave, unconditioned eyes (blue) compared to conditioned 2 × 5 minutes (red) ± SEM. For the b-wave the curves were significantly different (n = 4; P = 0.03, two-way ANOVA) (B). For the a-wave the difference between the two curves did not reach significance (n = 4, P > 0.05, two-way ANOVA) (C).
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×