June 2003
Volume 44, Issue 6
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Retina  |   June 2003
The Contribution of Glycolytic and Oxidative Pathways to Retinal Photoreceptor Function
Author Affiliations
  • Bang V. Bui
    From the Department of Optometry and Vision Sciences, University of Melbourne, Melbourne, Victoria, Australia; and the
  • Michael Kalloniatis
    From the Department of Optometry and Vision Sciences, University of Melbourne, Melbourne, Victoria, Australia; and the
    Department of Optometry Vision Science, University of Auckland, Auckland, New Zealand.
  • Algis J. Vingrys
    From the Department of Optometry and Vision Sciences, University of Melbourne, Melbourne, Victoria, Australia; and the
Investigative Ophthalmology & Visual Science June 2003, Vol.44, 2708-2715. doi:10.1167/iovs.02-1054
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      Bang V. Bui, Michael Kalloniatis, Algis J. Vingrys; The Contribution of Glycolytic and Oxidative Pathways to Retinal Photoreceptor Function. Invest. Ophthalmol. Vis. Sci. 2003;44(6):2708-2715. doi: 10.1167/iovs.02-1054.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To consider how aerobic and anaerobic metabolic processes limit posthypoxemic decay in retinal function, measured by electroretinogram (ERG).

methods. The hypothesis that lowering metabolic demand would prolong endogenous metabolic stores was tested by comparing the rate of ERG decay in rats in dark- (n = 5) versus light-adapted (15 minutes, 112 cd/m2, n = 5) conditions and with serial versus single (n = 5 at each of seven time points) light stimulation. Postmortem hypoxemia was induced by cervical dislocation. Glucose (10 and 100 mM) and glutamine or lactate (100 mM) were injected into the vitreous 10 minutes before hypoxemic insult, to consider glycolytic-oxidative versus oxidative metabolism, respectively.

results. Lowering the metabolic drain by light adaptation or serial stimulation significantly improved the photoreceptoral saturated amplitude during the first 5 to 7.5 minutes after postmortem hypoxemia. Increasing substrate availability with exogenous glucose preloading delayed the loss of the photoreceptoral response, thereby extending the delay constant from 4.8 to 10.9 minutes. Postreceptoral amplitudes were not improved by any exogenous substrate. Providing glucose at 5 minutes after hypoxemia provided no benefits. Similar to glucose, glutamine and lactate loading significantly delayed photoreceptoral decay over the first 7.5 minutes, after which time glucose was the more effective substrate.

conclusions. The postmortem decay of photoreceptoral function reflects depletion of both endogenous oxygen and carbon substrate reserves. The findings provide evidence that a transition between aerobic and anaerobic metabolism occurs after approximately 8 minutes of complete hypoxemia.

Hypoxemic insult results in rapid changes in retinal function 1 and in altered neurotransmitter metabolism. 2 Clinically, the consequences of acute retinal vascular occlusions can be devastating, resulting in irreversible vision loss. 3 4 It is therefore vital to understand the nature of posthypoxemic functional decay and its relation to the depletion of endogenous glucose and oxygen stores. Hypoxia or hypoglycemia, separately severely attenuate retinal function measured by electroretinogram (ERG). 1 5 However, the time course of such functional decay 6 7 does not appear to parallel the rapid depletion of endogenous oxygen reserves after hypoxemic insult. 8 Hence, we wanted to consider how endogenous metabolite stores might limit the decay of posthypoxemic retinal function. This knowledge would be useful in developing treatment options for acute hypoxemia, such as central retinal artery occlusion in humans. 
An important question for the clinician is whether the energy demands of the hypoxemic tissue can be attenuated. Phototransduction and the dark current account for a substantial proportion of retinal adenosine triphosphate (ATP) 1 9 and probably represent the largest drain on endogenous substrate stores. Consequently, it might be anticipated that light adaptation would significantly prolong posthypoxemic retinal function, because constant light is known to reduce oxygen 1 10 11 and glucose consumption significantly. 10 12 13  
Apart from lowering the dark current through lowered Na+K+-adenosine triphosphatase (ATPase) activity 6 constant light may partially stimulate retinal metabolism. 1 Moreover, light adaptation may favor different metabolic mechanisms, as evidenced by the exquisite sensitivity of rod-driven function to mild changes in glucose concentration compared with cones. 14 We consider an alternate approach to overcome these potential confounds. This technique is based on the rationale that serial stimulation can repetitively attenuate the dark current, whereas single stimuli maintain the retina in a heightened metabolic state for progressively longer intervals until stimulation. 
An additional set of experiments was conducted to consider the site at which we might be modulating metabolic stores. In particular, we attempted to differentiate between the constraints imposed by aerobic and anaerobic substrates on posthypoxemic function. First, we determined whether increasing substrate availability can prolong function by preloading the vitreous with glucose. Glucose is the major metabolic substrate that contributes to production of ATP through glycolysis, which in turn provides oxidative carbon skeletons. Retinal function is normally supported by both oxidative metabolism and glycolysis. 1 9 However, the contributions of these mechanisms are altered under hypoxemic conditions, as evidenced by the finding that retinal function can be partially maintained by glycolysis, which is upregulated to compensate for loss of oxidative ATP-producing pathways. 1 15 Hence, to consider the contributions of anaerobic and aerobic pathways we contrasted the functional benefits of glucose with substrates that only fuel oxidative metabolism. Glutamine 9 and lactate 16 support only retinal function in the presence of adequate oxygen because these compounds can be used to fuel the tricarboxylic acid cycle. 17 The differential effects on retinal function of glucose versus glutamine-lactate preloading would provide information about the constraints imposed by anaerobic and aerobic metabolic processes. 
Materials and Methods
Electroretinography
All experimental protocols in this study were approved by our institutional ethics committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were maintained in a 12-hour light–dark (40–130 lux, on at 8 AM) environment with normal rat chow and water available ad libitum. 
Electroretinograms were recorded from adult Long-Evans rats (aged 9 weeks) after overnight dark adaptation (>12 hours). Anesthesia was induced using a mixture of ketamine and xylazine (60:5 mg/kg, Troy Laboratories, Smithfield, New South Wales, Australia). Mydriasis (≥4 mm) was achieved with tropicamide (Mydriacyl 0.5%; Alcon Laboratories, Frenchs Forest, New South Wales, Australia) and corneal anesthesia with proxymetacaine (Ophthetic 0.5%; Allergan, Frenchs Forest, New South Wales, Australia). All animal manipulations were conducted under dim red light provided by a light-emitting diode (λmax = 650 nm). Flash ERGs (white) were recorded with custom silver–silver chloride (Ag-AgCl) electrodes placed on the cornea and in the mouth, referenced to a stainless steel electrode (Medelec, Richmond, Victoria, Australia) inserted in the tail. After electrode placement, a further 10-minute dark adaptation was allowed before signal acquisition. 
Signals were elicited using a commercially available photographic flash unit (285 V; Vivitar Photographics, Newbury Park, CA) delivered through a Ganzfeld sphere. Flash exposure was measured as previously described, 18 and yielded a luminous energy of 3.5 log cd-s/m2. We used a luminous energy of 2.5 log cd-s/m2 achieved using a calibrated neutral density filter (1.0 ND, Kodak Wratten; Eastman Kodak Co., Rochester, NY). This luminous energy gave a saturating a-wave amplitude. Responses were amplified (gain ×1000; −3 dB at 0.1 and 3000 Hz, P55; Grass Instruments, Inc., West Warwick, RI) and digitized at 4 kHz. 
ERG Protocol
Four baseline ERG signals were collected, after which animals were killed by cervical dislocation under dim red light. The time of death was noted when the heartbeat could no longer be detected. ERG signals were then serially recorded from groups of animals (n = 5) every 2.5 minutes for up to 40 minutes after death to observe postmortem functional decay. We considered whether reducing the metabolic drain on endogenous substrate reserves could prolong retinal function by comparing the dark-adapted postmortem condition to the less metabolically demanding light-adapted condition. 1 11 12 A group of five animals had ERGs collected under light-adapted conditions (112 cd/m2 beginning 15 minutes before experimentation). We also compared the effects of serial light stimulation to that returned by single stimulation. Single stimulation was achieved on different animals by applying only one flash of light at each of eight postmortem time points (1, 2, 4, 6, 8, 16, 32, and 64 minutes), which represent progressively longer times in the dark-adapted state. In contrast, serial stimulation repetitively interrupted the dark current, with sequential stimuli being delivered at baseline and at 2.5-minute intervals after insult (n = 5). The following section details the exogenous metabolic substrates used to determine the contribution of glycolytic and oxidative metabolic processes to posthypoxemia function. 
Exogenous Metabolic Substrates
We conducted a series of experiments to confirm that exogenous glucose can improve metabolic stores and, more important, prolong retinal function. Glucose (Sigma Chemical Co., St. Louis, MO) adjusted to a final vitreous concentration of either 10 or 100 mM was loaded into the vitreous chamber 10 minutes before hypoxemic insult. We compared the effect of increasing available substrate before and after insult by contrasting premortem (10 minutes) and postmortem (5 minutes after death) glucose loading. It is important to differentiate between the contribution of glucose to glycolytic and oxidative pathways. This was achieved by comparing the ameliorative effects of glucose with those of glutamine and lactate (Sigma Chemical Co.), which are purely tricarboxylic acid (TCA) cycle substrates. Consequently, glutamine and lactate (100 mM) were individually preloaded 10 minutes before insult (n = 5). 
All substrate concentrations represent the adjusted final vitreous concentration, which was calculated assuming a vitreous chamber volume of 40 μL, which represents an average based on previous studies 19 20 and our own calculations from published measurements. 21 Exogenous metabolic substrates were diluted in phosphate-buffered saline, and equalized approximately to pH 7.4, using 1 M hydrochloric acid and 1 M sodium hydroxide (BDH Chemicals, Kilsyth, Victoria, Australia). An injection volume between 2 to 5 μL has been shown to produce reliable results. 19 20 We adopt a 2-μL volume to minimize any affects of elevated intraocular pressure on the ERG. Substrates were injected with a sleeved 33-gauge needle, with 1.5 mm of the tip exposed, inserted 2 mm behind the limbus into the vitreous chamber at a 45° angle from the sclera. 
ERG Analysis
Phototransduction amplitude and sensitivity may be affected by hypoxemia, because ATP is essential for phototransduction and Na+K+-ATPase. 1 9 22 Hence, we applied the phototransduction model of Hood and Birch 23  
\[\mathrm{P3}(i,t)\ {=}\ \mathrm{Rm}_{\mathrm{P}_{3}}\ {\cdot}\ {[}1\ {-}\ e^{{-}i\ {\cdot}\ \mathrm{S}\ {\cdot}\ (t{-}t_{\mathrm{d}})^{2}}{]}\ \mathrm{for}t{>}t_{\mathrm{d}}\]
In equation 1 , P3 is the summed rod photocurrent as a function of stimulus exposure i (in candela-seconds per square meter) and time t (in seconds), and RmP3 (in microvolts) is the saturated amplitude of the photoreceptor. Sensitivity (S, in square meters per candela-cubic second) is scaled by i, whereas t d (seconds) allows for both biochemical and recording latencies. This model was fitted to the raw data up to the first minimum of each a-wave. Optimization was achieved by minimizing the sum-of-square error term on a spreadsheet (Excel; Microsoft Corp., Redmond, WA). Because cone intrusion in the rat ERG is minimal at our intensity, we apply the phototransduction model without rod isolation. 24 More complex formulations for phototransduction, which account for membrane capacitance were not used because the influence of hypoxemia on this parameter is unclear. 
Postreceptoral responses were extracted by digital subtraction of the modeled photoreceptoral response from the raw waveform, because both the photoreceptoral and postreceptoral components are simultaneously altered by hypoxemia, such that a modification of the photoreceptoral response may mask a postreceptoral change. Postreceptoral amplitudes were then calculated from baseline to the peak amplitude. For each animal, postmortem ERG parameters are normalized to their respective baseline measurements. In all cases, data are expressed as a mean (±SEM, n = 5) for each group as a function of time since death. 
To quantify the rate of functional decay postmortem we applied a simple exponential function (equation 2) . Normalized amplitude (y) is described as a function of time (t, minutes) by a maximum response (a), decay time constant (t c, minutes) and offset (y o), which accounts for the noise in the ERG.  
\[y\ {=}\ y_{\mathrm{o}}\ {+}\ (a\ {-}\ y_{\mathrm{o}})\ {\cdot}\ e^{\left({-}\ \frac{t}{t_{\mathrm{c}}}\right)}\]
 
Statistics
A nonparametric bootstrap was used to compare transition functions (equation 2) . This technique allows an unbiased comparison of effects by returning the 95% confidence limits for the parameters of the exponential decay function. 25 Raw data were compared by repeated-measures ANOVA, with an adjusted α of 0.01 to protect against type 2 errors. ANOVA outcomes are expressed as probabilities, and when nonsignificant results are shown, the power of the performed test is shown in brackets. 
Results
Postmortem hypoxemia resulted in a rapid reduction of the ERG, beginning with a loss of the postreceptoral b-wave, followed by impairment of the photoreceptoral a-wave (Fig. 1A) . The light-adapted retina has a smaller photoreceptoral and postreceptoral response (10% and 60% of dark-adapted amplitudes) consistent with a cone contribution. 24 Light adaptation reduced the relative level of photoreceptoral impairment at the early time points (Figs. 1B 1C) . Light-adapted photoreceptoral amplitude (unfilled circles) was significantly better than dark-adapted amplitudes (filled circles) at 2.5 minutes after death. However, the decay time constant (thin line, t c = 4.52 minutes) was not significantly prolonged (thick line, t c = 4.81 minutes). This probably reflects the cone contribution to the light-adapted ERG and the fact that cone metabolism responds more robustly to the effects of hypoxia. We tested this possibility by serial stimulation, which is described in the next section. Although a trend for postreceptoral improvement was seen, the effect was also not significant. This finding may reflect the fast decay in the b-wave and our limited sampling at early time points. 
Figure 2 considers the photoreceptoral model (P3) and the derived postreceptoral response obtained from serial stimulation and signals derived at single time points. Consistent with the light-adaptation experiment, normalized phototransduction saturated amplitude (RmP3) decayed significantly slower with signals elicited serially (filled circles, thick line, t c = 4.81 minutes) compared with single signals (unfilled circles, thin line, t c= 3.06 minutes). These outcomes are shown in Figure 2B and summarized in Table 1 . Similarly, phototransduction sensitivity (log S, Fig. 2C ) decay was slower for serial (t c= 3.45 minutes) than single signals (t c= 2.10 minutes). 
The data indicated that decreasing the metabolic drain on endogenous stores by light adaptation and serial stimulation delays functional loss. The following experiments examined the effect of increasing available substrate stores on maintaining retinal function. Preloading the vitreous with glucose slowed the loss of the photoreceptor response compared with the control (Fig. 3A) , which is limited to the photoreceptoral portion of the ERG response, because our technique could not accurately follow postreceptoral decay. As a consequence, we concentrated on receptoral changes in the following analysis. Figure 3B confirms that glucose preloading (unfilled circles) significantly improved RmP3 (P < 0.001) compared with the control (filled circles), with an improvement in the decay constant (Table 1) . A small improvement was observed in log S (Table 1)
Figure 4A shows that 100 mM glucose (unfilled circles) had functional effects similar to those induced by 10 mM glucose (shaded squares) on both RmP3 (Fig. 4A , Table 1 , P < 0.001) and log S (Fig. 4B , Table 1 , P = 0.30 [0.59]) compared with the control (filled circles). Both substrate concentration and the timing of delivery were important for functional survival (Figs. 4C 4D) . The benefits of glucose preloading were not replicated when 10 mM glucose was injected into the vitreous after 5 minutes of postmortem hypoxemia (shaded triangles). Indeed, RmP3 (Fig. 4C , P = 0.80[0.30], t c= 5.10 minutes) and log S (Fig. 4D , P = 0.98[0.14], t c= 4.18 minutes) for the 5-minute postmortem application were indistinguishable from the vehicle-treated control (filled circles). 
We injected glutamine and lactate into the vitreous to compare the effect of glucose vitreous loading with these aerobic substrates. 17 Figure 5 shows that providing oxidative substrates in the form of glutamine or lactate significantly improved RmP3 compared with the control. Both glutamine (Fig. 5A , unfilled squares, P < 0.001) and lactate (Fig. 5C , unfilled triangles, P < 0.01) significantly delayed the onset of decay at early time points, which manifests as a small increase in the delay constant (Table 1) . However, neither glutamine (Fig. 5B , P = 0.81 [0.29], t c= 3.41 minutes) nor lactate (Fig. 5C , P = 0.39 [0.53], t c= 3.79 minutes) was able to significantly improve log S (Figs. 5B 5C)
Figure 6 contrasts the benefits of the glycolytic–aerobic (glucose) and purely aerobic substrates (glutamine-lactate), by plotting the relative difference for RmP3 between the various substrates against the control. Both 10 and 100 mM exogenous glucose provided a maximum functional benefit of approximately 35% at 7.5 to 10 minutes after death (Fig. 6A) . In contrast, glucose injection at 5 minutes after death (Fig. 6B , unfilled circles) caused little improvement. Glutamine (Fig. 6C , unfilled squares) and lactate (Fig. 6D , unfilled triangles) both achieved improvements comparable to glucose (100 mM), reaching a peak between 5 and 7.5 minutes after death. In contrast to the glucose effect that continued to beyond 10 minutes, the palliative effect of the purely oxidative substrates was lost after this time. 
Discussion
Postmortem Retinal Function
Hypoxemic insult rapidly impaired retinal function, consistent with previous findings. 9 A rapid loss of the postreceptoral response was followed by a gradual photoreceptoral decay, as has been demonstrated in a range of mammalian species. 1 26 27 The rapid loss of retinal function is commensurate with the high retinal metabolic demand. 28 The metabolic needs of photoreceptors are particularly acute, 1 11 as might be expected from the high density of mitochondria and oxidative enzymes in these cells. 28 29 Large quantities of high-energy phosphates are needed to support ionic transport, particularly the Na+K+-ATPase 22 30 responsible for the dark current. 6 The inability of hypoxemic tissue to restore this circulating current can manifest as a loss of photoreceptoral function. 31 Because our method acutely impairs blood supply to both the inner retina and the choriocapillaris, the depletion of endogenous substrate reserves is likely to constrain the loss of retinal function. 
The underlying cause of the inner retinal functional 26 27 and morphologic 32 susceptibility to hypoxemia remains unclear. Indeed, such sensitivity is contrary to the greater oxygen consumption and metabolic demand of the outer retina. 1 11 An advantage might be conferred to photoreceptors from the metabolic buffering provided by their proximity to the retinal pigment epithelium and choriocapillaris, whereas metabolites from the inner retinal circulation are more rapidly depleted with hypoxemia. 11 Alternatively, the postreceptoral susceptibility has been attributed to reduced neurotransmission in the retina, 14 with a similar mechanism suggested in the brain. 33 In support of this thesis, hypoglycemia did not reduce the energy state of the brain, suggesting reduced energy consumption and/or an alternate energy source. 34 35 Glucose metabolism is metabolically coupled to the neurotransmitter cycle, 36 37 such that inhibiting neurotransmission can lower energy consumption and provide a TCA cycle substrate. 38 39 Consistent with this idea, glutamate, glutamine, and γ-aminobutyric acid levels are decreased, whereas aspartate and NH4 + are increased after metabolic impairment in the brain 35 and retina. 40 In addition, glutamate 41 and γ-aminobutyric acid 42 metabolism by the TCA cycle is increased when their respective concentrations are elevated. 
In our model, functional decay was constrained by ATP produced from remaining oxygen and carbon substrates, including glucose, glycogen stores, and alternate sources of carbon skeletons. Tornquist and Alm 43 showed that most of the glycolytic substrate is derived from the choroidal circulation. During ischemic insult, glucose consumption is increased. 12 Glucose passage through blood–retinal barriers can occur through Na+-dependent symport; however, most is mediated by facilitated diffusion. 44 Glucose transporter (GLUT)-1 is the main contributor to glucose entry and is prominent on the outer and inner blood–retinal barriers, photoreceptors, ganglion cells, and Müller cells. 44 45 During acute metabolic insult, GLUT-1 activity is reduced, 46 which critically constrains brain metabolism. 47  
Mobilization of astrocytic glycogen stores has been observed after increased neuronal activity 36 and during ischemia in the brain. 48 It is not surprising that glycogen depletion shows some correlation with EEG loss in the rat brain. 49 Furthermore, improved posthypoglycemia neuronal survival is achieved by increasing glycogen stores. 50 Retinal glycogen depletion after hypoxemic insult has a time course of 20 to 45 minutes. 51 52 In the rabbit retina, 50% of the glycogen stores is depleted after 15 minutes of hypoxemia, 53 whereas glycogen loss in the brain has a decay time constant of approximately 6 minutes. 54 Given this time course, endogenous glycogen is likely to be an important factor constraining postmortem functional decay in our model. 
Reduction of Metabolic Demand
By reducing metabolic demand, we were able to preserve endogenous pools of metabolic substrates and thereby prolong photoreceptoral function. Light adaptation reduces oxygen 11 and glucose consumption, 10 particularly by photoreceptor inner segments. 55 This effect occurs largely through inhibition of the Na+K+-ATPase–driven photoreceptoral dark current. 6 However, light adaptation also favors cone-mediated function, whose metabolic demand is lower than the rod system. 14 Moreover, cones have access to circulating glucose and glycogen stores, whereas rods lack glycogen and the rate-limiting glycogenolytic enzyme, glycogen phosphorylase. 56 57 Our findings are in accord with previous reports that rod responses show greater susceptibility to hypoglycemia than light-adapted cone responses. 14 58  
The functional improvement induced by light adaptation was restricted to the first 5 minutes. This limited effect may be due to the increased oxidative demand of cGMP turnover 1 22 and the acidosis, which follows from lactate build-up through heightened glycogenolysis and anaerobic glycolysis. 56 By using serial light stimulation to intermittently interrupt the dark current, we were able to isolate the rod response and prolong hypoxemic retinal function compared with single stimuli. One conjecture is that serial stimulation might interrupt ATP consumption of Na+K+-ATPase 1 9 without elevation of glycolysis and thereby acidosis. 
Exogenous Metabolic Substrates
The experiments described herein show that endogenous substrates are a critical rate determinant of posthypoxemic retinal function. 2 This was further confirmed by increasing substrate availability with glucose preloading, consistent with previous findings. 59 Both 10 and 100 mM glucose provided similar functional benefits as could be expected from the GLUT-1 K m value of approximately 1 mM. 44 45 60 Glucose injection at 5 minutes after death did not significantly delay functional loss, consistent with the impairment of glucose transport during hypoxemic insult. 46 Hence, beyond 5 minutes after death, retinal function is likely to be supported by retinal glycogen and alternate metabolic substrates such as glutamine 17 61 and lactate. 16 62  
Glutamine is firstly converted through phosphate-activated glutaminase to glutamate, which can be deaminated to form the TCA cycle carbon skeleton of α-ketoglutarate. 17 Glutamine also contributes to the production of other metabolic fuels such as glutathione and phosphocreatine. 61 Lactate is converted to pyruvate through lactate dehydrogenase, which then fuels the TCA cycle. 63 In addition, the reactions involving phosphate-activated glutaminase and lactate dehydrogenase produce the reducing equivalent nicotinamide adenine dehydrogenase (NADH) 63 which is needed for transporting electrons into the mitochondrial compartment. 64 Glutamine and lactate preloading significantly improved photoreceptoral function; however, this effect was limited to the first 7.5 minutes, whereas glucose was effective for up to 25 minutes. Although all three substrates can be incorporated into aerobic pathways, only glucose can generate ATP by anaerobic glycolysis. 17 Hence, the contrasting effects of glutamine and lactate versus glucose provide evidence that aerobic processes contribute to retinal function for the first 7.5 minutes, after which, function is supported by anaerobic glycolysis. In accordance with this idea, significant changes to highly ATP-dependent processes such as neurotransmitter recycling becomes significant at 8 minutes after death. 65  
The discrepancy between aerobic substrates and glucose may reflect differentially the failure of monocarboxylate transporters 66 and the A-system transporter GlnT/ATA1, 67 which mediate neuronal uptake of lactate and glutamine, respectively, compared with facilitative glucose transporters. 44 This may be expected, because transport of both lactate and glutamine is dependent on Na+ cotransport. An altered Na+ gradient resultant from Na+K+-ATPase failure after hypoxemia impairs lactate and glutamine transport, whereas glucose uptake can continue by facilitated diffusion. 44 Given that Na+K+-ATPase consume most oxidative ATP, 1 a failure of substrate transporters should mirror the impairment of oxidative metabolism. Hence, this mechanism is consistent with a transition to purely anaerobic metabolism 8 minutes after hypoxemia. 
Alternatively, exogenous aerobic substrates and glucose may have differential demands on endogenous oxygen stores. In particular, restoration of the transmembrane Na+ gradient after increased glutamine or monocarboxylate transporter activity requires increased ATP and oxygen usage. However, the exogenous glutamine is unlikely to elevate oxygen consumption appreciably, because glutamine transporters function at close to maximum capacity under normal conditions. 68 69 Similarly, it is doubtful that lactate transport substantially elevates oxygen consumption 10 minutes after substrate preloading, because most neuronal lactate uptake is mediated by an initial rapid equilibrative influx occurring within less than 1 minute. 70 71 Hence, elevated oxygen consumption is unlikely to underlie the limited functional benefit observed with glutamine and lactate compared with glucose. Moreover, a necessary corollary of accelerated oxygen depletion would be a more rapid functional decline, given the dependence of the photoreceptoral potential on oxidative metabolism. 1 9 Our findings are contrary to such an expectation and suggest that the metabolic yield from exogenous glutamine or lactate outweighs the energy cost of their transport and metabolism. 
Exogenous substrates were unable to improve the postreceptoral response. An ameliorative effect was anticipated with glutamine, which can contribute to neurotransmitter glutamate pools. 17 The absence of an effect provides some support for the idea that amino acids are metabolized into the TCA cycle, at the expense of synaptic transmission. However, this notion requires further investigation, and would benefit from a hypoxemic model with an extended time course together with measurement of the time course of oxygen depletion. 
In summary, after hypoxemic insult, retinal function is constrained by the availability of both aerobic and anaerobic substrates. Although endogenous oxygen remains, amino acids may be sequestered to metabolic pathways to support photoreceptoral function and may underlie the early loss of the inner retinal response. The differential effect of oxidative substrates compared with glucose suggests that a transition from aerobic to anaerobic metabolism occurred between 7.5 and 10 minutes after hypoxemia. Our results support the idea that the supply of glucose during acute ischemia would assist in maintaining retinal integrity. In addition, the provision must be made as soon as possible after occurrence of acute ischemia, and elevation of overall light levels would also have beneficial effects in maintaining retinal function. 
 
Figure 1.
 
Effect on retinal function of dark and light adaptation before postmortem hypoxemia. (A) Representative baseline (thick solid line) and postmortem (thin solid line) waveforms in a dark-adapted animal. (B) Representative baseline (thick solid line) and postmortem (thin solid line) waveforms in a light-adapted animal (112 cd/m2). (C) Normalized (mean ± SEM, n = 5) postmortem photoreceptoral light- and dark-adapted a- and b-wave amplitudes.
Figure 1.
 
Effect on retinal function of dark and light adaptation before postmortem hypoxemia. (A) Representative baseline (thick solid line) and postmortem (thin solid line) waveforms in a dark-adapted animal. (B) Representative baseline (thick solid line) and postmortem (thin solid line) waveforms in a light-adapted animal (112 cd/m2). (C) Normalized (mean ± SEM, n = 5) postmortem photoreceptoral light- and dark-adapted a- and b-wave amplitudes.
Figure 2.
 
Effect of serial and single stimuli on postmortem retinal function. (A) Representative raw ERG waveforms (□) are described using a phototransduction P3 model (solid line) at baseline and various times after death. Postreceptoral P2 responses (▪) were extracted by digital subtraction of the P3 model from the raw data. (B) Normalized (mean ± SEM, n = 5) decay of RmP3 with serial versus single signals. (C) Normalized phototransduction log S for serial versus single stimuli.
Figure 2.
 
Effect of serial and single stimuli on postmortem retinal function. (A) Representative raw ERG waveforms (□) are described using a phototransduction P3 model (solid line) at baseline and various times after death. Postreceptoral P2 responses (▪) were extracted by digital subtraction of the P3 model from the raw data. (B) Normalized (mean ± SEM, n = 5) decay of RmP3 with serial versus single signals. (C) Normalized phototransduction log S for serial versus single stimuli.
Table 1.
 
Decay Time Constants for Posthypoxemic Phototransduction Parameters for Various Conditions
Table 1.
 
Decay Time Constants for Posthypoxemic Phototransduction Parameters for Various Conditions
Phototransduction Saturated Amplitude (RmP3) Phototransduction Sensitivity (log S)
Mean, t c 2.5% CL 97.5% CL Mean, t c 2.5% CL 97.5% CL
Control (serial) 4.81 4.28 5.49 3.45 2.47 4.43
Control (single) 3.06* 2.73 3.48 2.10* 1.68 2.66
10 mM Glucose 10.91, † 7.85 13.99 4.69, † 4.25 5.12
100 mM Glucose (10-min preload) 10.06, † 8.99 13.88 5.57, † 4.63 5.78
100 mM Glucose (5-min postload) 5.01 4.19 6.21 4.18 3.73 4.62
100 mM Glutamine 9.01, † 7.50 11.30 3.41 2.83 3.99
100 mM Lactate 12.03, † 8.04 23.89 3.79 2.98 4.60
Figure 3.
 
Effect of glucose preloading on postmortem retinal function. (A) Representative waveforms showing the first 20 ms of the response. Photoreceptoral responses were described using a model of phototransduction (Equation 1) , which returns the phototransduction saturated amplitude (RmP3) and sensitivity (log S). Control and glucose preloaded waveforms are shown. (B) Normalized (± SEM, n = 5) postmortem RmP3 and postreceptoral amplitudes are described using an exponential decay.
Figure 3.
 
Effect of glucose preloading on postmortem retinal function. (A) Representative waveforms showing the first 20 ms of the response. Photoreceptoral responses were described using a model of phototransduction (Equation 1) , which returns the phototransduction saturated amplitude (RmP3) and sensitivity (log S). Control and glucose preloaded waveforms are shown. (B) Normalized (± SEM, n = 5) postmortem RmP3 and postreceptoral amplitudes are described using an exponential decay.
Figure 4.
 
Effect of concentration and time of delivery of exogenous glucose on postmortem photoreceptoral function. Normalized parameters (±SEM) are described using an exponential decay. (A) Control versus 100 and 10 mM glucose phototransduction RmP3 decay. (B) Control versus 100 and 10 mM glucose phototransduction sensitivity (log S) decay. (C) Postmortem RmP3 in control versus glucose preloaded 10 minutes before and postloaded 5 minutes after insult. (D) Postmortem log S in control versus preloaded and postloaded glucose.
Figure 4.
 
Effect of concentration and time of delivery of exogenous glucose on postmortem photoreceptoral function. Normalized parameters (±SEM) are described using an exponential decay. (A) Control versus 100 and 10 mM glucose phototransduction RmP3 decay. (B) Control versus 100 and 10 mM glucose phototransduction sensitivity (log S) decay. (C) Postmortem RmP3 in control versus glucose preloaded 10 minutes before and postloaded 5 minutes after insult. (D) Postmortem log S in control versus preloaded and postloaded glucose.
Figure 5.
 
Effect of glutamine and lactate preloading on postmortem photoreceptoral function. (A) Control versus glutamine phototransduction RmP3 decay. (B). Control versus glutamine phototransduction log S decay. (C) Control versus lactate phototransduction RmP3 decay. (D) Control versus lactate phototransduction log S decay.
Figure 5.
 
Effect of glutamine and lactate preloading on postmortem photoreceptoral function. (A) Control versus glutamine phototransduction RmP3 decay. (B). Control versus glutamine phototransduction log S decay. (C) Control versus lactate phototransduction RmP3 decay. (D) Control versus lactate phototransduction log S decay.
Figure 6.
 
Comparing metabolic substrate effects on posthypoxemic phototransduction RmP3. Data are shown as a mean (±SEM) percentage difference between vehicle and substrate-treated eyes (n = 5 for each group). (A) Comparison of preloading with 100 and 10 mM glucose. (B) Comparison of glucose (100 mM) preloaded 10 minutes before and postloaded 5 minutes after insult. (C) Comparison of preloaded glucose and glutamine. (D) Comparison of preloaded glucose and lactate.
Figure 6.
 
Comparing metabolic substrate effects on posthypoxemic phototransduction RmP3. Data are shown as a mean (±SEM) percentage difference between vehicle and substrate-treated eyes (n = 5 for each group). (A) Comparison of preloading with 100 and 10 mM glucose. (B) Comparison of glucose (100 mM) preloaded 10 minutes before and postloaded 5 minutes after insult. (C) Comparison of preloaded glucose and glutamine. (D) Comparison of preloaded glucose and lactate.
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Figure 1.
 
Effect on retinal function of dark and light adaptation before postmortem hypoxemia. (A) Representative baseline (thick solid line) and postmortem (thin solid line) waveforms in a dark-adapted animal. (B) Representative baseline (thick solid line) and postmortem (thin solid line) waveforms in a light-adapted animal (112 cd/m2). (C) Normalized (mean ± SEM, n = 5) postmortem photoreceptoral light- and dark-adapted a- and b-wave amplitudes.
Figure 1.
 
Effect on retinal function of dark and light adaptation before postmortem hypoxemia. (A) Representative baseline (thick solid line) and postmortem (thin solid line) waveforms in a dark-adapted animal. (B) Representative baseline (thick solid line) and postmortem (thin solid line) waveforms in a light-adapted animal (112 cd/m2). (C) Normalized (mean ± SEM, n = 5) postmortem photoreceptoral light- and dark-adapted a- and b-wave amplitudes.
Figure 2.
 
Effect of serial and single stimuli on postmortem retinal function. (A) Representative raw ERG waveforms (□) are described using a phototransduction P3 model (solid line) at baseline and various times after death. Postreceptoral P2 responses (▪) were extracted by digital subtraction of the P3 model from the raw data. (B) Normalized (mean ± SEM, n = 5) decay of RmP3 with serial versus single signals. (C) Normalized phototransduction log S for serial versus single stimuli.
Figure 2.
 
Effect of serial and single stimuli on postmortem retinal function. (A) Representative raw ERG waveforms (□) are described using a phototransduction P3 model (solid line) at baseline and various times after death. Postreceptoral P2 responses (▪) were extracted by digital subtraction of the P3 model from the raw data. (B) Normalized (mean ± SEM, n = 5) decay of RmP3 with serial versus single signals. (C) Normalized phototransduction log S for serial versus single stimuli.
Figure 3.
 
Effect of glucose preloading on postmortem retinal function. (A) Representative waveforms showing the first 20 ms of the response. Photoreceptoral responses were described using a model of phototransduction (Equation 1) , which returns the phototransduction saturated amplitude (RmP3) and sensitivity (log S). Control and glucose preloaded waveforms are shown. (B) Normalized (± SEM, n = 5) postmortem RmP3 and postreceptoral amplitudes are described using an exponential decay.
Figure 3.
 
Effect of glucose preloading on postmortem retinal function. (A) Representative waveforms showing the first 20 ms of the response. Photoreceptoral responses were described using a model of phototransduction (Equation 1) , which returns the phototransduction saturated amplitude (RmP3) and sensitivity (log S). Control and glucose preloaded waveforms are shown. (B) Normalized (± SEM, n = 5) postmortem RmP3 and postreceptoral amplitudes are described using an exponential decay.
Figure 4.
 
Effect of concentration and time of delivery of exogenous glucose on postmortem photoreceptoral function. Normalized parameters (±SEM) are described using an exponential decay. (A) Control versus 100 and 10 mM glucose phototransduction RmP3 decay. (B) Control versus 100 and 10 mM glucose phototransduction sensitivity (log S) decay. (C) Postmortem RmP3 in control versus glucose preloaded 10 minutes before and postloaded 5 minutes after insult. (D) Postmortem log S in control versus preloaded and postloaded glucose.
Figure 4.
 
Effect of concentration and time of delivery of exogenous glucose on postmortem photoreceptoral function. Normalized parameters (±SEM) are described using an exponential decay. (A) Control versus 100 and 10 mM glucose phototransduction RmP3 decay. (B) Control versus 100 and 10 mM glucose phototransduction sensitivity (log S) decay. (C) Postmortem RmP3 in control versus glucose preloaded 10 minutes before and postloaded 5 minutes after insult. (D) Postmortem log S in control versus preloaded and postloaded glucose.
Figure 5.
 
Effect of glutamine and lactate preloading on postmortem photoreceptoral function. (A) Control versus glutamine phototransduction RmP3 decay. (B). Control versus glutamine phototransduction log S decay. (C) Control versus lactate phototransduction RmP3 decay. (D) Control versus lactate phototransduction log S decay.
Figure 5.
 
Effect of glutamine and lactate preloading on postmortem photoreceptoral function. (A) Control versus glutamine phototransduction RmP3 decay. (B). Control versus glutamine phototransduction log S decay. (C) Control versus lactate phototransduction RmP3 decay. (D) Control versus lactate phototransduction log S decay.
Figure 6.
 
Comparing metabolic substrate effects on posthypoxemic phototransduction RmP3. Data are shown as a mean (±SEM) percentage difference between vehicle and substrate-treated eyes (n = 5 for each group). (A) Comparison of preloading with 100 and 10 mM glucose. (B) Comparison of glucose (100 mM) preloaded 10 minutes before and postloaded 5 minutes after insult. (C) Comparison of preloaded glucose and glutamine. (D) Comparison of preloaded glucose and lactate.
Figure 6.
 
Comparing metabolic substrate effects on posthypoxemic phototransduction RmP3. Data are shown as a mean (±SEM) percentage difference between vehicle and substrate-treated eyes (n = 5 for each group). (A) Comparison of preloading with 100 and 10 mM glucose. (B) Comparison of glucose (100 mM) preloaded 10 minutes before and postloaded 5 minutes after insult. (C) Comparison of preloaded glucose and glutamine. (D) Comparison of preloaded glucose and lactate.
Table 1.
 
Decay Time Constants for Posthypoxemic Phototransduction Parameters for Various Conditions
Table 1.
 
Decay Time Constants for Posthypoxemic Phototransduction Parameters for Various Conditions
Phototransduction Saturated Amplitude (RmP3) Phototransduction Sensitivity (log S)
Mean, t c 2.5% CL 97.5% CL Mean, t c 2.5% CL 97.5% CL
Control (serial) 4.81 4.28 5.49 3.45 2.47 4.43
Control (single) 3.06* 2.73 3.48 2.10* 1.68 2.66
10 mM Glucose 10.91, † 7.85 13.99 4.69, † 4.25 5.12
100 mM Glucose (10-min preload) 10.06, † 8.99 13.88 5.57, † 4.63 5.78
100 mM Glucose (5-min postload) 5.01 4.19 6.21 4.18 3.73 4.62
100 mM Glutamine 9.01, † 7.50 11.30 3.41 2.83 3.99
100 mM Lactate 12.03, † 8.04 23.89 3.79 2.98 4.60
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