Revival of light signalling in the postmortem mouse and human retina

Abstract

Death is defined as the irreversible cessation of circulatory, respiratory or brain activity. Many peripheral human organs can be transplanted from deceased donors using protocols to optimize viability. However, tissues from the central nervous system rapidly lose viability after circulation ceases^1,2, impeding their potential for transplantation. The time course and mechanisms causing neuronal death and the potential for revival remain poorly defined. Here, using the retina as a model of the central nervous system, we systemically examine the kinetics of death and neuronal revival. We demonstrate the swift decline of neuronal signalling and identify conditions for reviving synchronous in vivo-like trans-synaptic transmission in postmortem mouse and human retina. We measure light-evoked responses in human macular photoreceptors in eyes removed up to 5 h after death and identify modifiable factors that drive reversible and irreversible loss of light signalling after death. Finally, we quantify the rate-limiting deactivation reaction of phototransduction, a model G protein signalling cascade, in peripheral and macular human and macaque retina. Our approach will have broad applications and impact by enabling transformative studies in the human central nervous system, raising questions about the irreversibility of neuronal cell death, and providing new avenues for visual rehabilitation.

Extended Data Fig. 1 |. Changes after 45 min death-to-enucleation delay to metabolites related to cell health and synaptic signaling.

Example RGB maps of CMP staining of metabolites in the immediately fixed vs. 45-minute postmortem wild-type retina (a). τQE (Taurine, Glutamine, Glutamate) → RGB for both immediate and 45 min PM retinas. The density maps for each metabolite are also shown (greyscale). Scale bar represents 50 μm. Histograms of the mean levels of Taurine, Glutamine and Glutamate measured in retinas either after immediate enucleation (black) or 45 min enucleation delay (red, b), according to masked cell populations (n = 3 retinas from 3 animals). Data are presented as averages (lines) ± SEM (shaded areas).

Extended Data Fig. 2 |. Phototransduction sensitivity and kinetics in relation to enucleation delay.

Example plot showing how dim flash response sensitivity is calculated (a). Responses normalized to maximal response of retina plotted against the intensity of light stimulus, curve fits Naka-Rushton function (Eq. 2) to amplitude data, dim flash sensitivity is defined as response amplitude divided by R_max at constant intensity producing ~20% of the maximal response amplitude. Dim flash sensitivities of retinas from wild-type mouse retinas with different enucleation delays (b), and I_1/2 values from retinas of wild-type mice with different enucleation delays (c). Example response plot showing how I_1/2 was obtained (d), responses of retina to each intensity presented is plotted and a Naka-Rushton fit applied to the plot. I_1/2 is the intensity required to produce half the maximal response size. Amplification constant plotted against enucleation delay for each retina (e). Time-to-peak (t_p) and integral (T_i, inset) of dim responses for each retina plotted against the enucleation delay (f, *** indicates p < 0.001 compared to 0min). Exact p values: t_p: 0 vs 90 min p = 0.0002, 0 vs 120 min p = 0.002, 0 vs 180 min p = 0.001, T_i: 0 vs 90 min p = 0.002, 0 vs 120 min p = 0.0007, 0 vs 180 min p = 0.01). Example dim flash response showing how time-to-peak (t_p) and integration time (T_i) was calculated (g). Time-to-peak is the time from light stimulus onset to the peak of the dim flash response. Integral (T_i) is the area under the dim flash response curve (orange shaded area), divided by the response amplitude. Light stimulus is indicated with a shaded yellow area. For all plots, bar height indicates mean, error bars represent ± SEM. All comparisons are one-way ANOVA with Holm-Bonferroni means comparisons. (DF: 35 for all comparisons, F values: Ti = 8.4, t_p = 5.8, Dim flash sensitivity = 2.0, Amplification constant = 1.6, I_1/2 : 2.2).

Extended Data Fig. 3 |. Drawings of ex vivo ERG specimen holder for CNC machining from polycarbonate.

(exported from Fusion 360, Autodesk, used to design the specimen holder for CNC machining). Retina samples are placed on the dome (shaded in blue in b). The dome has electrode channel with Ø 0.5–2 mm diameter that determines the recording area. O-ring (a, black shading; b, pink shading) minimizes shunt currents to improve signal-to-noise ratio (SNR).

Extended Data Fig. 4 |. Comparison of freshly enucleated Macaque retina, and human organ and research donor maculas with longer enucleation delays.

Light responses of macular cones from freshly enucleated macaque (a, hatched squares, n = 5 maculae, from 3 animals) human DCD donor (b, filled triangles, top trace, n = 5 maculae from 3 donors) and research donor eyes (b, crosses lower trace, n = 23 maculae from 17 donors). (c) The maximal response obtained from maculae from each tissue type plotted against enucleation delay. Inset shows Ln of R_max from each macula, and linear fit in red with decay (τ = 74 min). (d) Example of how sensitivity, S (1/I_½), is calculated. Macular responses to different light intensities were plotted and a Hill curve fitted (see Eq. 3 in Methods) to determine the intensity required to produce a half-maximal response size. (e) Sensitivities of each macula plotted against enucleation delay, linear fit in red (no significant correlation between delay and S, ANOVA, DF =24, F value = 3.9, p = 0.06).

Extended Data Fig. 5 |. Contributing factors to the quality of light-evoked responses from human postmortem research donor retinas.

(a) Histogram of research donor ages, with numbers of donors represented within the bars. (b) Histogram of the primary cause of death of donors as noted on tissue donor forms. (c) The maximal response obtained from each donor eye plotted against donor age, with linear fit showing a positive correlation (n = 23 maculae, Pearson’s r = 0.61, adjusted R² = 0.37) (d) Average maximal amplitude obtained from each research donor according to the primary cause of death (Stroke: squares, n = 2 donors, ACE: circles, n = 4 donors, Sepsis: triangles, n = 7 donors), data are averages of responses obtained from each individual donor, with mean presented as a solid line. Maculae that did not produce any detectable responses were included as a response size of 0 μV. Donor information (e) for a single donor (donor #8) in which ex vivo ERG ON-bipolar cell responses were recorded to flash intensities of 29–32,500 photons μm⁻²(f). Yellow bar indicates the onset of flash stimuli.

Extended Data Fig. 6 |. Decay of cone photoreceptor and bipolar cell function in relation to the enucleation delay in Gnat1^−/− mice.

(a) The R_max of mouse cone photoreceptors (black circle) and bipolar cells (red circle) plotted as a function of enucleation delay, normalized to maximal response obtained with 0 min enucleation delay. Smooth traces plot exponential decay function fitted to mean data. Inset plots natural log amplitude data for individual retinas (τ = 53 min for photoreceptors, τ = 35 min for bipolar cells), and linear fit (Photoreceptor fit: Pearson’s r = −0.851 with adj. R² = 0.704, Bipolar cell fit: Pearson’s r = 0.905 with adj R² = 0.819). Sensitivity of retinas at different enucleation delays (b, 1/I_1/2), Pearson’s r = 0.52 with adj. R² = 0.27. For all plots, n (retinas, animals) is 0min = 7, 45 min = 5, 120 min = 3, responses from individual retinas are plotted as hollow circles.

Extended Data Fig. 7 |. Double-flash responses allow the isolation of cone responses from rod in both human macula (a) and periphery (b).

Representative responses from human donor macular (a, black) and peripheral (b, red) punches to double-flash light stimuli to isolate cone responses. Light flashes are indicated with yellow shading, rod saturating flash intensity was ~25,000, cone stimulating intensities used were 2800–147,000 photons (560 nm) μm⁻².

Extended Data Fig. 8 |. Comparison of Human Macular and Peripheral Cone sensitivity and kinetics.

Dim flash response time-to-peak (at intensity producing response size 15–20% of maximal retina response in ms) recorded from human macular and peripheral cone responses (c), individual responses are shown as diamonds, with line indicating mean. The average dim flash photoreceptor response normalized to maximal response recorded from each retina, plotted against time (d). Graph line shows average response with shaded area showing ± SEM.

Extended Data Fig. 9 |. Determination of dominant time constant (τ_D) for macaque cones.

Representative responses to flashes of light ranging from 220 to 183,000 photons (500 nm) μm⁻² from a macular (a) and peripheral (c) sample recorded using ex vivo ERG. A rod-saturating pre-flash (15,000 photons μm⁻² at 500 nm) was used to isolate cones in peripheral samples. Saturation times (T_sat) measured at 50% recovery (example red dashed line in a, c) plotted as a function of logarithmic light flash intensity (in photons μm⁻²) for induvial macular (b) and peripheral samples (d). e. Log response amplitudes plotted against light intensities in macaque maculae (black) and peripheral retina punches (red) with Hill function fits (Eq. 3 in Methods). n = 5 macular and n = 7 peripheral punches from 3 animals. (f) Table of statistics for slopes in (b, d), i.e. τ_D, mean ± SEM.

Fig. 1 |. Postmortem decay and recovery of light-evoked retinal responses under in vivo and ex vivo conditions.

a, Schematic of in vivo electroretinogram recordings isolating photoreceptor responses using DL-AP4 (Methods). b, Representative traces of in vivo ERG responses before death and between 20–200 s postmortem. c, Bipolar cell (hollow circles) or photoreceptor (hollow squares) light responses were detected every 5 s and normalized to the response taken before death to obtain normalized R_max. n = 4 mice. Inset, natural logarithm of normalized responses with a linear fit for photoreceptor (black line, τ = 2.2 min) and bipolar cell (red line, τ = 1.3 min) responses. d, Schematic of ex vivo ERG recording experiments (Extended Data Fig. 3, Methods). e, Example traces showing ex vivo restoration of light-evoked responses during recordings; responses from each retina were replicated at least three times. f, Ex vivo ERG responses from retinas with different death-to-enucleation delays normalized to maximal response size obtained from immediately enucleated eyes (normalized R_max). Inset, natural logarithm of normalized responses with linear fit up to 120 min for photoreceptor (black line, τ = 41 min) and bipolar cell (red line, τ = 20 min) responses. Dashed lines show fit data from c. For ex vivo recordings: 0 min, 8 retinas from 8 mice; 15 min, 6 retinas from 5 mice; 30 min, 5 retinas from 5 mice; 45 min, 4 retinas from 4 mice; 60 min, 4 retinas from 4 mice; 90 min, 3 retinas from 3 mice; 120 min, 3 retinas from 3 mice; 180 min, 3 retinas from 3 mice. BC, bipolar cell; PR, photoreceptor.

Fig. 2 |. Factors affecting recovered ERG b-wave amplitudes in postmortem mouse retinas.

a, Representative antibody labelling of activated caspase-3 (CASP3) in retinal sections with 0 and 45 min death-to-enucleation delay. Bottom right image shows an expanded view showing two retinal neurons with caspase-3 staining. Scale bars, 10 μm. Right, quantification of caspase-3-positive cells per slice, per mouse retina (diamonds) and the mean (horizontal lines). 0 min, 3 retinas; 45 min, 4 retinas; 180 min, 3 retinas; P23H^+/− positive control, 3 retinas. b, Top, decline of b-wave amplitudes during low-oxygen perfusion normalized to R_max with normal perfusion (n = 5 individual retinas from 3 mice represented as hollow circles; solid circles indicate mean); inset, representative responses from a single retina at different time points during hypoxia. Bottom, linear fit to plotted natural log of hypoxic b-waves. Decay rate (τ) is indicated. c, Top, decay of b-wave amplitudes measured during perfusion in low pH (6.8) Ames perfusion normalized to amplitude in physiological pH (7.4) Ames perfusion (n = 7 individual retinas plotted as hollow circles; solid circles indicate the mean). Inset, representative responses from a single retina at different time points during low pH perfusion. Bottom, linear fit of the natural log of the normalized b-wave responses. Decay rate is indicated. d, Decline of ON bipolar responses after incubation in low oxygen for 0–60 min (0–30 min, n = 6; 60 min, n = 7). Inset, natural log of b-wave responses versus time. Decay rate is indicated. e, Recovery of b-wave amplitudes after incubation in low pH Ames for 60–180 min (control, n = 9; 60 min, n = 6; 180 min, n = 4). n refers to both number of retinas and number of mice, unless otherwise indicated.

Fig. 3 |. ERG b-wave is preserved in freshly obtained retinas from human donors after brain death.

a, Schematic of process for maintaining optimal function of retinas from donors after brain and cardiac death (Methods). b, c, Example ex vivo ERG recordings from the parafoveal and perifoveal (b) and foveal (c) regions of the retina with responses in the absence (left) and presence (right) of DL-AP4. d, The peripheral retina shows larger response amplitudes both in the absence (left) and presence (right) of DL-AP4. e, Left, maximal b-wave amplitude measured from peripheral retina of organ donors after brain death plotted against the delay to enucleation (10 pieces of 8 eyes from 5 donors). Right, schematic showing approximate locations of peripheral (red) and macular (black) samples.

Fig. 4 |. Comparison of phototransduction in rods and cones of the macula and periphery of freshly obtained retinas from human donors.

a–d, Examples responses to different light intensities of photoreceptors from freshly enucleated human eyes. Insets show approximate locations from which samples were taken. a, Macular photoreceptors. b, Macular cones. c, Peripheral rods. d, Peripheral cones. Dashed red line indicates the amplitude at which the recovery from saturated response (T_sat) is measured (70% for rods and 50% for cones). e, Response amplitudes (R) normalized to R_max from rods and cones, plotted against flash intensity. Lines show fit to Hill function (Methods, equation (3)), used to calculate the photoreceptor sensitivity (I_½; values presented in Supplementary Table 2). Number of samples: macular rods, 6; macular cones, 6; peripheral rods, 7; peripheral cones, 7. f, Recovery times of macular and peripheral photoreceptors from saturation to the threshold level (T_sat) at different light intensities plotted against the natural log of flash intensity. Lines show linear fit (Methods, equation (4)) to determine the slope (τ_D), equivalent to the rate constant of the slowest phototransduction reaction (values presented in Supplementary Table 1). Number of samples: macular rods, 5; macular cones, 6; peripheral rods, 7; peripheral cones, 7, taken from 4 eyes of 2 donors.