NADH hyperoxidation correlates with enhanced susceptibility of aged rats to hypoxia

Abstract

Aging increases mitochondrial dysfunction and susceptibility to hypoxia. Previous reports have indicated an association between post-hypoxic hyperoxidation of intra-mitochondrial enzymes and delayed neuronal injury. Therefore we investigated the relationship between NADH fluorescence and neuronal function during and after hypoxia across the lifespan. Hippocampal slices were prepared from adult (1 to >22 months) F344 rats. NADH fluorescence, extracellular voltage and tissue PO(2) were recorded from the CA1 region during hypoxia (95% N(2)) of various lengths following onset of hypoxic spreading depression (hsd). Slices from younger rats recovered evoked neuronal responses to a greater degree and exhibited less hyperoxidation after a hypoxic episode, than slices from older rats. However, the use of Ca(2+) free-media in slices from >22 month old rats improved recovery and delayed NADH hyperoxidation (2.5 min hypoxia after hsd). Post-hypoxic decrease of NADH fluorescence (hyperoxidation) was age dependent and correlated with decreased neuronal recovery. Slices exposed to repeated hypoxic episodes yielded data suggesting depletion of the NAD(+) pool, which may have contributed to the deterioration of neuronal function.

Fig. 1

(A) Timeline for hypoxia experiments. The arrow labeled hsd indicates the approximate time after the initiation of hypoxia at which the hsd occurred. The times (in min) shown in brackets indicate the time remaining before reoxygenation. (B) Timeline for hypoxia experiments with 0% Ca²⁺. Normal ACSF is replaced with Ca²⁺-free ACSF at 10 min following the start of the experiment (as indicated by the grey shaded area). This is continued for 20 min before the start of hypoxia, during hypoxia and for 10 min following reoxygenation.

Fig. 2

(A) Series of NADH fluorescence images during the progression of reversible hypoxia (hsd + 15 s) in a hippocampal slice from a 20 months old rat. The top image shows an unsubtracted raw camera picture with the region of interest indicated by a gray rectangle in the SR of the CA1 region. The asterisk indicates the position of the recording electrode. The oxygen and stimulating electrodes are above and below the recording electrode, respectively. Scale bar indicates 500 μm. Hypoxia was initiated at −180 s (as shown in the right margin). Zero time marks the onset of hsd. The scale bar in the right margin indicates the scale for the difference images, with white representing a 30% increase above baseline and black, a 30% decrease. Note that the CA1 region is preferentially affected by hypoxia, with a marked increase in NADH signal. (B) Corresponding responses of NADH as well as PO₂, % fEPSP and dc voltage to reversible hypoxia (hsd + 15 s). The horizontal line indicates the hypoxic interval, while the dotted vertical line shows the onset of hsd. The PO₂ was measured at the nadir of the slice. To account for the drift in NADH over time, a regression line (dotted line) was created from the baseline data prior to hypoxia.

Fig. 3

(A) Series of NADH fluorescence images during the progression of irreversible hypoxia (hsd + 5 min) in a hippocampal slice from a 25 months rat. The top image shows an unsubtracted raw camera picture with the region of interest indicated by a gray rectangle in the SR of the CA1 region. The asterisk indicates the position of the recording electrode. The oxygen and stimulating electrodes are above and below the recording electrode, respectively. Scale bar indicates 500 μm. Image times are shown in the right margin. Hypoxia was initiated at −450 s. Zero time marks the onset of hsd. The scale bar in the right margin indicates the scale for the difference images, with white representing a 50% increase above baseline and black, a 50% decrease. Note that the CA1 region is preferentially affected by hypoxia, with a marked increase in NADH signal. (B) Responses of PO₂, NADH (% change), fEPSP (% change from baseline) and dc voltage to hypoxia (hsd + 5 min) in a 25 months rat hippocampal slice. The horizontal line indicates the hypoxic interval, while the dotted vertical line shows the onset of hsd. The PO₂ was measured at the nadir of the slice. A regression line (dotted line) was created from the baseline data to account for the drift in NADH over time.

Fig. 4

Maximal % increase of NADH fluorescence (i.e. reduction) during hypoxia 15 s or 10 min after hsd onset in slices from 1–2, 3–6, 12–20 and >22 months old rats. Significant differences were found between the NADH reduction during 15 s and 10 min hypoxia post-hsd in slices from 3–6 and >22 months old rats (***p < 0.001).

Fig. 5

Time taken for fEPSP amplitude to recover to 50% of control in hippocampal slices from 1–2 months old rats (n = 7), 3–6 months old rats (n = 8), 12–20 months old rats (n = 8) and >22 months old rats (n = 7) following 15 s hypoxia post-hsd. The fEPSPs of slices from >22 months old rats were significantly slower to recover compared to slices from the younger age groups (1–2 and 12–20 months *p < 0.05; 3–6 months **p < 0.01).

Fig. 6

Recovery of fEPSP amplitude in hippocampal slices from 1–2, 3–6, 12–20, >22 months old rats following varying lengths of hypoxia post-hsd (0.25, 2.5, 5 and 10 min). Following 2.5 min of hypoxia post-hsd, fEPSP recovery differed significantly between slices from 3–6 months old (116 ± 47%, n = 8) and >22 months old rats (40 ± 49%, n = 9) (*p < 0.05) while following 5 min hypoxia post-hsd, the recovery of fEPSP in slices from 1–2 months old rats (54 ± 32%, n = 8) was significantly greater than the recoveries in slices from all older age groups [(3–6 months, 14 ± 32%, n = 7, *p < 0.05); (12–20 months, 7 ± 15%, n = 6, **p < 0.01); (>22 months, 3 ± 8%, n = 9, ***p < 0.001)].

Fig. 7

Change in NADH fluorescence (%) in hippocampal slices from 1–2, 3–6, 12–20, >22 months old rats following varying lengths of hypoxia post-hsd (0.25, 2.5, 5 and 10 min). Following 2.5 min hypoxia post-hsd, NADH hyperoxidation was significantly greater in slices from >22 months old rats (n = 9) compared to slices from 3 to 6 months old rats (n = 7; *p < 0.05). This effect was amplified between these age groups at 5 min hypoxia (**p < 0.01). In addition, the percentage of hyperoxidation was greater following 5 min hypoxia in slices from >22 months old rats (n = 6) compared to 1–2 months old rats (n = 7; **p < 0.01). Ten minutes of hypoxia post-hsd resulted in the greatest amount of hyperoxidation in all age groups with slices from 1 to 2 months old rats (n = 4) exhibiting significantly higher amounts of hyperoxidation compared to slices from 3 to 6 months old rats (n = 4) and >22 months old rats (n = 6; *p < 0.05).

Fig. 8

Graphs of PO₂ and NADH following repeated episodes of hypoxia (10 min post-hsd) in slices from 1 to 2 months old rats (n = 4). Reoxygenation following the first episode of hypoxia resulted in a PO₂ overshoot (increase above baseline) and NADH hyperoxidation (decrease below baseline). During the second episode of hypoxia the NADH fluorescence increase was less than during the first, and the second event was not followed by hyperoxidation. The PO₂ overshoot was enhanced after the second episode.

Fig. 9

(A) Recovery of fEPSP amplitude following 2.5 min hypoxia post-hsd in 1.2 and 0 mM calcium conditions in slices from >22 months old rats. The removal of calcium from the ACSF improved neuronal recovery following hypoxia from 40 ±49% (n= 9) to 76 ±72% (n=8) however the effect was not significant. (B) NADH fluorescence change (%) measured at 15 min following 2.5 min hypoxia post-hsd and reoxygenation in 1.2 and 0 mM calcium conditions in slices from>22months old rats. Hyperoxidation was absent 15 min after reoxygenation in slices perfused with 0% calcium (+1.5 ± 0.9%) compared to slices with calcium (−1.8 ± 1%, *p < 0.05).