Mitochondrial Function and Energy Metabolism after Hypoxia—Ischemia in the Immature Rat Brain: Involvement of NMDA-Receptors

Abstract

Treatment after hypoxia—ischemia (HI) in immature rats with the N-methyl-d-aspartate receptor (NMDAR) antagonist dizocilpine maleate (MK-801) reduces areas with high glucose utilization and reduces brain damage. The object was to study the metabolic effects of MK-801 treatment after HI. Seven-day-old rats were randomized to the following groups: non-HI, HI, or HI plus MK-801 (0.5 mg/kg immediately after HI). In the parietal cortex, the mitochondrial respiration was measured in homogenates 1 to 4 hours, and the energy metabolites at 3 and 8 hours after HI. The energy use was calculated from changes in energy metabolites after decapitation at 3 hours after HI. State 3 respiration was reduced by 46%, 32%, and 25% after HI compared with non-HI with pyruvate plus malate, glutamate plus malate, or glutamate plus succinate as substrates, respectively. Uncoupler-stimulated but not state 4 respiration was similarly reduced. The MK-801 augmented pyruvate plus malate—supported state 3 respiration after HI by 42%. The energy utilization was not affected by HI but was reduced by MK-801 treatment in the ipsilateral cortex from 4.6 ± 2.3 to 2.6 ± 1.8 μmol ~P/min/g. The levels of ATP and phosphocreatine did not differ between the HI and HI plus MK-801 groups at 3 hours, but were lower in the HI than in the HI plus MK-801 group at 8 hours after HI. In conclusion, treatment with MK-801 reduced energy utilization and improved mitochondrial function and energy status after HI, suggesting a linkage between NMDAR activation and impaired energy metabolism during reperfusion.

Keywords

Neonatal Hypoxia—ischemia NMDA Mitochondria Metabolism

In the cerebral cortex of the immature rat, the early recovery period after hypoxia—ischemia (HI) is characterized by an incomplete restitution of ATP and phosphocreatine. There is a persistent increase in lactate levels and normal or low NAD⁺/NADH ratio. The cerebral metabolic rate of glucose (CMR_glc) is increased in relation to a normal or low energy utilization, but the CBF is unimpaired until 48 hours after the insult (Mujsce et al., 1990; Palmer et al., 1990; Yager et al., 1991; Yager et al., 1992; Vannucci et al., 1994; Gilland and Hagberg, 1996; Gilland et al., 1997). This has been interpreted as signs of an impaired oxidative metabolism, and a reduction in the activity of cytochrome aa₃ (cytochrome c oxidase), the final component of the electron transport chain, was observed in the hippocampus, but not in the cortex, early after HI in the immature rat (Nelson and Silverstein, 1994). In the newborn lamb, there is a reduction of ADP-stimulated mitochondrial respiration and of cerebral oxygen utilization after asphyxia (Rosenberg, 1986; Rosenberg et al., 1989), and an impairment of mitochondrial respiration has been seen after both focal and global ischemia in adult rats (Rehncrona et al., 1979; Hillered et al., 1984; Sims and Pulsinelli, 1987; Kuroda et al., 1996).

Treatment after HI with the N-methyl-d-aspartate receptor (NMDAR) antagonist dizocilpine maleate (MK-801) reduces subsequent morphologic brain damage in the immature rat (Hattori et al., 1989; Hagberg et al., 1994), and it also decreases the size of areas with increased CMR_glc 1 to 5 hours after HI (Gilland and Hagberg, 1996). In newborn humans with hypoxic—ischemic encephalopathy, regional increases of CMR_glc predicts a later appearance of neurologic deficits corresponding to damage in those brain areas (Blennow et al., 1995). The NMDAR activation after HI may contribute to the development of infarction by affecting the energy metabolism, which is seen as increased CMR_glc. The mechanisms for this are unknown and may reflect that NMDAR activation enhances the energy requirements of the tissue or aggravates mitochondrial function during reperfusion. In the current study, these alternatives are explored in the 7-day-old rat model of HI by posttreatment with MK-801.

MATERIALS AND METHODS

Animals and chemicals

Inbred Wistar F rat pups of either sex were purchased from Møllegaard Breeding & Research Centre A/S (Skensved, Denmark) and fed by their dams until the start of the experiments at 7 days of age. All chemicals and enzymes for determining tissue metabolites were obtained from Sigma-Aldrich Sweden (Göteborg, Sweden) and Boehringer Mannheim Scandinavia AB (Bromma, Sweden).

Protocol

Animals in each litter were randomized to one of three groups: (1) the non-HI control group; (2) the HI group, which underwent HI and received saline injections; and (3) the HI plus MK-801 group, which underwent HI and received 0.5 mg/kg MK-801 intraperitoneally immediately after HI. In the rats used for measurement of energy metabolites and cerebral energy utilization 3 hours after HI, the non-HI controls were from separate litters to the HI and HI plus MK-801 groups. Cerebral energy metabolites at 8 hours after HI were measured in separate litters from the 3-hour time point. These rats were kept at 33°C ambient temperature, and were separated from the dams between 1 and 8 hours after HI (to eliminate the mild hypothermic effect of MK-801 that is evident from 4 hours after HI if the pups are kept in room temperature with their dams [Gilland and Hagberg, 1997]).

The rectal temperature was measured with a thermistor probe (BAT-12, Physitemp Instruments, Clifton, NJ, U.S.A.) immediately after removal from the dam. In rats used for measurement of energy utilization, the temperature was measured before decapitation at 3 hours after HI, whereas in the rats used for measurement of mitochondrial respiration, temperature was measured 1 hour after HI and rats were put back with the dam.

Induction of hypoxia—ischemia

Anesthesia was induced and maintained with halothane in oxygen—nitrous oxide on snout mask. The left common carotid artery was cut between ligatures of prolene suture (Rice et al., 1981). Animals were allowed to recover from anesthesia for at least 1 hour. The HI was induced by exposure to 7.7% ± 0.01 % oxygen in nitrogen for 70 minutes in a humidified chamber at 36°C. This induces a reversible reduction of CBF and infarction in the left (ipsilateral) hemisphere, whereas there is no reduction of CBF or any gross infarction in the contralateral hemisphere (Hagberg et al., 1997). After the insult, the pups were returned to their dams. In each litter used for measurement of pyruvate plus malate—supported mitochondrial respiration, one rat in the HI group was decapitated at 14 days after HI, and the hemispheres were dissected out and weighed to determine the degree of brain damage (Hagberg et al., 1994).

Measurement of cerebral energy utilization

Cerebral energy utilization was measured by the changes in energy metabolites during 1 minute after decapitation (Lowry et al., 1964; Gatfield et al., 1966). Shutting of the blood supply converts the brain into a closed biological system and limits the chemical events to those that can occur anaerobically (Lowry et al., 1964). Two rats were paired and received the same treatment. At 3 hours after HI, both rats were decapitated. The head of one was immediately frozen in liquid nitrogen, whereas the head of the other was left for 1 minute in room temperature before it was frozen. Portions (10 to 50 mg) of the parietal cortex were dissected out at −20°C, weighed, and solubilized in 3 mol/L HClO₄ (Lowry and Passonneau, 1972; Gilland and Hagberg, 1996) at −10°C. After centrifugation at 5000 × g for 10 minutes, one volume of the supernatant was neutralized with 2.5 volumes of 2 mol/L KHCO₃ and again centrifuged to remove precipitated KHCO₃. The supernatant was used for enzymatic—fluorometric determination of ATP, glucose, and lactate (Lowry and Passonneau, 1972). Changes in metabolite levels were determined by subtracting the values of the second rat from the values of the first rat in each pair. Changes in high-energy phosphate bindings (Δ~P) were calculated according to the formula: Δ~P (μmol/min/g) = 2ΔATP + Δphosphocreatine + 2Δglucose + 1.45(Δlactate − 2Δglucose). Changes in ADP were neglected, since they are small in the immature brain during short time intervals (Duffy et al., 1975) and changes in glycogen were replaced by Δlactate − 2Δglucose (i.e., the excess lactate production over glucose disappearance [Gatfield et al., 1966]). All brains and specimens were stored at −80°C.

Measurement of mitochondrial respiration

Mitochondrial respiration in brain tissue homogenates were measured according to Sims and Blass (1986). Since each measurement took 30 minutes to perform, six rats from each litter that underwent HI (the HI and HI plus MK-801 groups) were decapitated successively 1 to 4 hours after HI and the results pooled, irrespective of time after HI. The cortex of the left hemisphere was dissected out and immersed in ice-cold 10 mmol/L Tris buffer (pH 7.4) containing 0.32 mol/L sucrose and 1 mmol/L ethylendiammine tetraacetic acid (EDTA; potassium salt). The tissue was rinsed and the surface cleared from meninges and pial vessels. The anterior and posterior poles were cut away, and a central portion (weighing 60 ± 12 mg) was homogenized in 1-mL buffer by hand (four up-and-down strokes with a total clearance of 0.12 mm and eight up-and-down strokes with a total clearance of 0.05 mm) in a 2-mL glass homogenizer (Kontes, Vineland, NJ, U.S.A.).

Respiratory activity was measured at 28°C in a closed and magnetically stirred glass chamber (volume 0.54 mL), using a Clark-type oxygen electrode (L. Escheweiler & Co., Kiel, Germany). A total of 90 μL of the homogenate was added to a reaction buffer (pH 7.4), which consisted of 100 mmol/L KCl, 75 mmol/L mannitol, 25 mmol/L sucrose, 5 mmol/L Trisphosphate, 0.05 mmol/L EDTA, 10 mmol/L Tris-HCl, and 0.1% bovine serum albumin. After 3 minutes, the chamber was closed, and, after baseline measurement, 10 μL of 0.28 mol/L pyruvate and 0.14 mol/L malate was added (final concentrations 5 mmol/L and 2.5 mmol/L, respectively). State 3 (+ADP) respiration was measured after addition of 1.5 μL of 50 mmol/L ADP, and state 4 (–ADP) respiration was determined after the ADP was depleted. Uncoupling was initiated by adding 1 to 2 μL of 0.9 mmol/L carbonyl cyanide m-chlorophenylhydrazone in 32% ethanol (final concentration 1.7 to 3.3 μmol/L). In separate rats randomized only to the non-HI control and the HI groups, glutamate and succinate—supported respiration was measured (after baseline measurement) by adding 10 μL of 0.55 mol/L glutamate and 0.14 mol/L malate (final concentrations 10 mmol/L and 2.5 mmol/L, respectively), whereupon state 4 respiration was measured, and 2 μL of 50 mmol/L ADP, whereupon state 3 respiration was measured. Addition of 1 μL of 1 mmol/L rotenone completely inhibited oxygen uptake until 10 μL of 0.55 mol/L succinate was added. State 3 and state 4 respiration was measured as described earlier. Between samples, the chamber was rinsed with ethanol, distilled water, and reaction buffer to avoid contamination of the next sample by rotenone. All substrates and EDTA were neutralized with KOH before use.

Statistics

Analysis of variance was used for comparisons between groups with Scheffe's post hoc test. Paired Student's t test was used for comparisons between hemispheres. Differences with P < 0.05 were considered statistically significant. Values are given as mean ± SD.

RESULTS

Physiologic variables

There was no difference in rectal temperature between the HI and HI plus MK-801 groups, either at 1 hour or 3 hours after HI and drug administration (Table 1). Blood lactate levels at 3 hours after HI were 0.80 ± 0.06 mmol/L (n = 5) in the HI plus MK-801 group, and 0.86 ± 0.05 mmol/L (n = 3) in the HI group. The brain damage in the HI group, expressed as weight deficit of the ipsilateral hemisphere compared with the contralateral hemisphere, was 38% ± 16% (n = 4) at 14 days after HI. There was no mortality during or after HI.

TABLE 1.

Physiologic variables after hypoxia-ischemia (HI) and in non-HI controls

	HI	HI + MK-801	Non-HI
Rectal temperature (°C) at 1 h (n = 10–12)	34.6 ± 0.65	34.4 ± 0.62	34.7 ± 1.0
Rectal temperature (°C) at 3 h (n = 8–13)	35.7 ± 0.91*	35.5 ± 1.5*	34.1 ± 0.74
Blood glucose (mmol/L) at 3 h (n = 4–8)	4.2 ± 0.39*	3.9 ± 0.33*	5.6 ± 0.92
Blood glucose (mmol/L) at 8 h (n = 9)	3.0 ± 0.30	4.2 ± 0.23†

Values represent mean ± SD.

P < 0.05 versus the non-HI group.

†

P < 0.05 versus the HI group.

Cerebral energy metabolites

In both the HI and HI plus MK-801 groups at 3 and 8 hours after HI, the ATP, phosphocreatine, and lactate levels in the ipsilateral parietal cortex were altered compared with their respective contralateral cortex and compared with the ipsilateral cortex of the non-HI controls (Table 2 and Table 3). Glucose levels were significantly higher in the contralateral cortex of the HI plus MK-801 group. There were no significant differences in the ATP, phosphocreatine, and lactate levels between the HI and HI plus MK-801 groups at 3 hours after HI, but at 8 hours after HI, the HI plus MK-801 group had higher concentrations of ATP, phosphocreatine, and glucose and lower lactate concentrations in the ipsilateral cortex than the HI group (Table 3; P = 0.04, 0.004, 0.001, and 0.001, respectively).

TABLE 2.

Metabolite levels (μmol/g) in pieces of the left/ipsilateral and right/contralateral cortex 3 hours after hypoxia-ischemia (HI), and in non-HI controls

	HI (n = 11)		HI + MK-801 (n = 13)		Non-HI (n = 8)

	Ipsilateral	Contralateral	Ipsilateral	Contralateral	Left	Right
ATP	1.8 ± 0.32*‡	2.4 ± 0.21	1.9 ± 0.51*‡	2.5 ± 0.24	2.4 ± 0.12	2.5 ± 0.15
Phosphocreatine	2.4 ± 0.61*‡	3.3 ± 0.39	2.4 ± 0.77*‡	3.1 ± 0.56	3.5 ± 0.30	3.5 ± 0.28
Glucose	2.1 ± 0.54	2.3 ± 0.28*	2.2 ± 0.49‡	2.8 ± 0.46*†	1.8 ± 0.23	1.8 ± 0.13
Lactate	2.1 ± 0.63*‡	1.3 ± 0.26	2.0 ± 0.82*‡	1.1 ± 0.24	1.0 ± 0.24	1.0 ± 0.31

Values represent mean ± SD.

P < 0.05 versus homolateral cortex in the non-HI group.

†

P < 0.05 versus homolateral cortex in the HI group.

‡

P < 0.05 versus contralateral cortex.

TABLE 3.

Metabolite levels (μmol/g) in pieces of the left/ipsilateral and right/contralateral cortex 8 hours after hypoxia-ischemia (HI)

	HI (n = 9)		HI + MK-801 (n = 9)

	Ipsilateral	Contralateral	Ipsilateral	Contralateral
ATP	1.5 ± 0.30‡	2.2 ± 0.22	2.0 ± 0.60†‡	2.4 ± 0.32
Phosphocreatine	1.1 ± 0.55‡	2.5 ± 0.97	2.3 ± 0.99†	2.8 ± 0.60
Glucose	0.93 ± 0.41	0.89 ± 0.44	2.0 ± 0.72†	1.9 ± 0.68†
Lactate	1.4 ± 0.27‡	0.84 ± 0.24	1.0 ± 0.46†‡	0.53 ± 0.18†

Values represent mean ± SD.

†

P < 0.05 versus homolateral cortex in the HI group.

‡

P < 0.05 versus contralateral cortex.

Cerebral energy utilization

There was no significant change of cerebral energy utilization in the HI group compared with the non-HI controls, although there was a tendency toward reduced values in the former (Table 4). The HI plus MK-801 group had reduced cerebral energy utilization in the ipsilateral cortex (Δ~P = 2.6 ± 1.8 μmol/min/g) compared with the contralateral cortex (4.2 ± 0.92 μmol/min/g; P = 0.004), with the ipsilateral cortex in the HI group (4.6 ± 2.3 μmol/min/g; P = 0.04), and with the left parietal cortex in the non-HI controls (4.9 ± 0.61 μmol/min/g; P = 0.03; Table 4). The cerebral energy utilization in the right (contralateral) cortex did not differ between the non-HI, HI, and HI plus MK-801 groups.

TABLE 4.

Metabolite fluxes (μmol/min per g) after decapitation in pieces of the left/ipsilateral and right/contralateral cortex 3 hours after hypoxia-ischemia (HI), and in non-HI controls

	HI (n = 11)		HI + MK-801 (n = 13)		Non-HI (n = 8)

	Ipsilateral	Contralateral	Ipsilateral	Contralateral	Left	Right
Energy use (Δ~P)	4.6 ± 2.3	4.8 ± 0.98	2.6 ± 1.8 ^*†‡	4.2 ± 0.92	4.9 ± 0.61	4.8 ± 0.97
ΔATP	0.53 ± 0.27^*‡	0.17 ± 0.19	0.099 ± 0.43†	0.13 ± 0.19	0.12 ± 0.12	0.14 ± 0.10
ΔPhosphocreatine	1.6 ± 0.72^*	2.3 ± 0.53	1.1 ± 0.81^*‡	2.0 ± 0.37^*	2.4 ± 0.38	2.6 ± 0.31
ΔGlucose	0.97 ± 0.39	1.1 ± 0.34	0.38 ± 0.56†‡	0.85 ± 0.36	0.85 ± 0.24	0.95 ± 0.18
ΔLactate	–2.0 ± 1.4	–2.1 ± 0.33	–1.2 ± 1.0‡	–1.9 ± 0.41	–2.1 ± 0.39	–1.9 ± 0.50

Values represent mean ± SD. Values are the differences between the inital values of Table 2 and values from rats that were left for 1 minute in room temperature before freezing. Energy utilization (Δ~P) = 2 ΔATP + Δphosphocreatine + 2Δglucose + 1.45(Δlactate-2Δglucose).

P < 0.05 versus homolateral cortex in the non-HI group.

†

P < 0.05 versus homolateral cortex in the HI group.

‡

P < 0.05 versus contralateral cortex.

Mitochondrial respiration

After HI, ADP-stimulated and uncoupled respiration was significantly reduced, regardless of substrate (Fig. 1; Table 5 and Table 6). Also, after HI, the +ADP respiration was approximately 54% (P < 0.0001, n = 14), 68% (P = 0.01, n = 5), and 75% (P = 0.007, n = 5) of the respiration in non-HI controls with pyruvate plus malate, glutamate plus malate, or glutamate plus succinate in the presence of rotenone as substrates, respectively. There was no linear change in mitochondrial +ATP pyruvate—supported respiration over time between 1 and 4 hours after HI (R² = 0.083). There were no significant differences in –ADP respiration for any of the substrates used between the HI and the non-HI groups. Treatment with MK-801 after HI caused a normalization of pyruvate-supported +ADP respiration to 77% of non-HI controls (P = 0.006 compared with HI group, n = 14; Table 5). There was a significant difference between the HI group (4.8 ± 1.1, n = 5) and the HI plus MK-801 group (8.1 ± 1.9, n = 6; P = 0.01), even if including only the non-HI controls and the rats of the HI and HI plus MK-801 groups that were decapitated between 1 to 2 hours after HI. Treatment with MK-801 also tended to increase pyruvate-supported uncoupled respiration (not significant; Table 5).

FIG. 1.

Pyruvate (5 mmol/L) plus malate (2.5 mmol/L)–supported mitochondrial oxygen consumption tracings from the left cortex of nonhypoxia—ischemia (non-HI) control (a), HI group 1 hour after HI (b), and 1 hour after HI in rat that received 0.5 mg/kg MK-801 immediately after HI (c). Arrows denote addition of (1) ADP (to 140 μmol/L) to induce state 3 respiration, and (2) carbonyl cyanide m-chlorophenyl-hydrazone (to 2.5 μmol/L) to induce uncoupling of respiration. All concentrations are final concentrations.

TABLE 5.

Pyruvate (5 mmol/L) + malate (2.5 mmol/L) supported mitochondrial oxygen consumption (nmol O₂/min/mg protein) in pieces of the left/ipsilateral cortex 1 to 4 hours after hypoxia-ischemia (HI), and in non-HI controls

	HI (n = 14)	HI + MK-801 (n = 14)	Non-HI (n = 13)
+ADP (140 μmol/L)	5.5 ± 1.9^*	7.8 ± 1.6^*†	10 ± 1.7
–ADP	2.0 ± 0.37	2.4 ± 0.41	2.5 ± 0.73
+CCCP (1.7–3.3 μmol/L)	7.7 ± 0.83^*(n = 8)	10 ± 2.8^*(n = 8)	14 ± 2.6 (n = 7)
RCR (+ADP/–ADP)	2.6 ± 0.64^*	3.4 ± 0.83^*†	4.1 ± 0.60

CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; RCR, respiratory control ratio. Values represent mean ± SD.

P < 0.05 versus the non-HI group.

†

P < 0.05 versus the HI group.

TABLE 6.

Mitochondrial oxygen consumption (nmol O₂/min/mg protein) in pieces of the left/ipsilateral cortex 1 to 4 hours after hypoxia-ischemia (HI), and in non-HI controls

Substrate		HI (n = 5)	Non-HI (n = 5)
10 mmol/L glutamate + 2.5 mmol/L malate	+ADP (180 μmol/L)	7.6 ± 1.8^*	11 ± 1.5
	–ADP	2.4 ± 0.33	2.1 ± 0.38
	RCR (+ADP/–ADP)	3.2 ± 0.81^*	5.3 ± 0.66
10 mmol/L succinate + 1.8 μmol/L rotenone	+ADP (180 μmol/L)	6.3 ± 1.1^*	8.4 ± 0.67
	–ADP	3.9 ± 0.55	3.9 ± 0.82
	+CCCP (2.5 μmol/L)	6.2 ± 0.99^*	8.6 ± 0.52
	RCR (ADP+/ADP–)	1.6 ± 0.18^*	2.2 ± 0.46

Values represent mean ± SD. All concentrations are final concentrations. CCCP, carbonyl cyanide m-chlorophenylhydrazone; RCR, respiratory control ratio.

P < 0.05 versus the non-HI group.

DISCUSSION

Energy metabolites and utilization

The parietal cortex is the area that is most likely to be damaged after HI in the 7-day-old rat (Hagberg et al., 1994). In this study, vehicle-treated rats that were allowed to survive until 2 weeks after HI had an average tissue reduction of 38% of the ipsilateral hemisphere, which agrees with a 38% weight deficit of the ipsilateral hemisphere in vehicle-treated rats in a recent neuroprotection study from our laboratory (Gilland and Hagberg, 1997). With this severity of insult, it is most likely that the tissue sampled from the parietal cortex in the HI group would eventually have undergone infarction, which also is supported by the secondary deterioration of the phosphocreatine levels. In the HI group, the levels of high-energy phosphate compounds in the brain were partially recovered at 3 hours after HI, and the ipsilateral phosphocreatine levels were 75% of the contralateral; however, at 8 hours after the insult, a secondary energy failure was observed with the ipsilateral phosphocreatine levels being only 47% of the contralateral. This secondary energy failure has been described in human newborns after asphyxia and in several animal models and probably reflects a progression of tissue infarction or necrosis (Azzopardi et al., 1989; Palmer et al., 1990; Yager et al., 1992; Lorek et al., 1994; Buchli et al., 1995).

In accordance with Vannucci and others (1994), HI and reperfusion did not increase the rate of energy utilization in the brain. On the contrary, there was a small tendency toward reduced energy use after HI (Table 4). Treatment with MK-801 after HI reduced the energy utilization in the ipsilateral parietal cortex to 57% of the rate in vehicle-treated rats after HI (Table 4) but did not affect the energy utilization in the contralateral cortex. This indicates that the metabolic rate is more dependent on NMDAR activation during reperfusion after HI than under normal conditions. The mechanism for this NMDAR dependency may relate to increased extracellular levels of excitatory amino acids during early reperfusion (Puka-Sundvall et al., 1996), and to enhanced sensitivity of the NMDAR. The latter effect can be caused by removal of the Mg²⁺ block from the receptor channel when intracellular energy levels are low (Novelli et al., 1988; Beal et al., 1993; Greene and Greenamyre, 1996) or by other changes in the function of the NMDAR after ischemia (Andiné et al., 1988; Hammond et al., 1994; Heurteaux et al., 1994; Miyazaki et al., 1994). These effects, which could act synergistically, cause increased influx of Ca²⁺ and Na⁺ through the NMDAR channels, resulting in elevated energy use for active ion transport. The lack of effect of MK-801 treatment on energy utilization in the contralateral cortex is in agreement with an unaltered CBF in the contralateral cortex after MK-801 treatment (Gilland and Hagberg, 1996). In contrast, MK-801 treatment reduces the CMR_glc also in the contralateral cortex (Gilland and Hagberg, 1996). This might reflect an increased use of lactate and ketone bodies as substrates for energy production in the contralateral hemisphere (Nehlig and Pereira de Vasconcelos, 1993; Vannucci et al., 1994).

Mitochondrial oxygen consumption

Hypoxia—ischemia induced a reduction of both NAD-linked, pyruvate plus malate—supported, and glutamate plus malate—supported respiratory rates and of FAD-linked, succinate-supported respiration (Table 5 and Table 6). Thus, the mitochondrial dysfunction cannot be explained by a selective inhibition of the pyruvate dehydrogenase complex or the NADH-CoQ reductase complex (complex I of the electron transport chain), as has been suggested in the adult rat (Zaidan and Sims, 1993). For all three substrates, the uncoupled respiration was reduced as much as +ADP respiration, precluding a selective reduction in ATP-synthetase (F₀-F₁-ATPase; complex V) activity.

Isolated brain mitochondria represent only 3% to 16% of the total pool of mitochondria in the tissue, and it cannot be excluded that isolation of mitochondria involves a selection of subgroups after HI (Sims and Blass, 1986). Measuring oxygen consumption by tissue homogenates does not involve any selection of mitochondria. However, after homogenization of the brain, both nonsynaptosomal, and synaptosomal mitochondria are present. The +ADP respiratory rate primarily measures the activity of nonsynaptosomal mitochondria, since ADP cannot enter synaptosomes, whereas the –ADP and uncoupler-stimulated respiratory rates measure the activity of the whole mitochondrial population. In the 7-day-old rat, we found a ratio of uncoupled to +ADP respiratory rates of 1.4, which is 25% lower than in the adult (Sims and Blass, 1986; Sims and Pulsinelli, 1987; Sims, 1991), in accordance with fewer dendrites and axons in the immature neuron (Kristt, 1978; Romijn et al., 1991). In adult rat homogenates, nonsynaptosomal mitochondria are of both neuronal and glial origin (Sims and Blass, 1986). In the immature rat, glia are less developed (Privat, 1975) and, presumably, a substantial part of the free mitochondria are of neuronal origin.

The respiratory control ratio (+ADP/–ADP), which reflects the coupling of respiration to ATP synthesis, was lowered after HI for all three substrates, but this did not reflect an uncoupling of the mitochondrial metabolism, since it was caused by a reduction of +ADP respiration and not by an increase in –ADP respiration. In addition, the energy metabolites in the ipsilateral cortex 1 to 4 after HI indicates an impaired flux of metabolites through the citric acid cycle, electron transport chain, or both, combined with lactate production. The levels of the glycolytic intermediates glucose, pyruvate, and lactate are increased, the cytoplasmic NAD⁺/NADH ratio is reduced, and the mitochondrial NAD⁺/NADH ratio is normal or reduced (Palmer et al., 1990; Yager et al., 1991; Yager et al., 1992; Gilland and Hagberg, 1996). The concentration of lactate in the ipsilateral hemisphere was higher than in the contralateral hemisphere and higher than the blood lactate levels. The high lactate permeability across the blood—brain barrier in the immature brain (Cremer et al., 1979) and the normal CBF (Gilland and Hagberg, 1996; Gilland et al., 1997) suggest that the increased lactate levels in the brain reflect lactate production and not decreased clearance of lactate accumulated during HI.

Relevance of the metabolic impairment

The decreased mitochondrial respiratory capacity and sustained energy utilization probably is an early step in the NMDAR-dependent injurious processes after HI and not secondary to tissue infarction as is outlined here. At 2 to 3 hours after HI, we did not find a difference in ATP and phosphocreatine concentrations between rats that had received MK-801 or vehicle after HI, implying that there was not a difference in the extent of tissue damage at this time. However, treatment with MK-801 induced a marked improvement in the mitochondrial respiratory capacity already at 2 hours and reduced the energy utilization and the CMR_glc at 3 hours after HI (current results; Gilland and Hagberg, 1996).

Mitochondrial impairment as an early step in the neurodamaging cascade after HI agrees well with data from adult rats after forebrain ischemia, where a reduction in mitochondrial function precedes, by several hours, changes in energy metabolite levels (Pulsinelli and Duffy, 1983; Sims, 1991).

There were, however, differences both in ATP and phosphocreatine concentrations between MK-801 and vehicle-treated rats at 8 hours after HI, suggesting that the effects on mitochondrial function and energy utilization are of functional importance for the energy state of the tissue, and that the secondary energy failure depends on NMDAR activation.

Stimulation of the NMDAR might cause mitochondrial damage through elevation of intracellular Ca²⁺, release of nitric oxide (NO·), and production of free radicals (Greene and Greenamyre, 1996). In vitro exposure of mitochondria to free radicals induces a similar reduction of +ADP and uncoupler stimulated respiration, as does HI (Hillered and Ernster, 1983), and treatment with a free radical scavenger after reversible focal ischemia attenuates a secondary mitochondrial dysfunction in adult rats (Kuroda et al., 1996). Hypothetically, the mitochondrial damage and impaired energy metabolism makes the cells more susceptible to NMDAR stimulation, which further impairs the mitochondrial function through increases in intracellular Ca²⁺, NO· formation and free radical production (Beal et al., 1993).

CONCLUSION

In conclusion, treatment with MK-801 after HI reduces energy utilization and attenuates the mitochondrial dysfunction and secondary energy failure. These results explain why NMDAR activation induces increased CMR_glc during reperfusion, and they may be relevant in understanding NMDAR involvement in the pathogenesis of HI injury in the immature brain.

Footnotes

Abbreviations used

Acknowledgment

The authors thank Neil R. Sims for providing the impetus for measuring mitochondrial respiration.

References

Andiné

Jacobson

Hagberg

(1988) Calcium uptake evoked by electrical stimulation is enhanced postischemically and precedes delayed neuronal death in CA1 of rat hippocampus: involvement of N-methyl-d-aspartate receptors. J Cereb Blood Flow Metab 8:799–807.

Azzopardi

Wyatt

Cady

Delpy

Baudin

Stewart

Hope

Hamilton

Reynolds

EOR

(1989) Prognosis of newborn infants with hypoxic—ischemic brain injury assessed by phosphorus magnetic resonance spectroscopy. Pediatr Res 25:445–51.

Beal

Hyman

Koroshetz

(1993) Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases? Trends Neurosci 16:125–31.

Blennow

Ingvar

Lagercrantz

Stone

Eriksson

Forssberg

Ericson

Flodmark

(1995) Early [18F]FDG positron emission tomography in infants with hypoxic—ischaemic encephalopathy shows hypermetabolism during the postasphyctic period. Acta Paediatr 84:1289–95.

Buchli

Rumpel

Maeder

Nedelcu

Martin

(1995) Hypoxic ischemic brain damage in neonatal rats monitored by 31P magnetic resonance spectroscopy (MRS). Pediatr Res 38:428.

Cremer

Cunningham

Partridge

Braun

Oldendorf

(1979) Kinetics of blood brain barrier transport of pyruvate, lactate and glucose in suckling, weanling and adult rats. J Neurochem 33:439–45.

Duffy

Kohle

Vannucci

(1975) Carbohydrate and energy metabolism in perinatal rat brain: relation to survival in anoxia. J Neurochem 24:271–6.

Gatfield

Lowry

Schulz

Passonneau

(1966) Regional energy reserves in mouse brain and changes with ischaemia and anaesthesia. J Neurochem 13:185–95.

Gilland

Bona

Hagberg

(1998) Temporal changes of regional glucose use, blood flow and microtubule-associated protein 2 immunostaining after hypoxia—ischemia in the immature rat brain. J Cereb Blood Flow Metab 18:222–228.

10.

Gilland

Hagberg

(1996) NMDA-receptor dependent increase of cerebral glucose utilization after hypoxia—ischemia in the neonatal rat. J Cereb Blood Flow Metab 16:1005–13.

11.

Gilland

Hagberg

(1997) Is MK-801 neuroprotection mediated by systemic hypothermia in the immature rat? Neuroreport 8:1603–05.

12.

Greene

Greenamyre

(1996) Bioenergetics and glutamate excitotoxicity. Prog Neurobiol 48:613–34.

13.

Hagberg

Bona

Gilland

Puka-Sundvall

(1997) Hypoxia—ischemia model in the 7-day-old rat: possibilities and shortcomings. Acta Paediatr 422:85–8.

14.

Hagberg

Gilland

Diemer

Andiné

(1994) Hypoxia—ischemia in the neonatal rat brain: histopathology after post-treatment with NMDA and non-NMDA receptor antagonists. Biol Neonate 66:206–13.

15.

Hammond

Crépel

Gozlan

Ben-Ari

(1994) Anoxic LTP sheds light on the multiple facets of NMDA receptors. Trends Neurosci 17:497–503.

16.

Hattori

Morin

Schwartz

Fujikawa

Wasterlain

(1989) Posthypoxic treatment with MK801 reduces hypoxic—ischemic damage in the neonatal rat. Neurology 39:713–8.

17.

Heurteaux

Lauritzen

Widmann

Lazdunski

(1994) Glutamate induced overexpression of NMDA receptor messenger RNAs and protein triggered by activation of AMPA/kainate receptors in rat hippocampus following forebrain ischemia. Brain Res 659:67–74.

18.

Hillered

Ernster

(1983) Respiratory activity of isolated rat brain mitochondria following in vitro exposure to oxygen radicals. J Cereb Blood Flow Metab 3:207–14.

19.

Hillered

Siesjö

Arfors

K-E

(1984) Mitochondrial response to transient forebrain ischemia and recirculation in the rat. J Cereb Blood Flow Metab 4:438–46.

20.

Kristt

(1978) Neuronal differentiation in somatosensory cortex of the rat. I. Relationship to synaptogenesis in the first postnatal week. Brain Res 150:467–86.

21.

Kuroda

Katsura

K-I

Hillered

Bates

Siesjö

(1996) Delayed treatment with α-phenyl-N-tert-butyl nitrone (PBN) attenuates secondary mitochondrial dysfunction after transient focal cerebral ischemia in the rat. Neurobiol Dis 3:149–57.

22.

Lorek

Takei

Cady

Wyatt

Penrice

Edwards

Peebles

Wylezinska

Owen-Reece

Kirkbride

Cooper

Aldridge

Roth

Brown

Delpy

Reynolds

EOR

(1994) Delayed (“secondary”) cerebral energy failure after acute hypoxia—ischemia in the newborn piglet: continuous 48-hour studies by phosphorus magnetic resonance spectroscopy. Pediatr Res 36:699–706.

23.

Lowry

Passonneau

(1972) A Flexible system of enzymatic analysis. New York, Academic Press.

24.

Lowry

Passonneau

Hasselberger

Schulz

(1964) Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. J Biol Chem 239:18–30.

25.

Miyazaki

Katayama

Furuichi

Kano

Yoshino

Tsubokawa

(1994) N-methyl-d-aspartate receptor-mediated prolonged after-discharges of CA1 pyramidal cells following transient cerebral ischemia in the rat hippocampus in vivo. Brain Res 657:325–9.

26.

Mujsce

Christensen

Vannucci

(1990) Cerebral blood flow and edema in perinatal hypoxic—ischemic brain damage. Pediatr Res 27:450–3.

27.

Nehlig

Pereira de Vasconcelos

(1993) Glucose and ketone body utilization by the brain of neonatal rats. Progr Neurobiol 40:163–221.

28.

Nelson

Silverstein

(1994) Acute disruption of cytochrome oxidase activity in brain in a perinatal rat stroke model. Pediatr Res 36:12–9.

29.

Novelli

Reilly

Lysko

Henneberry

(1988) Glutamate becomes neurotoxic via the N-methyl-d-aspartate receptor when intracellular energy levels are reduced. Brain Res 451:205–12.

30.

Palmer

Brucklacher

Christensen

Vannucci

(1990) Carbohydrate and energy metabolism during the evolution of hypoxic—ischemic brain damage in the immature rat. J Cereb Blood Flow Metab 10:227–35.

31.

Privat

(1975) Postnatal gliogenesis in the mammalian brain. Int Rev Cytol 40:281–323.

32.

Puka-Sundvall

Gilland

Bona

Lehmann

Sandberg

Hagberg

(1996) Development of brain damage after neonatal hypoxia—ischemia: excitatory amino acids and cysteine. Metab Brain Disease 11:109–23.

33.

Pulsinelli

Duffy

(1983) Regional energy balance in rat brain after transient forebrain ischemia. J Neurochem 40:1500–3.

34.

Rehncrona

Mela

Siesjö

(1979) Recovery of brain mitochondrial function after complete and incomplete cerebral ischemia. Stroke 10:437–46.

35.

Rice

Vannucci

Brierley

(1981) The influence of immaturity on hypoxic—ischemic brain damage in the rat. Ann Neurol 9:131–41.

36.

Romijn

Hofman

Gramsbergen

(1991) At what age is the developing cerebral cortex of the rat comparable to that of the full-term newborn human baby? Early Hum Dev 26:61–7.

37.

Rosenberg

(1986) Cerebral blood flow and O2 metabolism after asphyxia in neonatal lambs. Pediatr Res 20:778–82.

38.

Rosenberg

Parks

Murdaugh

Parker

(1989) Mitochondrial function after asphyxia in newborn lambs. Stroke 20:674–9.

39.

Sims

(1991) Selective impairment of respiration in mitochondria isolated from brain subregions following transient forebrain ischemia in the rat. J Neurochem 56:1836–44.

40.

Sims

Blass

(1986) Expression of classical mitochondrial respiratory responses in homogenates of rat forebrain. J Neurochem 47:496–505.

41.

Sims

Pulsinelli

(1987) Altered mitochondrial respiration in selectively vulnerable brain subregions following transient forebrain ischemia in the rat. J Neurochem 49:1367–74.

42.

Vannucci

Yager

Vannucci

(1994) Cerebral glucose and energy utilization during the evolution of hypoxic—ischemic brain damage in the immature rat. J Cereb Blood Flow Metab 14:279–88.

43.

Yager

Brucklacher

Vannucci

(1991) Cerebal oxidative metabolism and redox state during hypoxia—ischemia and early recovery in immature rats. Am J Physiol 261:H1102–H8.

44.

Yager

Brucklacher

Vannucci

(1992) Cerebral energy metabolism during hypoxia—ischemia and early recovery in immature rats. Am J Physiol 262:H672–H7.

45.

Zaidan

Sims

(1993) Selective reductions in the activity of the pyruvate dehydrogenase complex in the mitochondria isolated from brain subregions following forebrain ischemia in rats. J Cereb Blood Flow Metab 13:98–104.