Sage Journals: Discover world-class research

Abstract

Post-treatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduces hypoxic–ischemic brain injury in immature animals. To elucidate possible mechanisms, cerebral glucose utilization (CMR_glc) and cerebral blood flow (CBF) were measured 1–5 h after hypoxia–ischemia and administration of MK-801 in 7-day-old rats. After 100 min of unilateral hypoxia–ischemia, half of the pups were injected with MK-801. CMR_glc was assessed by the [¹⁴C]deoxyglucose (2-DG) method. The brains were analyzed either by autoradiography or for energy metabolites and chromatographic separation of 2-DG-6-phosphate and 2-DG. CBF was measured by the autoradiographic [¹⁴C]iodoantipyrine method. Mean CMR_glc in the cerebral cortex was increased ipsilaterally after hypoxia–ischemia to 15 ± 3.3 μmol 100 g⁻¹ min⁻¹ (p < 0.01) and areas with CMR_glc >20 μmol 100 g⁻¹ min⁻¹ amounted to 8.0 ± 7.7 mm² in the ipsilateral hemisphere compared with 1.2 ± 1.6 mm² contralateral (p < 0.001). Treatment with MK-801 decreased CMR_glc bilaterally (p < 0.05) and reduced ipsilateral areas with increased CMR_glc by 64% (p < 0.01). CBF was unaltered after hypoxia–ischemia and by MK-801 treatment. In conclusion, regional glucose hyper-utilization in the parietal cortex after hypoxia–ischemia was attenuated by MK-801; this may have relevance to the neuroprotective effect of NMDA-receptor antagonists in this model.

Keywords

Neonatal Hypoxia Ischemia NMDA Glucose metabolism Blood flow

Brain damage due to perinatal hypoxia–ischemia (HI) causes perinatal mortality and neurologic and psychological deficits in humans (Hagberg et al., 1993; Volpe, 1995). Evidence accumulating during recent years suggests that excitatory amino acids (EAAs) are important for the development of HI brain damage in the immature rat (Johnston and Silverstein, 1987; Hagberg et al., 1990; Hattori and Wasterlain, 1990; Barks and Silverstein, 1992). In the immature rat brain, EAA receptors of the N-methyl-D-aspartate (NMDA) type are “hyperactive,” [i.e., NMDA toxicity, receptor density, and excitability is enhanced compared with that of the adult (Greenamyre et al., 1987; Ben-Ari et al., 1988; Hamon and Heinemann, 1988; McDonald et al., 1988; Tremblay et al., 1988; Represa et al., 1989)]. Blocking of NMDA (McDonald et al., 1987; Hattori et al., 1989) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; Hagberg et al., 1994) receptors before or after HI offers neuroprotection in the immature rat. The mechanism for the neuroprotection offered by NMDA-receptor antagonists is unknown. High concentrations of EAAs are directly toxic if administered to brain cells in vitro (Choi, 1987), and high concentrations of glutamate analogues like NMDA and AMPA injected in vivo exert neurotoxicity in the immature rodent brain (Olney et al., 1971; McDonald et al., 1988; Ikonomidou et al., 1989; McDonald et al., 1992).

During HI, endogenous extracellular (ec) concentrations of EAAs increase considerably (Hagberg et al., 1987; Andiné et al., 1991; Silverstein et al., 1991), but the concentrations of EAAs return to levels only slightly higher than normal after the insult (Puka-Sundvall et al., 1995). These concentrations are unlikely to exert direct neurotoxicity and do not explain the neuroprotective effect of NMDA-receptor antagonists after HI. A moderately elevated excitatory activity (Chen et al., 1986; Puka-Sundvall et al., 1995), inhibited EAA re-uptake (Hu et al., 1991) and post-HI changes in the function of the NMDA receptors (Andiné et al., 1988; Kohmura et al., 1990; Hammond et al., 1994; Heurteaux et al., 1994) may contribute to the injurious cascade under conditions with reduced energy supply (Novelli et al., 1988; Simon and Shiraishi, 1990; Gill et al., 1992; Mies et al., 1993; Greene and Greenamyre, 1995). Indeed, it has been suggested that mitochondrial function is impaired during early recovery after HI in the 7-day-old rat, characterized by increased cerebral glucose utilization (CMR_glc), increased tissue lactate concentrations, decreased NAD⁺/NADH quotient, and decreased ATP levels despite normal or decreased energy utilization and normalized CBF (Mujsce et al., 1990; Yager et al., 1991; Vannucci et al., 1994). Impairment of oxidative metabolism may represent a generalized phenomenon applicable also to postasphyxia conditions in human infants as brain regions with enhanced 18-fluoro-deoxyglucose uptake (Blennow et al., 1996), accumulation of lactate (Groenendaal et al., 1994) and secondary depression of the phosphocreatine/orthophosphate quotient (Roth et al., 1992) appear to predict an unfavorable outcome.

The aim of this study was to obtain regional information on CMR_glc and CBF during the early reperfusion phase after immature HI and on how these parameters are affected by post-HI treatment with a neuroprotective dose of the NMDA-receptor antagonist dizocilpine (MK-801).

MATERIALS AND METHODS

Animals and chemicals

Inbred Wistar F rat pups of either sex were purchased from BK Universal, Stockholm, Sweden, housed at 24°C, and fed by their dams until the start of the experiments when 7 days old. 2-Deoxy-D-[U-¹⁴C]glucose (2-DG; specific activity, 230–330 mCi/mmol), 4-iodo[N-methyl-¹⁴C]-antipyrine (IAP; specific activity, 53 mCi/mmol), Hyperfilm βMax, and [¹⁴C]methacrylate standards were obtained from Amersham Sweden AB, Stockholm, Sweden. Chemicals for scintillation counting (Soluene-350, Permablend III, and Triton) were purchased from Packard Instrument B.V. Chemical Operations, Groningen, The Netherlands, and Sigma-Labkemi, Göteborg, Sweden.

Protocol

In our experiments, 152 pups from 21 litters were used. In each litter, except for the animals that did not undergo HI (non-HI controls) and animals used for 2-DG and IAP plasma/blood concentration curve estimates, half of the pups received 0.5 mg/kg of MK-801 intraperitoneally in a single dose immediately after HI (MK-801-treated rats), and the other half served as control animals (HI controls) and received the vehicle of the drug (saline) immediately after HI. Because of the catheterization procedure of intravenous injection, animals from each litter were consecutively, from 1–5 h after HI, injected with either 2-DG or IAP and subsequently decapitated. The rectal temperature was measured with a thermistor probe (BAT-12, Physitemp Instruments, Clifton, NJ, U.S.A.).

Induction of hypoxia–ischemia

Anesthesia was induced and maintained with halothane in oxygen/nitrous oxide in a snout mask. The left common carotid artery was cut between ligatures of Prolene suture (6–0; Rice et al., 1981). Animals were allowed to recover from anesthesia for at least 1 h. HI was induced by exposure to 7.7 ± 0.01% oxygen in nitrogen for 100 min in a humidified chamber at 36°C. After the insult, the pups were returned to their dams.

Measurement of regional cerebral glucose utilization, glucose, lactate, and ATP

Regional cerebral glucose utilization was assessed by the 2-DG method (Sokoloff et al., 1977), modified for the 7-day-old rat (Vannucci et al., 1989). Because these small rats offer methodologic problems, glucose utilization was measured in two different ways. In the first group, pups were reanesthetized after HI, and the femoral vein was catheterized with a polypropylene catheter (outer diameter, 200 μm) by a proximal femoral incision and ligation of the vein. The skin was lightly sutured but not closed. When the animals reacted with movement to tail pinching, 50–150 μCi/kg 2-DG in 30 μl saline was administered intravenously for 1 min, the catheter was removed, and the ligatures and sutures tied close. At 1, 2, 5, 10, 20, 30, 45, or 90 min later, blood was collected from severed neck vessels at decapitation. This method offered the advantage of injecting the 2-DG quickly and allowing 45 min of circulation time (Sokoloff et al., 1977; Mori et al., 1990) but necessitated 25–30 min of reanesthesia. In the other group, 2.5 μCi in 0.2 ml saline, ∼250 μCi/kg, was injected subcutaneously in fully awake rats, after which blood was collected at decapitation at 5, 10, 15, 20, 45, or 90 min after injection. This method impairs the rats' function less but requires a longer (90 min) circulation time of the 2-DG until measurement of glucose utilization (Vannucci et al., 1989).

A portion of the blood was used to determine glucose concentration by an enzymatic-photometric technique (Reflolux II M, Boehringer Mannheim GmbH, Mannheim, Germany), whereas the rest was centrifuged at 5,000 g for 15 min, after which 10 μl of plasma was added to 9 ml of scintillation fluid (0.75 L xylene, 6 g Permablend III, and 0.25 L Triton per liter). Samples were counted in a calibrated liquid scintillation counter (1215 Rackbeta, Wallac OY, Turku, Finland). This allowed calculations of the mean arterial plasma curves for 2-DG after either intravenous or subcutaneous injection (Fig. 1a, b). Because blood sampling from each rat could be done only once, the curve was fitted to the individual pup depending on the 2-DG plasma concentration at decapitation 45 (if intravenous injection) or 90 min (if subcutaneous injection) after injection, the times used for measurement of CMR_glc.

FIG. 1.

Individual values and fitted mean curve for (a) plasma concentrations of 2-deoxy-D-[¹⁴C]glucose after intravenous injection of 150 μCi/kg, (b) plasma concentrations of 2-deoxy-D-[¹⁴C]glucose after subcutaneous injection of 250 μCi/kg, and (c) blood concentrations of [¹⁴C]iodoantipyrine after subcutaneous injection of 500 μCi/kg.

If they were planned for autoradiography, the brains were dissected out after decapitation and in <1 min frozen in isopentane cooled by dry ice. Coronal sections were cut at −20°C, mounted on glass slides, dried, and autoradiographed on Hyperfilm βMax together with [¹⁴C]methacrylate standards. Autoradiographs were calibrated and analyzed by quantitative densitometry (NIH Image, NIH, Bethesda, MD, USA) by a blinded observer. CMR_glc was calculated by Sokoloff's original equation with values of the rate constants from measurements on adult rats (Sokoloff et al., 1977) and the lumped constant from mean of ipsi- and contralateral estimations, respectively (see the following). After film exposure, sections were stained with hematoxylin–eosin to correlate the CMR_glc to histologic damage.

When planned for measurement of metabolites and 2-deoxy-D-[¹⁴C]glucose-6-phosphate (2-DG-6-P), the rats were decapitated over liquid nitrogen and the heads immediately frozen. Portions (10–50 mg) of the parietal cortex were dissected out at −20°C, weighed, and solubilized in 3 M HClO₄ (Lowry and Passonneau, 1972) at ∼10°C. After centrifugation at 5,000 g for 25 min, one volume of the supernatant was neutralized with 2.5 volumes of 2 M KHCO₃ and again centrifuged to remove precipitated KHCO₃. The supernatant was divided into three portions, and 50–150 μl was added to 9 ml of scintillation fluid and counted as previously; 150 μl was used for enzymatic–fluorometric determination of ATP, glucose, and lactate (Lowry and Passonneau, 1972), and 300–600 μl was passed over an ion-exchange column formate form (AG1-X8 resin, Biorad Econocolumn, Biorad, Solna, Sweden) to retain 2-DG-6-P, whereas unphosphorylated 2-DG was eluted with 2.6 ml of distilled water. The remaining 2-DG-6-P was eluted with two portions of 3 ml 1 M HCl. Eluted portions were added to 9 ml of scintillation fluid and counted in a liquid scintillation spectrometer. The 2-DG-6-P activity in the tissue was used as the numerator in Sokoloff's original equation (Sokoloff et al., 1977; Vannucci et al., 1989). The lumped constant vas estimated from a nomogram for adult rat brain that allows calculation of the lumped constant from the brain and plasma glucose concentrations (Pardrige et al., 1982). All brains and specimens were stored at −80°C.

Measurement of local cerebral blood flow

CBF was measured by the autoradiographic IAP method (Sakurada et al., 1978), adapted to the immature rat (Lyons et al., 1987). First, 5–10 μCi IAP (500–1,000 μCi/kg) in 0.2 ml saline was injected subcutaneously. Then at 20, 30, 40, 50, 60, or 90 s after injection, the rats were decapitated, and 10 μl of blood was solubilized in 1 ml of Soluene-350 overnight, added to 9 ml of scintillation fluid and counted. The brain was dissected out in <40 s (Jay et al., 1988), frozen in isopentane cooled by dry ice, and processed for autoradiography as previously described. Because blood could be drawn from rats only at decapitation, the mean arterial blood concentration curve of IAP, calculated from rats decapitated at 20–90 s after injection (Fig. 1c), was fitted to the pups decapitated at 60 s and used for autoradiography and calculation of CBF; 0.944 ml/g was used as the brain-blood partition coefficient (Lyons et al., 1987).

Statistics

The Mann–Whitney U test was used for unpaired statistical analysis between HI controls and MK-801-treated rats, and the Wilcoxon test for paired analysis between ipsilateral and contralateral brain regions. Values are given as mean ± SD. Spearman's rank correlation coefficient (r_s) was used for rank correlation, and the least squares method, for linear regression.

RESULTS

Plasma curves and mode of injection

Individual values and mean 2-DG plasma concentration curves after intravenous and subcutaneous injection and blood IAP concentration curves after subcutaneous injection are shown in Fig. 1. The 2-DG-6-P/ total 2-DG quotients were similar after intravenous injection with 45-min circulation time (0.57 ± 0.16 ipsilaterally and 0.42 ± 0.09 contralateral; n = 8) and subcutaneous injection with 90 min circulation time (0.63 ± 0.14 ipsilaterally and 0.55 ± 0.10 contralateral; n = 15), both of which showed an interhemispheric difference. Calculated CMR_glc in the parietal cortex of HI controls did not differ between intravenously and subcutaneously injected rats (15 ± 3.6, n = 9, vs. 15 ± 4.1 μmol 100 g⁻¹ min⁻¹, n = 7, in the ipsilateral hemisphere and 11 ± 4.9, n = 9, vs. 10 ± 2.0 μmol 100 g⁻¹ min⁻¹, n = 7, in the contralateral hemisphere).

Temperature

There was no difference in rectal temperature at decapitation between HI controls (34.4 ± 1.3°C, n = 26) and 0.5 mg/kg of MK-801-treated rats after HI (34.3 ± 1.4°C, n = 20).

CMR_glc and metabolites in whole tissue

Table 1 shows data from portions of the parietal cortex in rats injected with 2-DG 1–5 h after HI. In HI controls, CMR_glc was higher ipsilaterally than contralateral (15 ± 3.3 vs. 9.4 ± 2.1 μmol 100 g⁻¹ min⁻¹; p = 0.002), accompanied by ipsilaterally decreased ATP and glucose and increased lactate concentrations, increased lumped constant, and increased 2-DG-6-P/total 2-DG ratio. Compared with non-HI controls, CMR_glc in the ipsilateral hemisphere was not significantly increased (p = 0.2; Table 1). CMR_glc in the contralateral hemisphere was, however, decreased when compared with that of non-HI controls (p = 0.01; Table 1). Compared with HI controls, rats treated with MK-801 had lower CMR_glc ipsilaterally (11 ± 5.3 μmol 100 g⁻¹ min⁻¹; p = 0.05) and contralateral (7.1 ± 2.1 μmol 100 g⁻¹ min⁻¹; p = 0.02), a tendency to increased glucose and decreased lactate ipsilaterally (not significant), and increased cerebral glucose concentration contralaterally. The lumped constant was not significantly lower ipsilaterally in MK-801-treated rats, but MK-801 treatment decreased the 2-DG-6-P/total 2-DG ratio ipsilaterally.

TABLE 1.

Metabolic variables from pieces of the parietal cortex in untreated and MK-801-treated rats 1–3 h after hypoxia–ischemia and non-hypoxia–ischemia-exposed controls

	Control (n = 12)			MK-801 (n = 8)

	Ipsilateral		Contralateral	Ipsilateral		Contralateral	Non HI (n = 4)
CMR_glc (μmol 100 g⁻¹ min⁻¹)	15 ± 3.3^a		9.4 ± 2.1	11 ± 5.3^b		7.1 ± 2.1^b	13 ± 2.3
ATP (μmol/g)	1.6 ± 0.36^a		2.3 ± 0.33	1.6 ± 0.31		2.3 ± 0.32	2.3 ± 0.07
Glucose (μmol/g)	2.1 ± 0.64^a		2.8 ± 0.42	2.5 ± 0.52		3.5 ± 0.39^c	1.4 ± 0.29
Lactate (μmol/g)	3.5 ± 2.13^a		1.6 ± 0.42	2.2 ± 0.97		1.4 ± 0.56	1.3 ± 0.30
Lumped constant	0.53 ± 0.08^d		0.46 ± 0.03	0.47 ± 0.04		0.42 ± 0.01^c	0.54 ± 0.04
2-DG-6-P/total 2-DG	0.70 ± 0.10^a		0.56 ± 0.09	0.57 ± 0.16^b		0.48 ± 0.14	0.78 ± 0.04
Blood glucose (M)		6.1 ± 0.92			5.8 ± 0.89		4.3 ± 0.90

Values represent mean ± SD.

CMR_glc, cerebral glucose utilization; HI, hypoxia–ischemia; DG, deoxyglucose.

p ≦ 0.01, significant difference between hemispheres in hypoxic–ischemic control group.

p ≦ 0.05, significant difference between control and treated groups.

p ≦ 0.01, significant difference between control and treated groups.

p ≦ 0.05, significant difference between hemispheres in hypoxic–ischemic control group.

There was a close linear correlation between CMR_glc as calculated by the original Sokoloff equation and as calculated with the 2-DG-6-P replacement and individually estimated lumped constants (modified CMR_glc = 2.6 + 0.80 × Sokoloff CMR_glc; r = 0.97; p < 0.0001; residual SD = 1.18; Fig. 2), with a tendency to differ at very low values of CMR_glc.

FIG. 2.

Linear correlation of cerebral glucose utilization (CMR_glc) in the ipsilateral cortex as calculated with Sokoloff's original equation (Sokoloff et al., 1977) and with mean lumped constants to CMR_glc calculated with 2-deoxy-D-[¹⁴C]glucose-phosphate (2-DG-6-P) measurement modification and with individually estimated lumped constants (Vannucci et al., 1989).

In the ipsilateral hemisphere, there was a negative rank correlation between CMR_glc and brain ATP (r_s = −0.36; p = 0.04), a positive correlation between CMR_glc and brain lactate (r_s = 0.48: p = 0.005), and a negative linear regression of brain glucose on CMR_glc [glucose (in micromols per gram) = 3.3 – 0.078 × CMR_glc (in micromols per 100 g per min); r = 0.59; p = 0.006) when analyzing HI controls together with MK-801-treated rats.

CMR_glc and CBF calculated from autoradiographs

CMR_glc values calculated from autoradiographs are shown in Table 2. In HI controls, mean CMR_glc in the parietal cortex was increased ipsilaterally compared with that in the contralateral hemisphere (14 ± 3.6 and 8.9 ± 3.3 μmol 100 g⁻¹ min⁻¹, respectively; p < 0.001), although CMR_glc in the ipsilateral hemisphere was very heterogeneous, with glucose-hypoutilizing areas corresponding to morphologically damaged areas (loss of acidophilia), and glucose-hyperutilizing areas corresponding to histologically intact areas surrounding damaged areas (Figs. 3 and 4). The glucose-hyperutilizing areas were larger ipsilaterally than contralaterally (8.0 ± 7.7 and 1.2 ± 1.6 mm², respectively, for CMR_glc > 20 μmol 100 g⁻¹ min⁻¹; p < 0.001; Table 2, Fig. 4). Treatment with MK-801 reduced CMR_glc ipsilaterally (to 10 ± 4.7 μmol 100 g⁻¹ min⁻¹; p = 0.016) and contralaterally (to 4.9 ± 2.6 μmol 100 g⁻¹ min⁻¹; p < 0.001) and correspondingly reduced the size of the glucose-hyperutilizing areas to 2.9 ± 3.8 mm² in the ipsilateral hemisphere (p = 0.008; Table 2, Fig. 4).

TABLE 2.

Mean blood glucose utilization (CMR_glc.) in parietal cortex and size of areas where CMR_glc >20 and 30 μmol 100 g⁻¹ min⁻¹, respectively, in whole hemisphere in midhippocampal sections in control and MK-801-treated rats 1–5 h after hypoxia–ischemia

	Control (n = 16)		MK-801 (n = 17)

	Ipsilateral	Contralateral	Ipsilateral	Contralateral
CMR_glc (μmol 100 g⁻¹ min⁻¹)	14 ± 3.6^a	8.9 ± 3.3	10 ± 4.7^b	4.9 ± 2.6^c
Area of CMR_glc >20 (mm²)	8.0 ± 7.7^a	1.2 ± 1.6	2.9 ± 3.8^d	0.13 ± 0.13^b
Area of CMR_glc >30 (mm²)	1.5 ± 1.8^a	0.1 ± 0.1	0.5 ± 0.8^b	0.0 ± 0.0^d

Values represent mean ± SD.

p ≦ 0.001, significant difference between hemispheres in hypoxic–ischemic control group.

p ≦ 0.05, significant difference between control and treated groups.

p ≦ 0.01, significant difference between control and treated groups.

p ≦ 0.001, significant difference between control and treated groups.

FIG. 3.

Representative 2-deoxy-D-[¹⁴C]glucose autoradiographs (calibrated in micromol per 100 g per minute) (a and c) and hematoxylin–eosin staining (b and d) of the same section from a and b) untreated and (c and d) MK-801-treated (0.5 mg/kg immediately after hypoxia–ischemia) rat 4 h after hypoxia–ischemia.

FIG. 4.

Representative 2-deoxy-D-[¹⁴C]glucose autoradiographs(calibrated in micromol per 100 g per minute) from (a) 0.5 mg/kg of MK-801 after hypoxia—ischemia-treated rats and (b) untreated controls after hypoxia–ischemia.

Mean CMR_glc values did not change with time between 1 and 5 h after HI either in the ipsilateral (CMR_glc = 15 — 0.009 × min after HI; r = 0.14; p = 0.5) or contralateral hemisphere (CMR_glc = 8.3 + 0.004 × min after HI; r = 0.03; p = 0.7) in HI controls (n = 16).

CBF in the parietal cortex of HI controls did not differ between the ipsilateral (51 ± 22 ml 100 g⁻¹ min⁻¹) and contralateral hemispheres (50 ± 25 ml 100 g⁻¹ min⁻¹) (Table 3, Fig. 5), but did show a slight heterogeneity corresponding to histologic damage. Treatment with MK-801 did not significantly affect CBF (73 ± 37 ml 100 g⁻¹ min⁻¹ ipsilaterally and 56 ± 27 ml 100 g⁻¹ min⁻¹ contralaterally; Table 3).

TABLE 3.

Mean CBF in parietal cortex from midhippocampal sections in control and MK-801–treated rats 1–5 h after hypoxia–ischemia and non-hypoxia–ischemia–exposed controls

	Control (n = 14)		MK-801 (n = 12)

	Ipsilateral	Contralateral	Ipsilateral	Contralateral	Non-HI (n = 3)
CBF (ml 100 g⁻¹ min⁻¹)	51 ± 22	50 ± 25	73 ± 37	56 ± 27	68 ± 11

Non-HI, non-hypoxia–ischemia.

Values represent mean ± SD. No significant differences were found.

FIG. 5.

[¹⁴C]lodoantipyrine autoradiograph (calibrated in milliliters per 100 g per minute) of hippocampal section 2 h after hypoxia–ischemia.

DISCUSSION

The predominant lesions in this model of immature HI brain damage were infarction and selective neuronal necrosis in the hemisphere ipsilateral to the carotid artery occlusion, affecting, in order of decreasing frequency the posterior cerebral cortex, hippocampus, thalamus, and striatum (Rice et al., 1981; Towfighi et al., 1991; Hagberg et al., 1994). The parietal cortex, which was focused on in this study, shows neuronal damage in 92–95% of HI controls, although there is a large individual variation in the extent of damage (Rice et al., 1981; Hagberg et al., 1994).

In this study we found no difference in CBF 1–5 h after HI between ipsi- and contralateral parietal cortex (Fig. 5), neither of which differed from non-HI controls. These values agree well with earlier published results both in non-HI immature rats (Lyons et al., 1987; Nehlig et al., 1989) and in post-HI 7-day-old rats (Mujsce et al., 1990), showing complete reperfusion up to 24 h after HI.

CMR_glc values calculated from autoradiographs were similar to those in calculations from pieces of brain tissue with estimated lumped constant and measured 2-DG-6-P/total 2-DG ratio (Tables 1 and 2), even though the rate constants used were calculated from data on the adult rat (Sokoloff et al., 1977). This was probably due to the time intervals used (45 and 90 min), when the influence of the rate constants is relatively minor (Mori et al., 1990). In the adult, there is a significant loss of 2-DG-6-P from the brain at 90 min (Mori et al., 1990), in the immature brain, the activities of glycolytic enzymes are ≦50% of the activities in the adult, which would make the dephosphorylation insignificant at 90 min (Vannucci et al., 1989). This is supported by the similar values of CMR_glc calculated after 45 and 90 min of circulation time, after intravenous and subcutaneous injection, respectively, in this study. The values for CMR_glc in the parietal cortex of non-HI controls agree well with those in earlier studies in the immature rat (Vannucci et al., 1989; Bômont et al., 1992; Nehlig and Pereirade Vasconcelos, 1993).

During HI in this model, there is a marked reduction of CBF to the ipsilateral hemisphere together with a reduction in CMR_glc, depletion of high-energy reserves, and decreased NAD⁺/NADH ratio of the cytoplasm (Welsh et al., 1982; Vannucci et al., 1988; Vannucci et al., 1989; Ringel et al., 1991; Yager et al., 1991; Yager et al., 1992).

After HI, CMR_glc values in the ipsilateral hemisphere were very heterogeneous, with hypoglucose-utilizing areas interspersed between areas with increased glucose uptake (CMR_glc > 20 μmol 100 g⁻¹ min⁻¹) (Fig. 3; i.e., at levels well above values from non-HI controls; Table 1). This heterogeneous glucose uptake was highly reproducible and seen in all animals 1–5 h after HI (Fig. 4). The area with increased glucose utilization was markedly larger in the ipsilateral hemisphere than in the contralateral (Table 2), and the mean CMR_glc value in the ipsilateral hemisphere after HI was higher than that in the contralateral hemisphere (Tables 1 and 2). Compared with non-HI controls, there was no significant increase of mean CMR_glc values in the ipsilateral cortex, although large areas showed CMR_glc above non-HI control levels. Compared with non-HI controls, there was a significant reduction in mean glucose utilization in the contralateral cortex (Table 1; Fig. 3). This has been shown previously and explained in terms of oxidative metabolism of lactate and ketone bodies, because it was not accompanied by any change in cerebral energy utilization, as determined from the changes in the concentrations of glucose, lactate, ATP, and phosphocreatine during 2 min after decapitation (Vannucci et al., 1994). In the same study, Vannucci et al. (1994) found normal levels of glucose uptake and decreased cerebral energy utlization in the ipsilateral hemisphere 1 h after HI, and increased glucose uptake and unaltered cerebral energy utilization 4 h after HI, when compared with those of non-HI controls. In this study, we did not see any change of mean CMR_glc during 1–5 h after HI. The differences between the studies might be due to differences in brain regions sampled, severity of the insult, and time points analyzed.

Even though energy utilization in whole tissue is not increased after HI (Vannucci et al., 1994), localized areas with increased energy utilization could exist. In the immature brain, spreading depressions are difficult or impossible to elicit chemically (Bures, 1957), but burst-suppression patterns on EEG have been recorded after HI (Chen et al., 1986). This makes it unclear if the increase in CMR_glc partly represents increased electrical activity in a penumbra surrounding an infarction (Astrup et al., 1981; Simon and Shiraishi, 1990; Gill et al., 1992; Mies et al., 1993). An increased electrical activity might be caused by leakage of EAA or potassium from nearby infarcted (glucose-hypoutilizing) areas (Hansen, 1985; Butcher et al., 1990), although only moderately increased levels of EAA have been measured in this model during recovery (Puka-Sundvall et al., 1995).

The combination of increased CMR_glc, decreased or normal cerebral energy utilization (Vannucci et al., 1994), incomplete restitution of ATP levels, and increased lactate concentrations [which because of the high blood-brain barrier permeability for lactate in the immature brain (Cremer et al., 1979) might reflect a substantial lactate production] suggests that a significant part of the energy demand is supplied by glycolysis. This cannot be explained by low oxygen availability because the blood flow was not reduced after HI (our results and those of Mujsce et al., 1990). Instead, these data suggest an impaired oxidative/mitochondrial metabolism, which has been suggested before and might be an early, pathogenically important change during reflow after HI (Brown and Brierly, 1973; Brown, 1977; Rehncrona et al., 1979; Siesjö, 1981; Linn et al., 1987; Sims and Pulsinelli, 1987; Vannucci, 1990; Nelson and Silverstein, 1994; Vannucci et al., 1994). This post-HI energy failure due to decreased capacity of the oxidative metabolism might be further impaired by reduced glucose-transport capacity of the blood-brain barrier (Vannucci et al., 1995), which would limit the maximal substrate availability and rate of glycolysis.

The pathogenic importance of impaired oxidative metabolism is supported by positron emission tomography and magnetic resonance spectroscopy observations in children with defective mitochondrial respiration, characterized by increased CMR_glc and tissue lactate concentrations in the CNS (Duncan et al., 1995). In addition, the secondary energy failure occurring in the brain after severe birth asphyxia (Roth et al., 1992) in infants appears to be preceded by enhancement of the CMR_glc (Blennow et al., 1996) and increased tissue lactate levels (Groenendaal et al., 1994); all three of these biochemical events predict brain damage and a poor clinical outcome. We found that the most pronounced increases in CMR_glc levels were seen in regions proximate to areas of early infarction (loss of acidophilia in routine stains and glucose hypoutilization; Fig. 3), suggesting that tissue with increased CMR_gfc is at a risk of irreversible damage.

Post-HI administration of 0.5 mg/kg MK-801 decreases brain damage in the 7-day-old rat when administered after HI (Hagberg et al., 1994). In this study, this treatment reduced CMR_glc bilaterally and substantially reduced the size of areas with increased CMR_glc after HI. In focal ischemia in the adult rat, it is suggested that NMDA-receptor antagonists exert their protective effect by reducing the number of periinfarct depolarizations, which reduces the metabolic stress on cells in areas with reduced CBF and thereby reduced energy-producing capacity (Simon and Shiraishi, 1990; Gill et al., 1992; Mies et al., 1993). In this model, there are no areas with reduced CBF early after HI (our results and those of Mujsce et al., 1990), and it is unclear whether there is increased electrical activity after HI. It is possible that MK-801 exerts its neuroprotective effect by reducing a normal or increased (Puka-Sundvall et al., 1995) excitatory tonus and thereby reducing the metabolic demands on cells with impaired energy metabolism (see the preceding). This is supported by the fact that MK-801 reduced CMR_glc bilaterally. Speculatively, reduction of energy requirements during reflow in a critical phase of metabolic impairment may enhance the possibilities of repair and recovery. Another possibility is that EAAs, through their actions on the NMDA channel with increased Ca²⁺ influx, free radical formation, possibly through nitric oxide (NO) synthesis and peroxynitrite formation (Beckman, 1991; Halliwell, 1992; Coyle and Puttfarcken, 1993), contribute to the damage of the mitochondrial membranes and the oxidative metabolism in a more direct way (Brown and Brierly, 1973; Brown, 1977; Rehncrona et al., 1979; Siesjö, 1981; Linn et al., 1987; Sims and Pulsinelli, 1987; Vannucci, 1990; Nelson and Silverstein, 1994; Vannucci et al., 1994). This hypothesis is further supported by the finding of increased glucose consumption by cultured neurons after NO exposure (Maiese et al., 1995).

In conclusion, there was an increase in regional CMR_glc in the ipsilateral hemisphere, being very marked in (penumbral) areas surrounding infarcted tissue. These glucose-hyperutilizing areas, and CMR_glc generally in the brain, were attenuated by post-HI treatment with a neuroprotective dose of the NMDA-receptor antagonist MK-801. These events and the effect of MK-801 were unrelated to changes in CBF.

Footnotes

Acknowledgment:

This work was supported by the Swedish Medical Research Council (grant no. 9455), the 1987 Foundation for Stroke Research, the Sven Jerring Foundation, the Åke Wiberg Foundation, the Åhlén Foundation, the Magnus Bergwall Foundation, the Frimurare Barnhus Foundation, the Göteborg Medical Society, Fonden för studerandet av läkarvetenskapen vid Sahlgrenska sjukhuset, the First-of-May Flower Annual Campaign, and the Medical Faculty of Göteborg, University of Göteborg. Special thanks to Carin Alminger for invaluable technical assistance.

Abbreviations used

References

Andiné

Jacobson

Hagberg

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

Andiné

Sandberg

Bågenholm

Lehmann

Hagberg

(1991) Intra- and extracellular changes of amino acids in the cerebral cortex of the neonatal rat during hypoxia–ischemia. Dev Brain Res 64:115–120

Astrup

Siesjö

Symon

(1981) Thresholds in cerebral ischemia—the ischemic penumbra. Stroke 12:723–725

Barks

Silverstein

(1992) Excitatory amino acids contribute to the pathogenesis of perinatal hypoxic–ischemic brain injury. Brain Pathol 2:235–243

Beckman

(1991) The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J Dev Physiol 15: 53–59

Ben-Ari

Cherubini

Krnjevic

(1988) Changes in voltage dependence of NMDA currents during development. Neurosci Lett 94:88–92

Blennow

Ingvar

Lagercrantz

Stone-Elander

Eriksson

Forsberg

Ericson

Flodmark

(1996) Early [18F]FDG positron emission tomography in infants with hypoxic–ischemic encephalopathy shows hypermetabolism during the post-asphyxtic period. Acta Paediatr 95:1289–1295

Bômont

Bilger

Boyet

Vert

Nehlig

(1992) Acute hypoxia induces specific changes in local cerebral glucose utilization at different postnatal ages in the rat. Dev Brain Res 66:33–45

Brown

(1977) Structural abnormalities in neurons. J Clin Pathol 30:155–169

10.

Brown

Brierly

(1973) The earliest alterations in rat neurones and astrocytes after anoxia-ischemia. Acta Neuropathol 23:9–22

11.

Bures

(1957) The ontogenetic development of steady potential differences in the cerebral cortex in animals. Electroencephalogr Clin Neurophysiol 9:121–130

12.

Butcher

Bullock

Graham

McCulloch

(1990) Correlation between amino acid release and neuropathological outcome in rat brain following middle cerebral artery occlusion. Stroke 21:1727–1733

13.

Chen

Silverstein

Aldrich

Johnston

(1986) Evolution of acute cortical EEG changes in experimental perinatal hypoxic–ischemic encephalopathy. Neurology S1:S86

14.

Choi

(1987) Ionic dependence of glutamate neurotoxicity. J Neurosci 7:369–379

15.

Coyle

Puttfarcken

(1993) Oxiditative stress, glutamate, and neurodegenerative disorders. Science 262:689–695

16.

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 33:439–445

17.

Duncan

Herholz

Kugel

Roth

Ruitenbeek

Heindel

Wienhard

Heiss

(1995) Positron emission tomography and magnetic resonance spectroscopy of cerebral glycolysis in children with congenital lactic acidosis. Ann Neurol 37:351–358

18.

Gill

Andiné

Hillered

Persson

Hagberg

(1992) The effect of MK-801 on cortical spreading depression in the penumbral zone following focal ischemia in the rat. J Cereb Blood Flow Metab 12:371–379

19.

Greenamyre

Penney

Young

Hudson

Silverstein

Johnston

(1987) Evidence for transient perinatal glutamatergic innervation of globus pallidus. J Neurosci 7: 1022–1030

20.

Greene

Greenamyre

(1995) Exacerbation of NMDA, AMPA, and L-glutamate excitotoxicity by the succinate dehydrogenase inhibitor malonate. J Neurochem 64:2332–2338

21.

Groenendaal

Veenhoven

Van der Grond

(1994) Cerebral lactate and N-acetyl-aspartate/choline ratios in asphyxiated full-term neonates demonstrated in vivo using proton magnetic resonance spectroscopy. Pediatr Res 35:149–151

22.

Hagberg

Olow

(1993) The changing panorama of cerebral palsy in Sweden. VI. Prevalence and origin during the birth year period 1983–1986. Acta Paediatr 82:387–393

23.

Hagberg

Andersson

Kjellmer

Thiringer

Thordstein

(1987) Extracellulare overflow of glutamate, aspartate, GABA and taurine in the cortex and basal ganglia of fetal lambs during hypoxia–ischemia. Neurosci Lett 78:311–317

24.

Hagberg

Andiné

Lehmann

(1990) Excitatory amino acids and hypoxic–ischemic damage in the immature brain. In: Cerebral Ischemia and Resuscitation ( Schurr

Rigor

, eds), Boca Raton, Florida, CRC Press, pp 115–120

25.

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 66:206–213

26.

Halliwell

(1992) Reactive oxygen species and the central nervous system. J Neurochem 59:1609–1623

27.

Hammond

Crépel

Gozlan

Ben-Ari

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

28.

Hamon

Heinemann

(1988) Developmental changes in neuronal sensitivity to excitatory amino acids in area CA1 of the rat hippocampus. Dev Brain Res 38:286–290

29.

Hansen

(1985) Effect of anoxia on ion distribution in the brain. Physiol Rev 65:101–147

30.

Hattori

Morin

Schwartz

Fujikawa

Wasterlain

(1989) Posthypoxic treatment with MK801 reduces hypoxic–ischemic damage in the neonatal rat. Neurology 39: 713–718

31.

Hattori

Wasterlain

(1990) Excitatory amino acids in the developing brain: Ontogeny, plasticity, and excitotoxicity. Pediatr Neurol 6:219–228

32.

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

33.

McDonald

Johnston

Silverstein

(1991) Excitotoxic brain injury suppresses striatal high-affinity glutamate uptake in perinatal rats. J Neurochem 56:933–937

34.

Ikonomidou

Mosinger

Shahis

Labruyere

Olney

(1989) Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methyl-D-aspartate neurotoxicity. J Neurosci 9:2809–2818

35.

Jay

Lucignani

Crane

Jehle

Sokoloff

(1988) Measurement of local cerebral blood flow with [14C]iodoantipyrine in the mouse. J Cereb Blood Flow Metab 8:121–129

36.

Johnston

Silverstein

(1987) Perinatal anoxia. In: Animal Models of Dementia ( Coyle

, ed), New York, Alan R. Liss, pp 223–251

37.

Kohmura

Yamada

Hayakawa

Kinoshitsa

Matsumoto

Mogami

(1990) Hippocampal neurons become more vulnerable to glutamate after subcritical hypoxia: An in vitro study. J Cereb Blood Flow Metab 10:877–884

38.

Linn

Paschen

Ophoff

Hossman

(1987) Mitochondrial respiration during recirculation after prolonged ischemia in cat brain. Exp Neurol 96:321–333

39.

Lowry

Passonneau

(1972) A Flexible System of Enzymatic Analysis. New York, Academic Press

40.

Lyons

Vasta

Vannucci

(1987) Autoradiographic determination of regional cerebral blood flow in the immature rat. Pediatr Res 2:471–476

41.

Maiese

Vannucci

Davis-Hill

Boccone

Simpson

(1995) Neuroprotection by peptide growth factors is dependent upon modulation of glucose transport during nitric oxide toxicity. J Cereb Blood Flow Metab 15(suppl 1):S454

42.

McDonald

Silverstein

Johnston

(1987) MK-801 protects the neonatal brain from hypoxic–ischemic damage. Eur J Pharmacol 140:359–361

43.

McDonald

Silverstein

Johnston

(1988) Neurotoxicity of N-methyl-D-aspartate is markedly enhanced in developing rat central nervous system. Brain Res 459:200–203

44.

McDonald

Trescher

Johnston

(1992) Susceptibility of brain to AMPA induced excitotoxicity transiently peaks during early postnatal development. Brain Res 583: 54–70

45.

Mies

Kohno

Hossman

K-A

(1993) MK-801, a glutamate antagonist, lowers flow threshold for inhibition of protein synthesis after middle cerebral artery occlusion of rat. Neurosci Lett 155:65–68

46.

Mori

Schmidt

Jay

Palombo

Nelson

Lucignani

Pettigrew

Kennedy

Sokoloff

(1990) Optimal duration of experimental period in measurement of local cerebral glucose utilization with the deoxyglucose method. J Neurochem 54:307–319

47.

Mujsce

Christensen

Vannucci

(1990) Cerebral blood flow and edema in perinatal hypoxic–ischemic brain damage. Pediatr Res 27:450–453

48.

Nehlig

De Vasconcelos

Boyet

(1989) Postnatal changes in local cerebral blood flow measured by the quantitative autoradiographic [14C]iodoantipyrine technique in freely moving rats. J Cereb Blood Flow Metab 9:579–588

49.

Nehlig

de Vasconcelos

Pereira A

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

50.

Nelson

Silverstein

(1994) Acute disruption of cytochrome oxidase activity in brain in a perinatal rat stroke model. Pediatr Res 36:12–19

51.

Novelli

Reilly

Lysko

Henneberry

(1988) Glutamate becomes neurotoxic via the N-methyl-D-aspartate receptor when intracellular energy levels are reduced. Brain Res 451:205–212

52.

Olney

Rhee

(1971) Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system. Exp Brain Res 14:61–76

53.

Pardrige

Crane

Mietus

Oldendorf

(1982) Nomogram for 2-deoxyglucose lumped constant for rat brain cortex. J Cereb Blood Flow Metab 2:197–202

54.

Puka-Sundvall

Sandberg

Bona

Gilland

Hagberg

(1995) Excitatory amino acids and cysteine in relation to brain damage in a neonatal rat model of hypoxic-ischemia. J Cereb Blood Flow Metab 15(suppl 1):S282

55.

Rehncrona

Mela

Siesjö

(1979) Recovery of brain mitochondrial function after complete and incomplete cerebral ischemia. Stroke 10:437–446

56.

Represa

Tremblay

Ben-Ari

(1989) Transient increase of NMDA-binding sites in human hippocampus during development. Neurosci Lett 99:61–66

57.

Rice

Vannucci

Brierley

(1981) The influence of immaturity on hypoxic–ischemic brain damage in the rat. Ann Neurol 9:131–141

58.

Ringel

Bryan

Vannucci

(1991) Regional cerebral blood flow during hypoxia–ischemia in the immature rat: Comparison of iodoantipyrine and iodoamphetamine as radioactive tracers. Dev Brain Res 59:231–235

59.

Roth

Edwards

Cady

Delpy

Wyatt

Azzopardi

Baudin

Townsend

Stewart

Reynolds

EOR

(1992) Relation between cerebral oxidative metabolism following birth asphyxia and neurodevelopmental outcome and brain growth at one year. Dev Med Child Neurol 34:285–295

60.

Sakurada

Kennedy

Jehle

Brown

Carbin

Sokoloff

(1978) Measurement of local cerebral blood flow with iodo[14C]antipyrine. Am J Physiol 231:H59–H66

61.

Siesjö

(1981) Cell damage in the brain: A speculative synthesis. J Cereb Blood Flow Metab 1:155–185

62.

Silverstein

Naik

Simpson

(1991) Hypoxia-ischemia stimulates hippocampal glutamate efflux in perinatal rat brain: An in vivo microdialysis study. Pediatr Res 30:587–590

63.

Simon

Shiraishi

(1990) N-metyl-D-aspartate antagonist reduces stroke size and regional glucose metabolism. Ann Neurol 27:606–611

64.

Sims

Pulsinelli

(1987) Altered mitochondrial respiration in selectively vulnerable brain subregions following transient forebrain ischemia in the rat. J Neurochem 49:1367–1374

65.

Sokoloff

Reivich

Kennedy

Des Rosiers

Patlak

Pettigrew

Sakurada

Shinohara

(1977) The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: Theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem 28:897–916

66.

Towfighi

Yager

Housman

Vannucci

(1991) Neuropathology of remote hypoxic–ischemic damage in the immature rat. Acta Neuropathol 81:578–587

67.

Tremblay

Roisin

Represa

Charriaut-Marlangue

Ben-Ari

(1988) Transient increased density of NMDA binding sites in the developing rat hippocampus. Brain Res 461:393–396

68.

Vannucci

(1990) Experimental biology of cerebral hypoxia–ischemia: Relation to perinatal brain damage. Pediatr Res 27:317–326

69.

Vannucci

Lyons

Vasta

(1988) Regional cerebral blood flow during hypoxia–ischemia in immature rats. Stroke 19: 245–250

70.

Vannucci

Christensen

Stein

(1989) Regional cerebral glucose utilization in the immature rat: Effect of hypoxia–ischemia. Pediatr Res 26:208–214

71.

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 14:279–288

72.

Vannucci

Mahler

Simpson

Lee

W-H

(1995) Effects of cerebral hypoxia–ischemia on glucose transporter expression in immature rat brain. J Cereb Blood Flow Metab 15(suppl 1):S287

73.

Volpe

(1995) Neurology of the Newborn. Philadelphia, WB Saunders

74.

Welsh

Vannucci

Brierley

(1982) Columnar alterations of NADH fluorescence during hypoxia–ischemia in immature rat brain. J Cereb Blood Flow Metab 2:221–228

75.

Yager

Brucklacher

Vannucci

(1991) Cerebral oxidative metabolism and redox state during hypoxia–ischemia and early recovery in immature rats. Am J Physiol 261: H1102–H1108

76.

Yager

Brucklacher

Vannucci

(1992) Cerebral energy metabolism during hypoxia–ischemia and early recovery in immature rats. Am J Physiol 262:H672–H677

NMDA Receptor–Dependent Increase of Cerebral Glucose Utilization after Hypoxia–Ischemia in the Immature Rat

Abstract

Keywords

MATERIALS AND METHODS

Animals and chemicals

Protocol

Induction of hypoxia–ischemia

Measurement of regional cerebral glucose utilization, glucose, lactate, and ATP

Measurement of local cerebral blood flow

Statistics

RESULTS

Plasma curves and mode of injection

Temperature

CMRglc and metabolites in whole tissue

CMRglc and CBF calculated from autoradiographs

DISCUSSION

Footnotes

Acknowledgment:

Abbreviations used

References

CMR_glc and metabolites in whole tissue

CMR_glc and CBF calculated from autoradiographs