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In experimental models of perinatal hypoxia-ischemia, antagonists of excitatory amino acid receptors are effective in reducing subsequent brain damage even when administered after the hypoxic-ischemic episode(1, 2). Excitatory amino acids act through three different ionotropic channel types: the NMDA,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors, and a metabotropic receptor(3). NMDA toxicity, receptor density, and excitability is enhanced in the immature brain, compared with that of the adult(3, 4). Blocking of NMDARs after hypoxia-ischemia in the immature rat offers neuroprotection even with a dose as low as 0.3 mg/kg MK-801(5).

Magnesium has several effects in the body, including regulation of vascular tone, blocking of voltage-dependent Ca2+ channels, being necessary for ATP regeneration(6–11), and giving a voltage-dependent and noncompetitive block of the NMDAR channel(12, 13), although this block is not as strong in the immature rat(14). In animal models, magnesium has proven to be neuroprotective after trauma and ischemia in the adult(15–17), and in the immature rat after either NMDA administration(18) or experimental hypoxic-ischemic insults. It appears to be effective either as monotherapy(19) or in combination with oxygen free radical scavengers(20). Epidemiologic data indicate that MgSO4 given in preeclampsia ameliorates cerebral injury with a reduced rate of cerebral paresis(21). A randomized controlled study of MgSO4 after severe perinatal asphyxia is currently recruiting babies in 16 countries (RAST study)(22).

In adult rats, noncompetitive and competitive NMDAR antagonists cause side effects that can be seen as increased glucose utilization, expression of HSP72, and neuronal vacuolization in corticolimbic areas. At high doses these areas undergo neuronal necrosis(23–29). This has limited the clinical developments of these drugs. This effect might, in part, be species-dependent, as e.g. MK-801 does not induce vacuolization in the cingulate cortex in the guinea pig (A. Lehman, personal communication).

The HSP72 is one of a family of stress-inducible proteins that is not normally present in cells(30), and its expression is used as a marker for cellular stress. HSP72 is visualized in neurons after ischemia in the adult rat(31). In immature rats, HSP72 expression has been detected as early as 1 h after hypoxia-ischemia with a persistent expression up to 24 h in neurons and endothelial cells(32, 33). In adult rats HSP72 is expressed in neurons in the posterior cingulate and retrosplenial cortices from 4 h to 7 d, with a maximum at 24 h, after MK-801 administration. These HSP72 immunoreactive cells also contain abnormal cytoplasmic vacuoles(27, 34).

The increased sensitivity to NMDAR stimulation and the marked neuroprotection offered by NMDAR antagonism in the immature brain during the reperfusion phase after hypoxia-ischemia strongly suggest that NMDAR-mediated mechanisms should be taken into account when constructing future neuroprotective strategies. It is, therefore, important to determine whether NMDAR antagonism has similar neurotoxic side effects in the immature brain, as it appears to have in that of the adult. Phencyclidine (30 mg/kg) and MK-801(1 mg/kg) do not induce HSP72 expression in 1- or 20-d-old rats(34), and MK-801 (1 or 5 mg/kg) administration does not cause neuronal vacuolization in 0.5- or 1-mo-old rats(35). The effects of NMDAR antagonism on glucose utilization and HSP72 expression has, however, not been studied in 7-d-old rats when NMDA toxicity, receptor density, and excitability are temporarily increased, and the neurodevelopmental maturation of the rat is similar to that of the near term human fetus(36).

The aim of this study was to evaluate the regional effects on glucose utilization and HSP72 expression in the immature rat brain of acute treatment with neuroprotective doses of either one of the noncompetitive NMDAR antagonists MK-801 or MgSO4, and thus indicate if noncompetitive NMDAR antagonists exert similar neurotoxicity in the immature brain as in the adult, and if MgSO4, which is in clinical use, differs in this aspect from other noncompetitive NMDAR antagonists.

METHODS

Animals and chemicals. Sprague-Dawley rat pups of either sex were purchased from Charles River, Uppsala, Sweden. 2-DG(specific activity 230-330 Ci/mol), Hyperfilm βMax, [14C]methacrylate standards, and the MAb against HSP72 (RPN 1197) were obtained from Amersham Sweden AB, Stockholm, Sweden. The avidin/biotin complex kit (Vectastain) was purchased from Vector Laboratories, Burlingame, CA, and the tyramide signal amplification kit (NEL 700) from DuPont NEN Research Products, Boston, MA. All substrates and enzymes for glucose measurement were bought from Boehringer Mannheim, Stockholm, Sweden. Addex-magnesium (Pharmacia, Sweden; MgSO4(7H2O), 246 g/mol) was used. To reduce uncertainty about dosing due to the heptahydrate form, which has about double the molecular weight of the nonhydrated form, the MgSO4 dosages were expressed in millimoles/kg.

Protocol. Rats were housed in 24 °C and fed by their dams. Seven-day-old rats were randomized to receive a single dose (about 0.3 mL/rat, isotonic) i.p. of either: saline, 0.5 mg/kg MK-801, 1.0 mg/kg MK-801, 2 mmol/kg MgSO4, or 4 mmol/kg MgSO4. Cerebral glucose utilization measurements were started 30 min (n = 8 in each group), 48 h(n = 10 in two groups), and 4 d (n = 8 in three groups) after drug administration. HSP72 expression was measured 4 h after drug injection (n = 4 in saline, 0.5 mg/kg MK-801 and 2 mmol/kg MgSO4 groups) and 24 h after injection (n = 9 in each group). At 4 h after injection all brains were fresh frozen, whereas at 24 h after injection five brains in each group were perfusion fixed. Separate pups were used as positive controls for HSP72 immunostaining and did not receive any drugs or vehicle. These rats underwent hypoxia-ischemia, which was induced by ligation of the left common carotid artery and exposure to 7.7% oxygen for 100 min at 36 °C(5, 37, 38). At 4 h after hypoxia-ischemia five pups were killed and the brains frozen, and at 24 h six pups were killed, of which four were frozen and two perfusion fixed. Animal experiments were approved by the ethical committee of Göteborg (no. 19-96).

Measurement of regional cerebral glucose utilization. Regional cerebral glucose utilization was assessed by the 2-DG method(39) modified for the 7-d-old rat(40, 41). 2-DG, 2.5 μCi in 0.2 mL of saline, about 200 μCi/kg, was injected s.c. in fully awake rats that were kept in a temperature-controlled chamber. Blood was collected at decapitation 90 min after injection. A portion of the blood was used to determine glucose concentration with an enzymatic-fluorometric technique(42), whereas the rest was centrifuged at 5000 ×g for 15 min after which 25 μL of plasma were added to 9 mL of scintillation fluid (0.75 L of xylene, 6 g of Permablend III, and 0.25 L of Triton per L). Samples were counted in a calibrated liquid scintillation counter (1215 Rackbeta, Wallac OY, Turku, Finland). A mean arterial 2-DG plasma concentration curve, made from separate animals decapitated at various intervals after injection, was fitted to the individual pup, depending upon amount injected and plasma radioactivity at decapitation(40). In 11-d-old rats, i.e. rats decapitated at 4 d after injection, the plasma levels of 2-DG reached a peak later, and the initial decline was slower, but the total rate of decrease in plasma 2-DG was substantially higher than in the younger animals (curves not shown).

The brains were dissected out after decapitation and frozen in isopentane chilled 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 with a CCD72 video camera and digitizing unit (Dage-MTI, Michigan City, IN) and a Macintosh Quadra 900 computer with National Institutes of Health Image 1.52 (National Institutes of Health, Bethesda, MD) by a blinded observer in six different coronal planes (5, 4.5, 4, 3, 2, and 0.5 mm behind bregma)(43). Regional cerebral glucose utilization was calculated by Sokoloff's original equation with values of the rate constants from measurements on adult rats(39) and the lumped constant from estimations in the 7-d-old rat(40).

The rectal temperature was measured with a thermistor probe (BAT-12, Physitemp Instruments, Clifton, NJ). All brains and specimens were stored at-80 °C.

Immunohistochemical staining. Perfusion fixation was performed, after an i.p. injection of methohexital (250 mg/kg), through the left ventricle with 5 mL of PBS (pH 7.4, 0.08 M), followed by 20 mL of 4% paraformaldehyde in PBS. Brains were left in 4% paraformaldehyde in PBS for 4 h, cryoprotected with 10% sucrose in PBS, and subsequently frozen on dry ice. Sections, 10-μm, were cut at -20 °C. The slides were put into citrate buffer, pH 6.0, and boiled twice for 3 min each in a microwave oven.

When the brains were fresh frozen, rats were decapitated, and brains were dissected out and frozen on dry ice; 10-μm sections were cut at -20 °C. Slides were fixed during 10 min of immersion in 4% paraformaldehyde in PBS.

After rinsing in PBS, nonspecific binding was blocked for 30 min with 4% horse serum in PBS followed by incubation with HSP72 antibody diluted 1:4000 in PBS with 0.2% Triton X-100 and 1% horse serum for 60 min. The sections were rinsed in PBS and incubated with a biotinylated horse-anti-mouse antibody for 60 min. Endogenous peroxidase activity was blocked by incubation in 0.6% H2O2 in methanol for 5 min. The tyramide signal amplification kit was used according to the manufacturer's instructions; sections were rinsed in TNT buffer, pH 7.5, incubated with Tris-NaCl-blocking-buffer, pH 7.5, for 30 min followed by the avidin/biotin complex kit for 30 min. Slides were rinsed in TNT, incubated with biotinyl tyramide for 3 min, then rinsed in TNT followed by streptavidin-horseradish peroxidase for 30 min. After rinsing in TNT the slides were placed in sodium acetate buffer (pH 6.0, 0.1 M) for 10 min. Finally the immunoreactivity was visualized with 50 mg of 3,3-di-aminobenzidine enhanced with 1.5 g of ammonium nickel sulfate, 200 mg of β-D-glucose, 40 mg of ammonium chloride, and 1 mg of β-glucose oxidase dissolved in 100 mL of sodium acetate buffer as previously described(44).

Negative controls were made using the same procedure as above but excluding the primary antibody. These sections were devoid of specific immunoreactivity. Positive controls were 7-d-old rat pups exposed to hypoxia-ischemia. All brains were evaluated by two observers in four different coronal planes.

Statistics. ANOVA with Fisher's post hoc test was used for all comparisons between groups.

RESULTS

Physical parameters. There was no significant difference in rectal temperature between the groups (Table 1). Plasma glucose was higher in animals that had received 4 mmol/kg MgSO4, and these rats had higher 2-DG levels in plasma at decapitation 2 h after injection (Table 1). MK-801 dose-dependently reduced weight gain at 4 d after injection. There was no mortality during the study.

Table 1 Physical parameters at decapitation in rats used for autoradiography at 30 min, 48 h, and 4 d after drug injection
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Glucose utilization. At 30 min after injection, 0.5 and 1.0 mg/kg MK-801 and 4 mmol/kg MgSO4 induced decreased glucose uptake in the caudoputamen, motor parietal cortex, somatosensory parietal cortex, the posterior cingulate cortex, and CA1 and CA3 subfields of the hippocampus (Table 2; Fig. 1). Both doses of MK-801 lowered glucose utilization in the retrosplenial cortex. Although MgSO4 did not affect glucose uptake in the retrosplenial cortex compared with controls, there was a significant difference between the two doses. Neither drug significantly affected the glucose uptake in the subiculum or the entorhinal cortex, although with both doses of MK-801 and the higher dose of MgSO4 there was a tendency toward reduced glucose utilization in these areas. The lower dose (2 mmol/kg) of MgSO4 showed a slightly higher glucose uptake than that of controls in all brain areas measured, but the differences never reached statistical significance (Table 2). At 48 h after injection, pups treated with 0.5 mg/kg MK-801 had reduced glucose utilization compared with controls in five of the nine structures examined(Table 3). At 4 d after drug injection, there were no significant differences in the regional glucose utilization between controls and rats treated with 0.5 mg/kg MK-801 or 4 mmol/kg MgSO4(Table 3).

Table 2 Mean regional glucose utilization(μmol/100g/min), measured by the 2-DG method, in different cerebral areas 30 min after drug injection
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Figure 1
figure 1

2-DG autoradiographs (cerebral glucose utilization; calibrated in μmol/100 g/min) from 7-d-old rats 30 min after injection with: (a) vehicle (control), (b) 0.5 mg/kg MK-801,(c) 1.0 mg/kg MK-801, (d) 2 mmol/kg MgSO4, and(e) 4 mmol/kg MgSO4. Neither the NMDAR antagonist MK-801, nor MgSO4 induced any regional increase of the glucose utilization in the limbic system.

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Table 3 Mean regional glucose utilization(μmol/100g/min), measured by the 2-DG method, in different cerebral areas 48 h and 4 d after drug injection
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The changes in tissue radioactivity were similar to the changes in glucose utilization, the only major difference being a significant reduction in tissue radioactivity caused by both doses of MK-801 in the subiculum at 30 min after injection (data not shown).

HSP72 expression. HSP72-positive neurons were found in all brains after hypoxia-ischemia. Four hours after hypoxia-ischemia there was an expression in scattered neurons and endothelial cells throughout the ischemic hemisphere, whereas at 24 h after hypoxia-ischemia HSP72 expression was found mainly in hippocampal neurons but with some expression in cortical neurons (Fig. 2).

Figure 2
figure 2

Expression of the HSP72 in 7- or 8-d-old rats:(a) 4 h after hypoxia-ischemia, (b) 24 h after hypoxia-ischemia, (c) 4 h after administration of 0.5 mg/kg MK-801,(d) 24 h after administration of 1.0 mg/kg MK-801, (e) 4 h after administration of 2 mmol/kg MgSO4, and (f) 24 h after administration of 4 mmol/kg of MgSO4. Neither of the drugs induced HSP72 immunostaining, in contrast to hypoxia-ischemia, which was used as a positive control and induced HSP72 immunostaining in all brains examined.

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There was no neural or glial HSP72 expression in any of the controls, nor in the brains from rats treated with 0.5 mg/kg MK-801, 1.0 mg/kg MK-801, 2 mmol/kg MgSO4, or 4 mmol/kg MgSO4, irrespectively of whether they were fresh frozen or perfusion fixed, and analyzed 4 or 24 h after drug administration (Fig. 2).

DISCUSSION

In this study a modification of the 2-DG method for measurement of glucose utilization(39) has been used due to the small size of the 7-d-old rat. This has been validated and gives accurate measurements of glucose utilization(40, 41). The close agreement between changes in brain radioactivity and calculated glucose utilization(data not shown) further supports the reliability of the findings. The metabolic effects of MK-801 are not due to temperature differences, because the rats were kept in a strictly temperature-controlled environment, and there were no differences in rectal temperature (Table 1), which correlates well with brain temperature in these rats(45).

Both doses of MK-801 markedly reduced cerebral glucose utilization at 30 min after injection, whereas only the higher MgSO4 dose significantly affected glucose utilization. It has been suggested that the attenuation of regionally increased glucose utilization is an important part of the neuroprotection offered after hypoxia-ischemia(40), because in the posthypoxic-ischemic brain it reflects decreased energy utilization and retained mitochondrial function with subsequently improved tissue energy status(52). A dose of 2 mmol/kg MgSO4 partly reduces brain damage after intrastriatal NMDA injections(18). A similar, or twice as high, dose of MgSO4(500 mg/kg) reduces brain damage after hypoxia-ischemia in the 7-d-old rat(19), whereas 300 mg/kg + 100 mg/kg/h did not reduce neuronal injury in fetal lambs(46). A study in postasphyxial children has shown 400 mg/kg MgSO4 to induce hypotension(22), and a dosage of 2 mmol/kg MgSO4 is proposed in the new clinical intervention MgSO4 study (RAST).

The complete absence of neuronal or glial HSP72 expression at two time points, when HSP72 is expressed in the immature rat after hypoxia-ischemia and in the adult rat after NMDAR antagonist exposition, after treatment with two different noncompetitive NMDAR antagonists at two dosages each, indicates that HSP72 is not expressed in the immature rat after NMDAR antagonism. HSP72 expression is used as a marker for cellular stress after NMDAR antagonism and ischemia in the adult and immature rat(27, 31, 32, 34). The absence of HSP72 expression, therefore, suggests absence of cellular injury after NMDAR antagonist exposition in the immature rat.

In adult rats, NMDAR antagonists cause regionally increased glucose utilization. This probably has a pathologic significance, because it is a sign of the cellular stress that induces expression of HSP72, neuronal vacuolization, and at high doses and late time points neuronal necrosis(47, 48). Olney and Farber(29) have found that these effects can be blocked by several classes of drugs and have proposed a circuitry of several excitatory pathways ending in the posterior cingulate/retrosplenial cortex, involving glutamatergic neurons stimulating inhibitory γ-aminobutyric acidergic neurons and noradrenergic neurons, with the final transmittors being neuropeptide Y, glutamate (through non-NMDARs), acetylcholine, and norepinephrine acting at α2-receptors. The fact that NMDAR antagonists do not induce neuronal vacuolization and HSP72 expression(34, 35) (present results), and also do not induce increased glucose utilization (present results), suggests that this circuitry is not developed in the immature brain. In fact, the thalamocortical afferents to the cingulate cortex begin to develop in the 1st wk after birth and are not fully developed until 3 wk of age in the rat(49). It has been suggested that higher doses might be needed for damage in the immature infant(34). The present results show that the effect of NMDAR antagonists on glucose use in the corticolimbic system is qualitatively different in the immature brain and that the difference is probably not a question of dosage. In the adult rat as little as 0.05 mg/kg and up to 5 mg/kg MK-801 induces a marked increase in glucose utilization in the posterior cingulate cortex(25), whereas 0.5 and 1.0 mg/kg MK-801 in this study caused a decreased glucose use. The latter dosage being double or triple of what is required for neuroprotection after hypoxia-ischemia in the immature rat(5). The decrease in glucose utilization induced by these drugs was temporary, and at 4 d after injection there was no longer any differences in glucose utilization between groups(Table 3).

It is not possible to extrapolate these data directly to the human situation. The neurotoxicity in human newborns will most likely depend on the maturation of the neuronal circuitry in the corticolimbic system at the actual gestational age. NMDAR antagonists produce psychotomimetic symptoms in adult humans, which seem to be a direct behavioral correlate to the increased metabolism in the rodent corticolimbic system [for review, see Olney and Farber(29), Evinson et al.(47), and Olney(48)]. Human infants, in parallel to immature rats, show a different response to NMDAR antagonism compared with adults. Almost half of adults over 30 y of age exhibit delirium or excitement, or experience visual disturbances upon awakening after ketamine anesthesia. The incidence of such adverse psychologic experiences is much lower in children(50). The present results concern acute administration of NMDAR antagonists, which is the likely situation after perinatal asphyxia. In contrast, prolonged, or repeated, administration of NMDAR antagonists very likely affects the normal neurodevelopmental maturation(51).

In conclusion, neuroprotective doses of MK-801 and magnesium reduce glucose use and do not induce expression of the neuronal stress marker HSP72 in the immature rat brain, which is compatible with the concept that NMDAR antagonists lack direct toxicity in the immature brain.