Journal of Cerebral Blood Flow and Metabolism

10:27�276 © 1990 Raven Press, Ltd., New York

Regional Neuroprotective Effects of the NMDA Receptor Antagonist MK-801 (Dizocilpine) in Hypoglycemic Brain Damage

M. P. Papagapiou and R. N. Auer Departments of Pathology and Clinical Neurosciences, Health Sciences Center, University of Calgary, Calgary, Alberta, Canada

Summary: Current evidence points to an important role of N-methyl-D-aspartate (NMDA) receptor activation in the pathogenesis of hypoglycemic neuronal death. MK801 {dizocilpine maleate, (+ )-5-methyl-10,1l-dihydro5H-di[a,d]cyclohepten-5,lO-imine} is an anticonvulsant compound also known to be a potent noncompetitive an­ tagonist at NMDA receptors, readily crossing the blood­ brain barrier after parenteral administration. Treatment of rats with dizocilpine (1.5-5.0 mg/kg) injected intrave­ nously during profound hypoglycemia (blood glucose lev­ els 1.5-2. 0 mM) at the stage of B-wave (1-4 Hz) slowing of the EEG mitigated selective neuronal necrosis in the hip­ pocampus and striatum, assessed histologically after 1week survival. The degree of neuroprotection in the stri­ atum and in the CAl pyramidal cells of the hippocampus was dose dependent. Because of concern for a possible

hypothermic mechanism of brain protection by MK-801, core temperature was closely monitored and was found not to decrease significantly. Since CBF is normal or in­ creased in hypoglycemia, a fall in brain temperature dur­ ing hypoglycemia is unlikely to play a role in the mecha­ nism of the neuroprotection seen with the drug. The find­ ings indicate that in profound hypoglycemia, intravenous administration of the NMDA antagonist dizocilpine, even after the appearance of B-wave EEG slowing, can reduce the number of necrotic neurons in several brain regions and suggest that the neuroprotective effect of MK-801 is not related to hypothermia. Key Words: Brain damage­ Excitatory amino acid-Hypoglycemia-N-Methyl-D­ aspartate antagonist-MK-801 (dizocilpine)-Neuronal necrosis.

Evidence has recently accumulated that hypogly­ cemia causes neuronal death in the brain not by simply and directly depriving neurons of glucose and energy. Rather, experimental results implicate activation of neuronal excitatory receptors (Auer et aI., 1985c) of the N-methyl-D-aspartate (NMDA) subtype (Wieloch, 1985) in mediating hypoglycemic neuronal death. One of the most potent, recently discovered (Clineschmidt et aI., 1982a) NMDA an­ tagonists is MK-801 (Wong et aI., 1986). In addition to protecting against NMDA neurotoxicity (Foster et aI., 1987; Olney et aI., 1987), MK-801 protects against hypoxic-ischemic neuronal injury when given either before (Gill et aI., 1987; McDonald et

aI., 1987, Olney et aI., 1989a) or after (Gill et aI., 1988; Park et aI., 1988; Rod and Auer, 1989) the insult. In tissue culture, cortical neurons are protected against glucose deprivation neuronal injury by MK801 (Monyer et aI., 1989). The present study was undertaken to test the efficacy in vivo of intrave­ nous administration of the potent NMDA antago­ nist dizocilpine (MK-80l) in protecting the major brain regions damaged by hypoglycemia. Because of results obtained in ischemia suggesting that MK801 may be neuroprotective partly via an induced hypothermia (Buchan and Pulsinelli, 1990), temper­ ature was closely monitored. The results demon­ strate a regional temperature-independent neuro­ protection by MK-801 in hypoglycemia in several brain regions.

Received August 1, 1989; revised September 21 , 1989; ac­ cepted September 25, 1989. Address correspondence and reprint requests to Dr. R. N. Auer at Neuropathology, Health Sciences Center, 3330 Hospital Dr. N. W. , Calgary, Alberta, Canada T2N 4Nl . Abbreviations used: BP, blood pressure; NMDA, N-methyl-o­ aspartate.

MATERIALS AND METHODS Male Wistar rats (Charles River, St. Constant, Quebec, Canada) weighing 222-466 g were fasted overnight with

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MK-801 (DIZOCILPINE) IN HYPOGLYCEMIC BRAIN DAMAGE

free access to tap water. Four groups of six rats were studied: Three treatment groups were given MK-801 {di­ zocilpine]maleate, (+ )-5-methyl-1O, ll-dihydro-5H-di­ [a,d]cyclohepten-5,IO-imine} in intravenous doses of 1.5, 2.8, and 5.0 mg/kg, injected during the B-wave (1-4 Hz) stage of EEG slowing; control rats were given intrave­ nous saline. The detailed description of the hypoglycemia model used can be found in a previous publication (Auer et al., 1984a). Briefly, rats were given 10-15 IV/kg i.p. regular insulin, and after 45 min were anesthetized in 3.0% halo­ thane, intubated, and ventilated with 1.0% halothane in a 2:1 N20/02 mixture. A tail vein and tail artery were can­ nulated, allowing suxamethonium paralysis and continu­ ous blood pressure (BP) recording, respectively. The BP was automatically recorded onto a computer disk every 6 s. Arterial blood glucose levels, pH, Pc02, and P02 were monitored. A heparinized central venous catheter was inserted via the right jugular vein for the purpose of con­ trolling the BP by withdrawal or reinfusion of the blood as required. A homeothermic heating blanket system with a rectal thermistor probe maintained the core temperature at 37-38°C. The bipolar interhemispheric EEG was digi­ tized on a computer in real time, representative 5-s strips being regularly stored. After surgery, the halothane was discontinued and electrocerebral silence was awaited. During the stage of high-amplitude slowing of the EEG to the B-range (blood glucose levels in the range of 1.5-2. 0 mM or 27-36 mg/100 ml), the rats were given intravenously either normal sa­ line or dizocilpine in the above doses. Following 30 min of electrocerebral silence, 1. 0 ml of 25% glucose was given by hand over 2 min, followed by 25% glucose infusion at a rate (0.056--0.078 mlImin) such that blood glucose levels rapidly ascended to a plateau between 5. 6 and 10.5 mM . When the rats began to awaken, they were taken off the ventilator, extubated, returned to their cages, and given oxygen and intravenous glucose for 10-12 h post surgical recovery. At 1 week of survival, transcardiac perfusion-fixation with phosphate-buffered 4% formaldehyde was done un­ der 3.0% halothane in 2:1 N20/02• The brains were re­ moved, processed in graded alcohols, embedded in par­ affin, subserially sectioned at 200-lLm intervals at a thick-

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ness of 8 ILm, and double-stained with 1% acid fuchsin and 0. 1% cresyl violet. The quantification of neuronal necro­ sis was carried out blindly by direct visual counting of acidophilic neurons under the microscope. Such neurons were known from previous work in hypoglycemia to be irreversibly damaged or necrotic (Auer et al., 1985a). Brain damage was graded according to the degree of se­ lective neuronal necrosis, as infarcts were uniformly ab­ sent. In the cerebral cortex, animals were rated according to the number of necrotic neurons per section, at the coronal leval of the subfornical organ, into the following grades of damage: 1,000 necrotic cells. In the hippocampus, where total cell counts were avail­ able from previous quantitative studies (Auer et al. , 1984a), the percentage neuronal necrosis in the dentate gyrus and CAl pyramidal cells was calculated at each level of the hippocampus along its septotemporal axis. Last, in the caudate nucleus, two laterally located 500-lLm microscopic fields, representing the most damaged area of the rat striatum in hypoglycemia (Auer et al. , 1984b), were counted and the percentage neuronal necrosis cal­ culated. For comparing the percentages of neuronal necrosis between groups, the arcsine transformation was per­ formed (Zar, 1974), to permit subsequent parametric anal­ ysis using a one-way analysis of variance with Dunnett's t test. The core temperatures measured were compared by analysis of variance. Finally, in comparing the number of damaged and undamaged hemispheres with controls, Fisher's exact probability was calculated.

RESULTS Physiologic parameters showed no significant dif­ ferences between the groups, apart from mild hy­ pertension (Table 1) in the MK-801-treated groups. This was likely due to the sympathomimetic action of MK-801 (Clineschmidt et aI., 1982b). A significant clinical difference was seen between the untreated rats and the rats treated with dizo­ cilpine. All three doses of MK-801 produced a cat-

TABLE 1. Physiologic measures during insulin-induced hypoglycemia and in recovery period following glucose administration MK-801 (mg/kg i. v. ) Control MABP (iso; mm Hg) MABP (post; mm Hg) Body temperature eC) Blood glucose (iso; mM) Blood glucose (post; mM) pH (iso) pH (post) Peo2 (iso; mm Hg) Peo2 (post; mm Hg) P02 (iso; mm Hg) P02 (post; mm Hg)

117.3 ± 5.8 108.9 ± 2.5 37.5±0.1 0.9 ± 0.0 7.6 ± 1.0 7.38 ± 0.02 7.37 ± 0.02 31.4 ± 2.2 35.8 ± 1.7 101.0± 2.3 108.0 ± 3.2

1.5 118.7 143.3 37.3 1.0 5.5 7.44 7.35 25.6 34.9 109.0 109.0

± ± ± ± ± ± ± ± ± ± ±

7.0 2.6a 0.1 0.1 0.6 0.03 0.02 1.2 1.7 9.1 4.7

2.8

5.0

106.3 ± 10.5 140.2±4.3a 37.2 ± 0.3 0.8±0.1 7.9 ± 1.1 7.46 ± 0.03 7.36±0.03 26.9 ± 1.8 30.4±1.2 114.0 ± 10.5 119.0± 5.8

117.3 ± 6.5 135.9 ± 3.6Q 37.5±0.0 1.0±0.0 9.2±1.1 7.42±0.02 7.35±0.01 30.3± 1.5 35.0±1.2 137.0± 5.7Q 131.0±5.8a

iso, taken during period of isoeIectric EEG; post, taken every 30 min for 3 h post hypoglycemia, beginning 30 min after recovery with glucose. Q p < 0.05 vs. control (one-way analysis of variance with Dunnett's t test).

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FIG. 1. Percentage (means ± SEM) neuronal necrosis in the CA1 sector of the hippocampus following injection of MK-801 (n = 6, all groups). Percentages were transformed according to the standard arcsine transformation (Zar, 1974) to allow one-way analysis of variance with Dunnett's t test. *p < 0.05; **p < 0.01.

aleptic state (Koek et al., 1988) of wakeful immo­ bility, the rats lying on their side with eyes open, unable to right (Whishaw and Auer, 1989). They did not respond to pain and could not be roused from this state. The catalepsy graduaJly subsided over the course of the first 2-3 days after the administra­ tion of MK-801 (Whishaw and Auer, 1989). Control rats were sluggish during this time period, but were otherwise normal. NeuropathologicaJly, MK-801 decreased hypo­ glycemic brain damage in the hippocampus and stri­ atum, but not the cortex. The mitigation of damage in the CAl sector was dose dependent (Fig. O. His­ tologicaJly, the reduction in neuronal necrosis in the CAl pyramidal ceJl band was striking (Fig. 2). In the dentate gyrus, all three doses of MK-801 mitigated neuronal death (Fig. 3), but the dose dependency of the protective effect seen in the CAl zone was not observed. Nevertheless, the characteristic neuronal

necrosis in the dentate granule cell band, also seen in human cases of hypoglycemia (Auer et aI., 1989), was frequently eliminated entirely (Fig. 4). In the lateral caudate nucleus, neuronal necrosis was strikingly reduced from 75% of the neuronal population to < 10% in animals treated with 5 mg/kg of MK-801 (Fig. 5). Complete histologic protection against neuronal necrosis was commonly seen at this dose of dizocilpine (Fig. 6). The cortex showed significant protection only in the group receiving 2.8 mg/kg of MK-801 (Fig. 7). No vacuolation or other changes suggestive of a histotoxic effect of MK-801 were seen in neurons of the retrosplenial or cingulate cortex (Olney et aI., 1989b). To examine the possibility that dizocilpine acts by a hypothermic mechanism, the continuously monitored core temperature was examined care­ fuJly in all four groups. All animals had a slight de­ crease in core body temperature at the onset of EEG isoelectricity (Fig. 8), but there was no corre­ lation between the dose of MK-801 and the magni­ tude of the temperature decrease. The only group in which the body temperature was significantly lower than control was the 1.5-mg/kg group (p < 0.01), the group showing the least robust neuroprotection his­ tologically. DISCUSSION The most recent significant advance in the study of brain damage due to low blood sugar has been the new understanding that hypoglycemic neuronal death is mediated by excitatory receptors (Auer et aI., 1985c; Wieloch, 1985). The subtype of excitato­ ry neuronal receptor involved is named after its most favored agonist, NMDA, evidenced by the

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FIG. 2. Hippocampal neuronal necrosis in rats subjected to 30 min of cerebral isoelectricity (flat EEG) due to profound hypo­ glycemia. Untreated rats show dense neuronal necrosis along the CA1 pyramidal cell band (A). but rats treated with 5 mg/kg i. v. MK-801 often show complete neuronal preservation (8). 0.1% cresyl violet and 1% acid fuchsin. Bars, 200 f.Lm.

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MK-801 (DIZOCILPINE) IN HYPOGLYCEMIC BRAIN DAMAGE

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FIG. 3. Percentage (means ± SEM) neuronal necrosis in the dentate gyrus of the hippocampus following injections of MK-801 (n = 6, all groups). Percentages were transformed according to the standard arcsine transformation (Zar, 1974) to allow one-way analysis of variance with Dunnett's t test. *p < 0.05; **p < 0.01.

demonstration that hypoglycemic neuronal damage can be reduced by intracerebral injection of the spe­ cific NMDA antagonist aminophosphonohep­ tanoate (Wieloch, 1985). The fact that neurons die via a receptor-mediated process, rather than by glu­ cose starvation and energy failure per se, allows therapeutic intervention using appropriate receptor antagonists. The present findings indicate that neu­ ronal necrosis in the brain may be reduced by in­ travenous administration of the centrally penetrat­ ing NMDA antagonist MK-801 at the stage of pro­ found hypoglycemia accompanied by a-wave slowing of the EEG, even if coma (isoelectric EEG, electrocerebral silence) ensues. MK-801 is currently the most potent NMDA re­ ceptor antagonist known. The compound was first

273

described in 1982 to have anticonvulsant, sympa­ thomimetic, and anxiolytic activity (Clineschmidt et aI., 1982a-c). Being a substituted dibenzocyclohep­ tenimine derivative, it readily crosses the blood­ brain barrier, raising the attractive clinical pos­ sibility of parenteral administration. A single intravenous dose of MK-801 in the rat causes neu­ robehavioral changes lasting 2-4 days (Whishaw and Auer, 1989), in spite of a plasma half-life of only 1 h (Hucker et aI., 1983; Scheller et aI., 1989). This probably relates to the very slow exit of the mole­ cule from the NMDA receptor-associated ion chan­ nel (Huettner and Bean, 1988). Reversible neuronal vacuolation has been reported several hours after MK-801 administration, probably related to tempo­ rary sublethal cellular perturbations associated with a phencyclidine agonist effect (Olney et aI., 1989b). MK-801 binds with great affinity to isolated brain fractions (Wong et aI., 1986, 1988; McKernan et aI., 1989), and a high density of binding has been shown in the hippocampus, amygdala, entorhinal cortex, and neocortex of the rodent (Bowery et aI., 1988) and human (Kornhuber et aI., 1989) brain. Physio­ logically, MK-801 effectively blocks NMDA recep­ tor-mediated processes in the hippocampus (Coan et aI., 1987), a region of high NMDA receptor den­ sity (Greenamyre et aI., 1985; Monaghan et aI., 1985). Thus, anatomic, physiologic, and pharmaco­ logic evidence indicates MK-801 is a specific and potent NMDA antagonist in the brain regions dam­ aged by hypoglycemia. These results, taken together with the finding in culture that MK-801 protects against glucose depri­ vation neuronal injury (Monyer et aI., 1989), sug-

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FIG. 4. The dentate gyrus in an untreated rat (A) and a rat treated with MK-801 5 mg/kg i.v. (8). The crest of the gyrus is visible on the right of each photomicrograph, with the medial portion of the internal and external blades in the superior and inferior portion. Necrotic neurons have pyknotic, shrunken nuclei, seen in the crest and outer blade of the untreated rat 1 week after 30 min of cerebral EEG isoelectricity due to profound hypoglycemia. The typical rat treated with 5 mg/kg i.v. MK-801 shows essentially no neuronal necrosis at a comparable septotemporal level of the hippocampus, although a few high-dose rats did show a few necrotic neurons in the dentate. 0.1% cresyl violet and 1% acid fuchsin. Bars, 125 j-Lm.

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Acknowledgment: The authors thank Ms. E. Tingle for typing the manuscript. This work was supported by the Juvenile Diabetes Foundation International.

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REFERENCES

FIG. 7. Hemispheres exhibiting the indicated numbers of ne­ crotic neurons in the control and three MK-801-treated groups (n = 6, all groups). There was a statistically signifi­ cant difference in the number of undamaged versus dam­ aged hemispheres between the control and the 2.8-mg/kg MK-801 group (Fisher's exact probability, p < 0.02).

enhancement in the ability of the brain to utilize glucose during hypoglycemia should have been manifest as a delay in the onset of EEG silence in the MK-801-treated groups. This argues against any MK-801-induced augmentation of glucose utiliza­ tion as a mechanism of neuroprotection in hypogly­ cemia. The identity of the endogenously produced brain excitotoxin causing hypoglycemic neuronal necro­ sis has not been established. Hypoglycemia causes a marked increase in levels of aspartate in whole­ brain tissue (Agardh et aI., 1981) and in the extra­ cellular space (Sandberg et aI., 1986) where neuro­ nal receptors are exposed to the high local concen­ trations. Brain extracellular glutamate levels are also increased in hypoglycemia, but to a lesser de­ gree than aspartate (Sandberg et aI., 1986; Butcher

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37.8

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Agardh C-D, Kalimo H, Olsson Y, Siesj6 BK (1981) Hypogly­ cemic brain injury: metabolic and structural findings in rat cerebellar cortex during profound insulin-induced hypogly­ cemia and in the recovery period following glucose admin­ istration. J Cereb Blood Flow Metab 1:71-84

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- CONTROL 1.5 mg/kg -- 2.8 mg/kg ..... 5.0 mg/kg

38.0

Abdul-Rahman A, Agardh C-D, Siesj6 BK (1980) Local cerebral blood flow in the rat during severe hypoglycemia and in the recovery period following glucose injection. Acta Physiol Scand 109:307-314

-120

o

-60

Time to

EEG

60

120

180

isoelectricity (min)

FIG. 8. Core body temperature as a function of time in rela­ tion to the onset of cerebral EEG silence (time 0 on abscissa). Recordings were taken at 30-min intervals from 3 h prior to isoelectricity to a maximum of 3 h of recovery. Only the tem­ perature fall in the 1.5-mg/kg group was significantly differ­ ent from control, and only at one point in time, the onset of isoelectricity. One-way analysis of variance with Dunnett's t test. *p < 0.05.

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