(Stroke. 1996;27:743-746.)
© 1996 American Heart Association, Inc.
Articles |
Effect of Age and Sex on N-Methyl-D-Aspartate Antagonist-Induced Neuronal Necrosis in Rats
From the Department of Pathology, University of Calgary (Alberta, Canada).
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Abstract |
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Background and Purpose Although N-methyl-D-aspartate (NMDA) antagonism may be a useful therapeutic approach in stroke treatment, it has been found that these pharmacological agents cause neuronal necrosis in restricted cortical regions of the rodent brain.
Methods To test the hypothesis that age and sex influence NMDA antagonist-induced neuronal necrosis, male and female rats were studied at 2 months (young), 12 months (middle-aged), and 24 months (old) of age. A dose of 5 mg/kg MK-801 was administered, followed by quantitation of neuronal necrosis at nine coronal levels in the cingulate and retrosplenial cortex at 1 week of survival.
Results Mortality was dependent on age but not sex and was higher in the old rats (P<.01). The number of necrotic neurons per hemisphere was greater in female than in male rats at all ages (P<.0001). Female rats also showed increasing neuronal necrosis with age (P<.05).
Conclusions The results indicate a major sex difference in neuronal cytotoxicity caused by NMDA antagonists and a minor increase in susceptibility with increasing age in females. The findings may be relevant to development of drugs with NMDA antagonist properties for use in human stroke.
Key Words: aging • gender • neuronal death • N-methyl-D-aspartate • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although potentially useful in the treatment of stroke,1 N-methyl-D-aspartate (NMDA) antagonists nevertheless have some neurotoxicity of their own.2 3 4 Morphologically, this neuronal toxicity is apparent as early microvacuolation of neurons in restricted areas of the rodent brain, ie, the cingulate and retrosplenial cortex.4 5 The vacuolation, seen only in aldehyde-fixed tissue,5 thus signifies a neuron that is not lethally injured but rather is "at risk." In a proportion of neurons, the vacuolation is the harbinger of neuronal necrosis.6 7
In experimental and clinical studies of stroke treatment, including treatment with NMDA antagonists, individuals of either sex and varying age might be treated. Often, stroke models comprise experimental therapeutic trials in young animals, far below the age at which clinical stroke is commonly seen. Likewise, pharmacological testing in young animals may be inappropriate with respect to neurological and neuropathologic evaluation of compounds with NMDA antagonist properties. Since molecules with NMDA antagonist properties are being developed for clinical use in stroke and other clinical conditions, it was deemed worthwhile to examine the effect of age and sex on quantitated neuropathologic necrosis with the potent, prototype noncompetitive NMDA antagonist MK-801. The results demonstrate the importance of age and sex not only with respect to studies of efficacy but also toxicity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 75 Wistar and Sprague-Dawley rats were used in the present study. The experiments complied with institutional guidelines for animal research. To examine potential sex differences in drug susceptibility, male and female rats were studied separately. No strain differences were found in subsequent separate analyses by strain (see "Results"), and therefore rats of either strain were analyzed together. To study the effect of age in male rats, three age groups were examined: 2 months (young; mean weight, 330 g; range, 321 to 345 g), 12 months (middle-aged; mean weight, 481 g; range, 382 to 620 g), and 24 months (old; mean weight, 738 g; range, 533 to 933 g). Likewise, in female rats three age groups were studied: 2 months (young; mean weight, 275 g; range, 262 to 284 g), 12 months (middle-aged; mean weight, 481 g; range, 382 to 620 g), and 24 months (old; mean weight, 623 g; range, 443 to 760 g). There were 10 young females and 10 young males, 20 middle-aged females and 10 middle-aged males, and 13 old females and 12 old males.
Since side effects were being studied, all animals were administered the relatively high dose of 5 mg/kg MK-801 IP. They were observed clinically, and mortality was recorded. Only surviving rats were included for histological analysis, done after perfusion fixation at 1 week of survival. The perfusion was performed on intubated, ventilated rats under 3% halothane anesthesia. The thorax was opened, and a cannula dripping normal saline was inserted into the left ventricle. The descending aorta was clamped, and the cannula was advanced into the ascending aorta. After a 20-second rinse of the cerebral circulation with saline, the perfusate was changed to fixative, consisting of a phosphate-buffered solution of 4% formaldehyde, at pH 7.32 and osmolarity of 287 mOsm. The brains were left in situ for several hours, removed, and placed in fixative. The next day they were cut into 3-mm-thick slices.
After histological processing, the brains were
sectioned from bregma -1.8 to bregma -7.3 mm.8
The 8-µm paraffin sections were doubled stained with hematoxylin and
eosin, and necrotic neurons were quantitated by direct visual counting
at high magnification (x400), sweeping across successive fields until
the entire cortex was counted, with the use of a light microscope.
Neuronophagic figures where the acidophilic perikaryon could be clearly
visualized, indicating cell body phagocytosis, were counted.
Acidophilic neurons are known to be necrotic from previous studies by
their electron microscopic appearance and their disappearance from
neural tissue in longitudinal temporal studies.9 10 Also
counted were figures of dendritic phagocytosis, where the microglial
rod cells were seen perpendicular to the pial surface (Fig 1
).
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The total number of necrotic neurons was obtained for the left and
right hemispheres at nine coronal levels between bregma -1.8 and
bregma -7.3. Almost all coronal levels were obtained
histologically in each rat, for a total of 714 sections
(1441 hemispheres) examined. The average number of necrotic neurons per
hemisphere was then determined for each rat. Intergroup differences
were analyzed with one-way ANOVA followed by
Scheffé's post hoc test. Mortality was compared with the
2 test. Significance was accepted at the
P<.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Results showed no differences between Wistar and Sprague-Dawley rats on separate analysis for each strain, and therefore all results are presented together. All animals remained in a cataleptic state for 2 to 3 days after administration of MK-801.11 12 Older animals tolerated the drug poorly compared with young animals. Mortality was 76% (75% in males, 76% in females; P=NS) in older animals, 43% in middle-aged animals (30% in males, 50% in females; P=NS), and 0% in young animals (P<.01). Animals died in a state of extreme immobility, with shallow breathing. There were no seizures. Two old females receiving the drug appeared cortically blind on observation and examination: they ran into the cage walls, did not react to confrontational threat, and did not anticipate the edge of the table top as they crawled toward it, often simply falling off the table.
Necrotic neurons were observed in cortical tissue without evidence of
injury to the neuropil or other cell types (Fig 1
). Females showed more
necrotic neurons than males (P<.0001) at all ages (Fig 2). In addition, females were more susceptible to
neuronal necrosis as they aged (P<.05). No such trend was
seen in males. The intracerebral distribution of
cortical neuronal necrosis along the anteroposterior axis is given in
Fig 3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neuronal necrosis due to NMDA antagonism is a recently described phenomenon.4 7 The early morphological changes involve vacuolation of neurons in the cingulate and retrosplenial cortex.4 A number of compounds resembling phencyclidine are capable of inducing this change, and it is preventable by anticholinergics, diazepam, and barbiturates.4 The early vacuolating change is seen only in aldehyde fixed material and is unaccompanied by changes in the intracellular redistribution of calcium.5
Focal brain hypermetabolism underlies the necrosis,13 14 15 16 and although it may seem paradoxical that an antagonist causes hypermetabolism, this may be related to disinhibition of cholinergic systems in the Papez circuit by NMDA blockade.2 17 The pattern of metabolic alteration caused by competitive antagonists is different from that caused by noncompetitive NMDA antagonists in its distribution, and fewer brain regions show hypermetabolism.15 The presence of cerebral necrosis accompanying hypermetabolism in the face of unimpaired blood flow is not without precedent in the rodent brain18 19 and may relate to neuronal rundown of energy stores and ion gradients in affected brain regions.
The biological basis for such a profound sex difference in a drug acting on the brain remains a mystery. A hormonal basis in the periphery or drug elimination rates seem worthy of consideration.20 However, the disappearance of sex differences in rat liver drug metabolism with aging21 argues against differences in drug elimination as the explanation for the present results. Sex differences have been reported with respect to ischemic vulnerability to neuronal death, with females being less sensitive.22 23 Females have been reported to be more sensitive than males to metabolic encephalopathy,24 hypothalamic glutamate neurotoxicity,25 and many classes of drugs.26 27 28 29 30 Behaviorally, females are more sensitive than males to NMDA antagonists such as MK-801,31 32 33 phencyclidine,20 and ketamine.34 Accordingly, the possibility that differences in brain synapses or circuitry between males and females could provide a novel explanation of sex differences must be entertained. The specific finding of increased neuronal NMDA sites35 and NMDA response36 with estradiol treatment provides a potential mechanism for enhanced sensitivity to NMDA effects in females.
Alternatively, the sex differences seen in this form of drug-induced hypermetabolic neuronal necrosis may simply be related to the decreased size of the female cortex compared with the male,37 with a corresponding higher rate of cerebral metabolism due to higher neuronal density in females.38 However, specific examination of the mesial occipital cortex of rats for neuronal39 and synaptic density40 has failed to show increased synaptic or neuronal density in females. It would be worthwhile to examine whether the hypermetabolism underlying NMDA antagonist-induced neuronal necrosis is equally present in males and females and whether any differences are abolished by hormones. In view of the enhanced NMDA receptor function with aging,41 it would also be worthwhile to determine whether increases in metabolism due to NMDA blockade are uniform throughout adult life. Such experiments might shed further light on differences in circuitry and metabolism activated by drugs in males and females and in young and old and might be of basic neurobiological interest, as well as having clinical import.
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Acknowledgments |
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This study was supported by the Medical Research Council of Canada (MT-9935). The author wishes to thank Yen Tang for excellent technical assistance.
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Footnotes |
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Reprint requests to Roland N. Auer, MD, PhD, FRCPC, Department of Pathology, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta, Canada T2N 4N1. E-mail rauer@acs.ucalgary.ca.
Received November 2, 1995; revision received December 5, 1995; accepted January 10, 1996.
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