Tatyana Tenkova

Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain

Recently, it was reported that anesthetizing infant rats for 6 h with a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) caused widespread apoptotic neurodegeneration in the developing brain, followed by lifelong cognitive deficits. It has also been reported that ketamine triggers neuroapoptosis in the infant rat brain if administered repeatedly over a period of 9 h. The question arises whether less extreme exposure to anesthetic drugs can also trigger neuroapoptosis in the developing brain. To address this question we administered ketamine, midazolam or ketamine plus midazolam subcutaneously at various doses to infant mice and evaluated the rate of neuroapoptosis in various brain regions following either saline or these various drug treatments. Each drug was administered as a single one‐time injection in a dose range that would be considered subanesthetic, and the brains were evaluated by unbiased stereology methods 5 h following drug treatment. Neuroapoptosis was detected by immunohistochemical staining for activated caspase‐3. It was found that either ketamine or midazolam caused a dose‐dependent, statistically significant increase in the rate of neuroapoptosis, and the two drugs combined caused a greater increase than either drug alone. The apoptotic nature of the neurodegenerative reaction was confirmed by electron microscopy. We conclude that relatively mild exposure to ketamine, midazolam or a combination of these drugs can trigger apoptotic neurodegeneration in the developing mouse brain.

Neurotransmitters and apoptosis in the developing brain

In the immature mammalian brain during a period of rapid growth (brain growth spurt/synaptogenesis period), neuronal apoptosis can be triggered by the transient blockade of glutamate N-methyl-d-aspartate (NMDA) receptors, or the excessive activation of gamma-aminobutyric acid (GABA(A)) receptors. Apoptogenic agents include anesthetics (ketamine, nitrous oxide, isoflurane, propofol, halothane), anticonvulsants (benzodiazepines, barbiturates), and drugs of abuse (phencyclidine, ketamine, ethanol). In humans, the brain growth spurt period starts in the sixth month of pregnancy and extends to the third year after birth. Ethanol, which has both NMDA antagonist and GABA(A) agonist properties, is particularly effective in triggering widespread apoptotic neurodegeneration during this vulnerable period. Thus, maternal ingestion of ethanol during the third trimester of pregnancy can readily explain the dysmorphogenic changes in the fetal brain and consequent neurobehavioral disturbances that characterize the human fetal alcohol syndrome. In addition, there is basis for concern that agents used in pediatric and obstetrical medicine for purposes of sedation, anesthesia, and seizure management may cause apoptotic neuronal death in the developing human brain.

Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain.

Recent studies have shown that administration of ethanol to infant rats during the synaptogenesis period (first 2 weeks after birth), triggers extensive apoptotic neurodegeneration throughout many regions of the developing brain. While synaptogenesis is largely a postnatal phenomenon in rats, it occurs prenatally (last trimester of pregnancy) in humans. Recent evidence strongly supports the interpretation that ethanol exerts its apoptogenic action by a dual mechanism--blockade of NMDA glutamate receptors and hyperactivation of GABA(A) receptors. These findings in immature rats represent a significant advance in the fetal alcohol research field, in that previous in vivo animal studies had not demonstrated an apoptogenic action of ethanol, had not documented ethanol-induced cell loss from more than a very few brain regions and had not provided penetrating insight into the mechanisms underlying ethanols neurotoxic action. To add to the mechanistic insights recently gained, it would be desirable to examine gene-regulated aspects of ethanol-induced apoptotic neurodegeneration, using genetically altered strains of mice. The feasibility of such research must first be established by demonstrating that appropriate mouse strains are sensitive to this neurotoxic mechanism. In the present study, we demonstrate that mice of the C57BL/6 strain, a strain frequently used in transgenic and gene deletion research, are exquisitely sensitive to the mechanism by which ethanol induces apoptotic neurodegeneration during the synaptogenesis period of development.

Apoptotic neurodegeneration induced by ethanol in neonatal mice is associated with profound learning/memory deficits in juveniles followed by progressive functional recovery in adults.

Administration of ethanol to rodents during the synaptogenesis period induces extensive apoptotic neurodegeneration in the developing brain. This neurotoxicity may explain the reduced brain mass and neurobehavioral disturbances in human Fetal Alcohol Syndrome (FAS). Here, we report binge-like exposure of infant mice to ethanol on a single postnatal day triggered apoptotic death of neurons from diencephalic structures that comprise an extended hippocampal circuit important for spatial learning and memory. The ethanol exposure paradigm yielding these neuronal losses caused profound impairments in spatial learning and memory at 1 month of age. This impairment was significantly attenuated during subsequent development, indicating recovery of function. Recovery was not associated with increased neurogenesis, suggesting plastic reorganization of neuronal networks compensated for early neuronal losses. We hypothesize that neuroapoptotic damage in homologous regions of human brain underlies cognitive deficits in FAS and the human brain of FAS victims has a similar capacity to effect functional recovery.

Ethanol-induced caspase-3 activation in the in vivo developing mouse brain.

Recently several methods have been described for triggering extensive apoptotic neurodegeneration in the developing in vivo mammalian brain. These methods include treatment with drugs that block NMDA glutamate receptors, drugs that promote GABA(A) neurotransmission, or treatment with ethanol, which has both NMDA antagonist and GABAmimetic properties. A single intoxication episode induced by any of these agents is sufficient to cause widespread neurodegeneration throughout many brain regions. The cell death process transpires rapidly from early to late stages within several hours. As the neurons die, they become TUNEL positive and show, by both light and electron microscopy, all of the classical morphological characteristics of apoptosis. In the present study, using immunocytochemical methods, we document that ethanol intoxication of 7-day-old infant mice causes a widespread pattern of caspase-3 activation corresponding to the pattern of apoptotic neurodegeneration that is occurring simultaneously.

Distinguishing excitotoxic from apoptotic neurodegeneration in the developing rat brain.

Much confusion has arisen recently over the question of whether excitotoxic neuronal degeneration can be considered an apoptotic phenomenon. Here, we addressed this question by using ultrastructural methods and DNA fragmentation analysis to compare a prototypic apoptotic in vivo central nervous system cell death process (physiologic cell death in the developing rat brain) with several central nervous system cell death processes in the in vivo infant rat brain that are generally considered excitotoxic (degeneration of hypothalamic neurons after subcutaneous administration of glutamate and acute neurodegeneration induced by hypoxia/ischemia or by concussive head trauma). We found by ultrastructural analysis that glutamate induces neurodegenerative changes in the hypothalamus that are identical to acute changes induced in the infant rat brain by either hypoxia/ischemia or head trauma, and that these changes are fundamentally different both in type and sequence from those associated with physiologic cell death (apoptosis). In addition, we show by ultrastructural analysis that concussive head trauma induces both excitotoxic and apoptotic neurodegeneration, the excitotoxic degeneration being very acute and localized to the impact site, and the apoptotic degeneration being delayed and occurring in regions distant from the impact site. Thus, in the head trauma model, excitotoxic and apoptotic degeneration can be distinguished not only by ultrastructural criteria but by their temporal and spatial patterns of expression. Whereas ultrastructural analysis provided an unambiguous means of distinguishing between excitotoxic and apoptotic neurodegeneration in each example analysed in this study, DNA fragmentation analysis (TUNEL staining or gel electrophoresis) was of no value because these tests were positive for both processes. J. Comp. Neurol. 408:461–476, 1999.

Apoptosis in the in vivo mammalian forebrain.

Apoptosis is a word originally introduced by Kerr, Wyllie, and colleagues for a cell death process they defined in terms of its ultrastructural appearance in nonneuronal cells from various tissues. There are very few studies providing detailed ultrastructural criteria for recognizing neuronal apoptosis in the in vivo mammalian brain. In the absence of such criteria, the Kerr/Wyllie description pertaining to nonneuronal cells has served as a reference standard. However, contemporary neurobiologists typically rely on cell culture models for studying neuronal apoptosis, and these models are rarely validated ultrastructurally; rather they are assumed to be appropriate models based on unvalidated biochemical tests for apoptosis. Relying on evidence generated in such cell culture models or on nonspecific cytochemical tests applied to brain tissue, many authors have recently suggested that an apoptotic mechanism may mediate neuronal death in a wide variety of human neurodegenerative diseases. Whether the cell death process in neurodegenerative diseases meets ultrastructural criteria for apoptosis has been given very little consideration. Recently, several methods have been described for triggering extensive apoptotic neurodegeneration in the developing in vivo mammalian brain. These methods include head trauma or treatment with several types of drugs (NMDA antagonists, GABAA agonists, or ethanol). We have performed an ultrastructural analysis of the neuronal cell death process triggered in the cerebral cortex and thalamus by these several methods and compared it with physiological cell death (PCD), a prototypic example of neuronal apoptosis that occurs naturally in the developing brain. Our findings, which are reviewed herein, demonstrate that the types and sequence of changes induced by each of the above methods are identical to those that characterize PCD. This confirms that each of these methods produces bona fide in vivo apoptotic neurodegeneration, and it signifies that our description of this neuronal apoptotic process, which differs in some respects from the Kerr/Wyllie description of nonneuronal apoptosis, can serve as a useful reference standard for recognizing the characteristic changes that in vivo neurons undergo when they are dying by an apoptotic mechanism.

Disseminated Corticolimbic Neuronal Degeneration Induced in Rat Brain by MK-801: Potential Relevance to Alzheimer's Disease☆

Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors by MK-801 induces neuronal degeneration in the posterior cingulate/retrosplenial cortex and other corticolimbic regions although damage in the latter has not been adequately characterized. This disseminated corticolimbic damage is of interest since NMDA hypofunction, the mechanism that triggers this neurodegenerative syndrome, has been postulated to play a role in the pathophysiology of Alzheimers disease (AD). Several histological methods, including electron microscopy, were used to evaluate the neurotoxic changes in various corticolimbic regions of rat brain following MK-801 or a combination of MK-801 plus pilocarpine. We found that MK-801 triggers neuronal degeneration in a widespread pattern similar to that induced by phencyclidine and that females showed more damage than males. The neurotoxic reaction involved additional brain regions when muscarinic receptors were hyperactivated by administering pilocarpine with MK-801. Ultrastructural evaluation revealed that a major feature of the neurotoxic action involves degeneration of dendritic spines which entails loss of synaptic complexes. The ultrastructural appearance of degenerating neurons was generally inconsistent with an apoptotic mechanism, although evidence equivocally consistent with apoptosis was observed in some instances. The cell death process evolved relatively slowly and was still ongoing 7 days posttreatment. Relevance of these results to AD is discussed.

Excitotoxic Versus Apoptotic Mechanisms of Neuronal Cell Death in Perinatal Hypoxia / Ischemia

Hypoxic/ischemic (H/I) neuronal degeneration in the developing central nervous system (CNS) is mediated by an excitotoxic mechanism, and it has also been reported that an apoptosis mechanism is involved. However, there is much disagreement regarding how excitotoxic and apoptotic cell death processes relate to one another. Some authors believe that an excitotoxic stimulus directly triggers apoptotic cell death, but this interpretation is largely speculative at the present time. Our findings support the interpretation that excitotoxic and apoptotic neurodegeneration are two separate and distinct cell death processes that can be distinguished from one another by ultrastructural evaluation. Here we review evidence supporting this interpretation, including evidence that H/I in the developing CNS triggers two separate waves of neurodegeneration, the first being excitotoxic and the second being apoptotic. The first (excitotoxic) wave destroys neurons that would normally provide synaptic inputs or synaptic targets for the neurons that die in the second (apoptotic) wave. Since neurons, during the developmental period of synaptogenesis, are programmed to commit suicide if they fail to achieve normal connectivity, this explains why neuroapoptosis occurs following H/I in the developing CNS. However, it does not support the interpretation that H/I directly triggers apoptotic neurodegeneration. Rather, it documents that H/I directly triggers excitotoxic neurodegeneration, and apoptotic neurodegeneration ensues subsequently as the natural response of developing neurons to a specific kind of deprivation - loss of the ability to form normal synaptic connections.

A Modified Silver Technique (de Olmos Stain) for Assessment of Neuronal and Axonal Degeneration

Silver impregnation histological techniques yield excellent visualization of degenerating neurons and their processes in animal models of neurological diseases. These methods also provide a particularly valuable complement to current immunocytochemical techniques for recognition of axon injury in the setting of brain or spinal cord trauma, ischemia, or neurodegenerative diseases. Despite their utility, silver methods are not commonly used because of complex preparation requirements and inconsistent results obtained by inexperienced histologists. This chapter details a modification of the de Olmos amino-cupric-silver protocol, which has been adapted for efficient processing of large numbers of mouse or rat brains. One author (T.I.T.) has used this method for several years to identify degenerating neurons in adult and neonatal rodent brains. A detailed protocol is provided, with attention to the most critical variables in tissue fixation and solution preparation. Examples are shown of axon injury in the rat brain after focal ischemia.