Krikor Dikranian

Antiepileptic drugs and apoptotic neurodegeneration in the developing brain.

Epilepsy is the most common neurological disorder of young humans. Each year 150,000 children in the United States experience their first seizure. Antiepileptic drugs (AEDs), used to treat seizures in children, infants, and pregnant women, cause cognitive impairment, microcephaly, and birth defects. The cause of unwanted effects of therapy with AEDs is unknown. Here we reveal that phenytoin, phenobarbital, diazepam, clonazepam, vigabatrin, and valproate cause apoptotic neurodegeneration in the developing rat brain at plasma concentrations relevant for seizure control in humans. Neuronal death is associated with reduced expression of neurotrophins and decreased concentrations of survival-promoting proteins in the brain. β-Estradiol, which stimulates pathways that are activated by neurotrophins, ameliorates AED-induced apoptotic neurodegeneration. Our findings present one possible mechanism to explain cognitive impairment and reduced brain mass associated with prenatal or postnatal exposure of humans to antiepileptic therapy.

Diffusion Tensor Imaging Reliably Detects Experimental Traumatic Axonal Injury and Indicates Approximate Time of Injury

Traumatic axonal injury (TAI) may contribute greatly to neurological impairments after traumatic brain injury, but it is difficult to assess with conventional imaging. We quantitatively compared diffusion tensor imaging (DTI) signal abnormalities with histological and electron microscopic characteristics of pericontusional TAI in a mouse model. Two DTI parameters, relative anisotropy and axial diffusivity, were significantly reduced 6 h to 4 d after trauma, corresponding to relatively isolated axonal injury. One to 4 weeks after trauma, relative anisotropy remained decreased, whereas axial diffusivity “pseudo-normalized” and radial diffusivity increased. These changes corresponded to demyelination, edema, and persistent axonal injury. At every time point, DTI was more sensitive to injury than conventional magnetic resonance imaging, and relative anisotropy distinguished injured from control mice with no overlap between groups. Remarkably, DTI changes strongly predicted the approximate time since trauma. These results provide an important validation of DTI for pericontusional TAI and suggest novel clinical and forensic applications.

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.

Detection of traumatic axonal injury with diffusion tensor imaging in a mouse model of traumatic brain injury

Traumatic axonal injury (TAI) is thought to be a major contributor to cognitive dysfunction following traumatic brain injury (TBI), however TAI is difficult to diagnose or characterize non-invasively. Diffusion tensor imaging (DTI) has shown promise in detecting TAI, but direct comparison to histologically-confirmed axonal injury has not been performed. In the current study, mice were imaged with DTI, subjected to a moderate cortical controlled impact injury, and re-imaged 4-6 h and 24 h post-injury. Axonal injury was detected by amyloid beta precursor protein (APP) and neurofilament immunohistochemistry in pericontusional white matter tracts. The severity of axonal injury was quantified using stereological methods from APP stained histological sections. Two DTI parameters--axial diffusivity and relative anisotropy--were significantly reduced in the injured, pericontusional corpus callosum and external capsule, while no significant changes were seen with conventional MRI in these regions. The contusion was easily detectable on all MRI sequences. Significant correlations were found between changes in relative anisotropy and the density of APP stained axons across mice and across subregions spanning the spatial gradient of injury. The predictive value of DTI was tested using a region with DTI changes (hippocampal commissure) and a region without DTI changes (anterior commissure). Consistent with DTI predictions, there was histological detection of axonal injury in the hippocampal commissure and none in the anterior commissure. These results demonstrate that DTI is able to detect axonal injury, and support the hypothesis that DTI may be more sensitive than conventional imaging methods for this purpose.

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.

Propofol-induced apoptosis of neurones and oligodendrocytes in fetal and neonatal rhesus macaque brain

BACKGROUND Exposure of the fetal or neonatal non-human primate (NHP) brain to isoflurane or ketamine for 5 h causes widespread apoptotic degeneration of neurones, and exposure to isoflurane also causes apoptotic degeneration of oligodendrocytes (OLs). The present study explored the apoptogenic potential of propofol in the fetal and neonatal NHP brain. METHOD Fetal rhesus macaques at gestational age 120 days were exposed in utero, or postnatal day 6 rhesus neonates were exposed directly for 5 h to propofol anaesthesia (n=4 fetuses; and n=4 neonates) or to no anaesthesia (n=4 fetuses; n=5 neonates), and the brains were systematically evaluated 3 h later for evidence of apoptotic degeneration of neurones or glia. RESULTS Exposure of fetal or neonatal NHP brain to propofol caused a significant increase in apoptosis of neurones, and of OLs at a stage when OLs were just beginning to myelinate axons. Apoptotic degeneration affected similar brain regions but to a lesser extent than we previously described after isoflurane. The number of OLs affected by propofol was approximately equal to the number of neurones affected at both developmental ages. In the fetus, neuroapoptosis affected particularly subcortical and caudal regions, while in the neonate injury involved neocortical regions in a distinct laminar pattern and caudal brain regions were less affected. CONCLUSIONS Propofol anaesthesia for 5 h caused death of neurones and OLs in both the fetal and neonatal NHP brain. OLs become vulnerable to the apoptogenic action of propofol when they are beginning to achieve myelination competence.

Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration.