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Many routes may lead to the transition from a healthy to a diseased phenotype. However, there are not so many routes to travel in the opposite direction; that is, therapy for different diseases. The following pressing question thus remains: what are the pathogenic routes and how can be they counteracted for therapeutic purposes? Human cells contain >500 protein kinases and nearly 200 protein phosphatases, acting on thousands of proteins, including cell growth factors. We herein discuss neurotrophins with pathogenic or metabotrophic abilities, particularly brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), pro-NGF, neurotrophin-3 (NT-3), and their receptor Trk (tyrosine receptor kinase; pronounced "track"). Indeed, we introduced the word trackins , standing for Trk-targeting drugs, that play an agonistic or antagonistic role in the function of TrkB BDNF , TrkC NT-3 , TrkA NGF , and TrkA pro-NGF receptors. Based on our own published results, supported by those of other authors, we aim to update and enlarge our trackins concept , focusing on (1) agonistic trackins as possible drugs for (1a) neurotrophin-deficiency cardiometabolic disorders (hypertension, atherosclerosis, type 2 diabetes mellitus, metabolic syndrome, obesity, diabetic erectile dysfunction and atrial fibrillation) and (1b) neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, and multiple sclerosis), and (2) antagonistic trackins, particularly TrkA NGF inhibitors for prostate and breast cancer, pain, and arrhythmogenic right-ventricular dysplasia. Altogether, the druggability of TrkA NGF , TrkA pro-NGF , TrkB BDNF , and TrkC NT-3 receptors via trackins requires a further translational pursuit. This could provide rewards for our patients.

The arachnoid barrier delineates the border between the central nervous system and dura mater. Although the arachnoid barrier creates a partition, communication between the central nervous system and the dura mater is crucial for waste clearance and immune surveillance 1,2 . How the arachnoid barrier balances separation and communication is poorly understood. Here, using transcriptomic data, we developed transgenic mice to examine specific anatomical structures that function as routes across the arachnoid barrier. Bridging veins create discontinuities where they cross the arachnoid barrier, forming structures that we termed arachnoid cuff exit (ACE) points. The openings that ACE points create allow the exchange of fluids and molecules between the subarachnoid space and the dura, enabling the drainage of cerebrospinal fluid and limited entry of molecules from the dura to the subarachnoid space. In healthy human volunteers, magnetic resonance imaging tracers transit along bridging veins in a similar manner to access the subarachnoid space. Notably, in neuroinflammatory conditions such as experimental autoimmune encephalomyelitis, ACE points also enable cellular trafficking, representing a route for immune cells to directly enter the subarachnoid space from the dura mater. Collectively, our results indicate that ACE points are a critical part of the anatomy of neuroimmune communication in both mice and humans that link the central nervous system with the dura and its immunological diversity and waste clearance systems., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies., (© 2021 Mikhailik et al.)

Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.

Fifteen years ago Olney and colleagues began using animal models to evaluate the effects of anesthetic and sedative agents (ASAs) on neurodevelopment. The results from ongoing studies indicate that, under certain conditions, exposure to these drugs during development induces an acute elevated apoptotic neurodegenerative response in the brain and long-term functional impairments. These animal models have played a significant role in bringing attention to the possible adverse effects of exposing the developing brain to ASAs when few concerns had been raised previously in the medical community. The apoptotic degenerative response resulting from neonatal exposure to ASAs has been replicated in many studies in both rodents and non-human primates, suggesting that a similar effect may occur in humans. In both rodents and non-human primates, significantly increased levels of apoptotic degeneration are often associated with functional impairments later in life. However, behavioral deficits following developmental ASA exposure have not been consistently reported even when significantly elevated levels of apoptotic degeneration have been documented in animal models. In the present work, we review this literature and propose a rodent model for assessing potential functional deficits following neonatal ASA exposure with special reference to experimental design and procedural issues. Our intent is to improve test sensitivity and replicability for detecting subtle behavioral effects, and thus enhance the translational significance of ASA models., (Copyright © 2018 Elsevier Inc. All rights reserved.)

When axonal fibres approach or leave the cortex, their trajectories tend to closely follow the cortical convolutions. To quantify this tendency, we propose a three-dimensional coordinate system based on the gyral geometry. For every voxel in the brain, we define a "radial" axis orthogonal to nearby surfaces, a "sulcal" axis along the sulcal depth gradient that preferentially points from deep white matter to the gyral crown, and a "gyral" axis aligned with the long axis of the gyrus. When compared with high-resolution, in-vivo diffusion MRI data from the Human Connectome Project, we find that in superficial white matter the apparent diffusion coefficient (at b = 1000) along the sulcal axis is on average 16% larger than along the gyral axis and twice as large as along the radial axis. This is reflected in the vast majority of observed fibre orientations lying close to the tangential plane (median angular offset < 7°), with the dominant fibre orientation typically aligning with the sulcal axis. In cortical grey matter, fibre orientations transition to a predominantly radial orientation. We quantify the width and location of this transition and find strong reproducibility in test-retest data, but also a clear dependence on the resolution of the diffusion data. The ratio of radial to tangential diffusion is fairly constant throughout most of the cortex, except for a decrease of the diffusivitiy ratio in the sulcal fundi and the primary somatosensory cortex (Brodmann area 3) and an increase in the primary motor cortex (Brodmann area 4). Although only constrained by cortical folds, the proposed gyral coordinate system provides a simple and intuitive representation of white and grey matter fibre orientations near the cortex, and may be useful for future studies of white matter development and organisation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Diffusion MRI allows us to make inferences on the structural organisation of the brain by mapping water diffusion to white matter microstructure. However, such a mapping is generally ill-defined; for instance, diffusion measurements are antipodally symmetric (diffusion along x and -x are equal), whereas the distribution of fibre orientations within a voxel is generally not symmetric. Therefore, different sub-voxel patterns such as crossing, fanning, or sharp bending, cannot be distinguished by fitting a voxel-wise model to the signal. However, asymmetric fibre patterns can potentially be distinguished once spatial information from neighbouring voxels is taken into account. We propose a neighbourhood-constrained spherical deconvolution approach that is capable of inferring asymmetric fibre orientation distributions (A-fods). Importantly, we further design and implement a tractography algorithm that utilises the estimated A-fods, since the commonly used streamline tractography paradigm cannot directly take advantage of the new information. We assess performance using ultra-high resolution histology data where we can compare true orientation distributions against sub-voxel fibre patterns estimated from down-sampled data. Finally, we explore the benefits of A-fods-based tractography using in vivo data by evaluating agreement of tractography predictions with connectivity estimates made using different in-vivo modalities. The proposed approach can reliably estimate complex fibre patterns such as sharp bending and fanning, which voxel-wise approaches cannot estimate. Moreover, histology-based and in-vivo results show that the new framework allows more accurate tractography and reconstruction of maps quantifying (symmetric and asymmetric) fibre complexity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research., Competing Interests: The authors declare no competing financial interests.

During human heart failure, the balance of cardiac energy use switches from predominantly fatty acids (FAs) to glucose. We hypothesized that this substrate shift was the result of mitochondrial degeneration; therefore, we examined mitochondrial oxidation and ultrastructure in the failing human heart by using respirometry, transmission electron microscopy, and gene expression studies of demographically matched donor and failing human heart left ventricular (LV) tissues. Surprisingly, respiratory capacities for failing LV isolated mitochondria (n = 9) were not significantly diminished compared with donor LV isolated mitochondria (n = 7) for glycolysis (pyruvate + malate)- or FA (palmitoylcarnitine)-derived substrates, and mitochondrial densities, assessed via citrate synthase activity, were consistent between groups. Transmission electron microscopy images also showed no ultrastructural remodeling for failing vs. donor mitochondria; however, the fraction of lipid droplets (LDs) in direct contact with a mitochondrion was reduced, and the average distance between an LD and its nearest neighboring mitochondrion was increased. Analysis of FA processing gene expression between donor and failing LVs revealed 0.64-fold reduced transcript levels for the mitochondrial-LD tether, perilipin 5, in the failing myocardium (P = 0.003). Thus, reduced FA use in heart failure may result from improper delivery, potentially via decreased perilipin 5 expression and mitochondrial-LD tethering, and not from intrinsic mitochondrial dysfunction.-Holzem, K. M., Vinnakota, K. C., Ravikumar, V. K., Madden, E. J., Ewald, G. A., Dikranian, K., Beard, D. A., Efimov, I. R. Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts., (© FASEB.)

The great majority of acute brain injury results from trauma or from disorders of the cerebrovasculature, i.e. ischaemic stroke or haemorrhage. These injuries are characterized by an initial insult that triggers a cascade of injurious cellular processes. The nature of these processes in spontaneous intracranial haemorrhage is poorly understood. Subarachnoid haemorrhage, a particularly deadly form of intracranial haemorrhage, shares key pathophysiological features with traumatic brain injury including exposure to a sudden pressure pulse. Here we provide evidence that axonal injury, a signature characteristic of traumatic brain injury, is also a prominent feature of experimental subarachnoid haemorrhage. Using histological markers of membrane disruption and cytoskeletal injury validated in analyses of traumatic brain injury, we show that axonal injury also occurs following subarachnoid haemorrhage in an animal model. Consistent with the higher prevalence of global as opposed to focal deficits after subarachnoid haemorrhage and traumatic brain injury in humans, axonal injury in this model is observed in a multifocal pattern not limited to the immediate vicinity of the ruptured artery. Ultrastructural analysis further reveals characteristic axonal membrane and cytoskeletal changes similar to those associated with traumatic axonal injury. Diffusion tensor imaging, a translational imaging technique previously validated in traumatic axonal injury, from these same specimens demonstrates decrements in anisotropy that correlate with histological axonal injury and functional outcomes. These radiological indicators identify a fibre orientation-dependent gradient of axonal injury consistent with a barotraumatic mechanism. Although traumatic and haemorrhagic acute brain injury are generally considered separately, these data suggest that a signature pathology of traumatic brain injury-axonal injury-is also a functionally significant feature of subarachnoid haemorrhage, raising the prospect of common diagnostic, prognostic, and therapeutic approaches to these conditions., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

Alzheimer's disease is characterized in part by extracellular aggregation of the amyloid-β peptide in the form of diffuse and fibrillar plaques in the brain. Electron microscopy (EM) has made an important contribution in understanding of the structure of amyloid plaques in humans. Classical EM studies have revealed the architecture of the fibrillar core, characterized the progression of neuritic changes, and have identified the neurofibrillary tangles formed by paired helical filaments (PHF) in degenerating neurons. Clinical data has strongly correlated cognitive impairment in AD with the substantial synapse loss observed in these early ultrastructural studies. Animal models of AD-type brain amyloidosis have provided excellent opportunities to study amyloid and neuritic pathology in detail and establish the role of neurons and glia in plaque formation. Transgenic mice overexpressing mutant amyloid precursor protein (APP) alone with or without mutant presenilin 1 (PS1), have shown that brain amyloid plaque development and structure grossly recapitulate classical findings in humans. Transgenic APP/PS1 mice expressing human apolioprotein E isoforms also develop amyloid plaque deposition. However no ultrastructural data has been reported for these animals. Here we show results from detailed EM analysis of amyloid plaques in APP/PS1 mice expressing human isoforms of ApoE and compare these findings with EM data in other transgenic models and in human AD. Our results show that similar to other transgenic animals, APP/PS1 mice expressing human ApoE isoforms share all major cellular and subcellular degenerative features and highlight the identity of the cellular elements involved in Aβ deposition and neuronal degeneration.

To identify quantitative MRI indices of injury in the brain following neonatal hypoxic-ischemic brain injury, we subjected mouse pups to hypoxia-ischemia on postnatal day 7 and obtained conventional and diffusion-weighted in vivo images of the brain 24 h later followed by histological assessment. T(2)-weighted images showed increased signal intensity in the CA1 and CA2 regions of the hippocampus ipsilateral to the injury and adjacent white matter. In contrast, diffusion imaging showed reduced apparent diffusion coefficient (ADC) values in CA1 and CA2, but increased values in the adjacent white matter. Histological analysis showed widespread gliosis with degenerating oligodendrocytes in the ipsilateral hippocampus. In addition, white matter areas that were abnormal by MRI showed an increase in the number of activated microglia (CD45 positive cells). Activated caspase-3 immunostaining showed a marked increase in neurons in the hippocampal regions corresponding to those with reduced ADC, and a quantitative measure of staining showed a statistically significant correlation with the ADC. In contrast, ADC was higher in adjacent white matter, where histology showed activation of microglia and reactive oligodendrocytes but not caspase-3 activation. These results suggest that the ADC response differs between areas of neuronal injury as compared with those showing glial changes without marked cell death., (Copyright © 2011 Wiley-Liss, Inc.)

Repetitive mild or "concussive" traumatic brain injury (TBI) can cause substantial neurologic impairment, but the pathological features of this type of injury are not fully understood. We report an experimental model of TBI in which the closed skulls of anesthetized male C57BL/6J mice are struck with an electromagnetically controlled rubber impactor twice with an interval of 24 hours between impacts. The mice had deficits in Morris water maze performance in the first week after injury that only partially resolved 7 weeks later. By routine histology, there was no apparent bleeding, neuronal cell loss, or tissue disruption, and amyloid precursor protein immunohistochemistry demonstrated very few immunoreactive axonal varicosities. In contrast, silver staining revealed extensive abnormalities in the corpus callosum and bilateral external capsule, the ipsilateral cortex and thalamus, and the contralateral hippocampal CA1 stratum radiatum and stratum oriens. Electron microscopy of white matter regions demonstrated axonal cytoskeletal disruption, intra-axonal organelle compaction, and irregularities in axon caliber. Reactive microglia were observed in the same areas as the injured axons by both electron microscopy and Iba1 immunohistochemistry. Quantitative analyses of silver staining and Iba1 immunohistochemistry at multiple time points demonstrated transient cortical and thalamic abnormalities but persistent white matter pathology as late as 7 weeks after injury.Thus, prominent and long-lasting abnormalities in this TBI model were underestimated using conventional approaches. The model may be useful for mechanistic investigations and preclinical assessment of candidate therapeutics.

During human brain development, the cerebral cortex undergoes substantial folding, leading to its characteristic highly convoluted form. Folding is necessary to accommodate the expansion of the cerebral cortex; abnormal cortical folding is linked to various neurological disorders, including schizophrenia, epilepsy, autism, and mental retardation. Although this process requires mechanical forces, the specific force-generating mechanisms that drive folding remain unclear. The two most widely accepted hypotheses are as follows: (1) Folding is caused by differential growth of the cortex and (2) folding is caused by mechanical tension generated in axons. Direct evidence supporting either theory, however, is lacking. Here we show that axons are indeed under considerable tension in the developing ferret brain, but the patterns of tissue stress are not consistent with a causal role for axonal tension. In particular, microdissection assays reveal that significant tension exists along axons aligned circumferentially in subcortical white matter tracts, as well as those aligned radially inside developing gyri (outward folds). Contrary to previous speculation, however, axonal tension is not directed across developing gyri, suggesting that axon tension does not drive folding. On the other hand, using computational (finite element) models, we show that differential cortical growth accompanied by remodeling of the subplate leads to outward folds and stress fields that are consistent with our microdissection experiments, supporting a mechanism involving differential growth. Local perturbations, such as temporal differences in the initiation of cortical growth, can ensure consistent folding patterns. This study shows that a combination of experimental and computational mechanics can be used to evaluate competing hypotheses of morphogenesis, and illuminate the biomechanics of cortical folding.

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.

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.

Acute, transient exposure to ethanol causes a widespread pattern of caspase-3 activation and neuroapoptosis in the developing rodent brain. To determine whether caspase-3 activation is an essential step in ethanol-induced developmental neuroapoptosis, we treated homozygous caspase-3 knockout mice or wild-type mice on postnatal day 7 with an apoptosis-inducing dose of ethanol and examined the brains at appropriate survival times for evidence of apoptotic neurodegeneration. In caspase-3 knockout mice, the cell death process evolved more slowly than in wild-type mice, and morphological changes observed were not those typically associated with apoptosis. However, neuronal cell counts performed 2 weeks post-treatment revealed that the extent of neuron loss was similar in wild-type and caspase-3-deficient mice. We conclude that absence of functional caspase-3 alters the time course and morphological characteristics of the neurodegenerative process but does not prevent ethanol-induced neuron death.

For three decades since the fetal alcohol syndrome (FAS) was first described, researchers have been keenly interested in understanding the mechanism(s) by which ethanol damages or disrupts development of the human fetal brain. It has been reported repeatedly that exposure of infant rats to ethanol causes a reduction in brain mass and loss of cerebellar Purkinje cells, but the mechanisms underlying these effects have remained elusive. In a recent series of studies, we have demonstrated that exposure of infant rats or mice to ethanol on a single occasion during the synaptogenesis period of development causes neurons in many regions of the developing central nervous system to commit suicide (die by apoptosis), but the cerebellum was not among the brain regions focused upon in these studies. Here we show in infant rats and mice that one-time exposure to ethanol triggers acute neurodegeneration of Purkinje cells and other neurons in the cerebellar cortex, deep cerebellar nuclei, and two related brainstem nuclei (nucleus pontis, inferior olivary complex). We also describe the time course of neurodegeneration and window of vulnerability for each of these neuronal cell types and demonstrate that the cell death process in each case is unequivocally apoptotic. We conclude that exposure of infant rats or mice to ethanol on a single occasion during synaptogenesis can kill Purkinje cells, and many other neuronal populations at all levels of the developing neuraxis, and in each case the mechanism of cell death is apoptosis.

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.

N-Methyl-D-aspartate (NMDA) glutamate receptor antagonists are being developed as therapeutic agents for several clinical conditions. However, the ability of these agents to produce neurotoxicity and psychosis can compromise their clinical usefulness. In addition, an NMDA receptor hypofunction (NRHypo) state may play a role in neurodegenerative and psychotic disorders. A better understanding of the mechanism underlying these adverse effects should allow for the safer use of these agents and might clarify mechanisms underlying certain clinical disorders. NRHypo neurotoxicity is mediated by a complex disinhibition mechanism in which NMDA antagonists abolish GABAergic inhibition, resulting in the simultaneous excessive release of acetylcholine and glutamate onto the vulnerable retrosplenial cortex (RSC) neurons. Systemically administered GABAergic agents are potent protectors against NRHypo neurotoxicity. To determine where in brain GABAergic agents could be acting to protect against NRHypo neurotoxicity, we injected the GABAergic agonist, muscimol, into different brain regions of rats treated systemically with a neurotoxic dose of the potent NMDA antagonist, MK-801. We report that muscimol injections into the anterior thalamus or diagonal band of Broca provide substantial protection, suggesting that disinhibition of neurons in these regions underlies NRHypo neurotoxicity. Muscimol injections into the RSC also provide substantial protection possibly by directly inhibiting the vulnerable RSC neuron. Injections of muscimol into other areas known to project to the RSC (ventral orbital cortex, anterior cingulate cortex and subiculum) provide only minimal protection. We conclude that GABAergic agents prevent NRHypo neurotoxicity mainly by activating GABA receptors in the anterior thalamus, diagonal band of Broca and RSC.

Purpose: Ethanol is known to have deleterious effects on the human fetal nervous system (fetal alcohol syndrome), including components of the visual system, but only modest progress has been made in understanding these effects. The authors have recently demonstrated that, during the period of synaptogenesis, a single episode of ethanol intoxication lasting for several hours triggers a massive wave of apoptotic neurodegeneration in several regions of the developing rat or mouse forebrain. The present study was undertaken to determine to what extent the developing visual system is vulnerable to the apoptogenic effects of ethanol., Methods: Infant rats and mice at ages from birth to 21 days were treated subcutaneously with a single dose of ethanol or with two doses, 2 hours apart, on a single day. Blood alcohol levels were determined, and the retinas and visual centers in the brain were examined by light and electronmicroscopy at various times from 4 to 24 hours after treatment., Results: Retinal ganglion cells and neurons in the lateral geniculate nucleus, superior colliculus, and visual cortex were all highly susceptible to ethanol's apoptogenic action, the period of peak sensitivity being postnatal days 1 to 4 for ganglion cells and 4 to 7 for the other visual neurons. A transient elevation of blood alcohol to approximately 120 mg/dL was sufficient to activate the cell death program in visual neurons., Conclusions: During synaptogenesis, a single ethanol intoxication episode triggers apoptotic cell death of neurons at all levels of the visual system from retina to the visual cortex.

We describe a new molecular mechanism of cell death by excitotoxicity mediated through nuclear transcription factor kappa B (NF kappa B) in rat embryonic cultures of dopaminergic neurons. Treatment of mesencephalic cultures with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) resulted in a number of changes that occurred selectively in dopaminergic neurons, including persistent elevation in intracellular Ca(2+) monitored with Fura-2, and a significant increase in intramitochondrial oxidation of dihydrorhodamine 123, probably associated with transient increase of mitochondrial permeability, cytochrome c release, nuclear translocation of NF kappa B, and transcriptional activation of the oncogene p53. Interruption of any of these steps by specific antagonists prevented neurite pruning and programmed cell death. In contrast, cell death was not prevented by caspase antagonists and only partly prevented by nitric-oxide synthase inhibitors. This signal transduction pathway might be a contributing mechanism in ongoing neuronal death in Parkinson disease.

Recently it was demonstrated that exposure of the developing brain during the period of synaptogenesis to drugs that block NMDA glutamate receptors or drugs that potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. All currently used general anesthetic agents have either NMDA receptor-blocking or GABA(A) receptor-enhancing properties. To induce or maintain a surgical plane of anesthesia, it is common practice in pediatric or obstetrical medicine to use agents from these two classes in combination. Therefore, the question arises whether this practice entails significant risk of inducing apoptotic neurodegeneration in the developing human brain. To begin to address this problem, we have administered to 7-d-old infant rats a combination of drugs commonly used in pediatric anesthesia (midazolam, nitrous oxide, and isoflurane) in doses sufficient to maintain a surgical plane of anesthesia for 6 hr, and have observed that this causes widespread apoptotic neurodegeneration in the developing brain, deficits in hippocampal synaptic function, and persistent memory/learning impairments.

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. beta-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.

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., ((c)2002 Elsevier Science (USA).)

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 ethanol's 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.

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.

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.

The deleterious effects of ethanol on the developing human brain are poorly understood. Here it is reported that ethanol, acting by a dual mechanism [blockade of N-methyl-D-aspartate (NMDA) glutamate receptors and excessive activation of GABA(A) receptors], triggers widespread apoptotic neurodegeneration in the developing rat forebrain. Vulnerability coincides with the period of synaptogenesis, which in humans extends from the sixth month of gestation to several years after birth. During this period, transient ethanol exposure can delete millions of neurons from the developing brain. This can explain the reduced brain mass and neurobehavioral disturbances associated with human fetal alcohol syndrome.

Breathing air with a high oxygen tension induces an inflammatory response and injures the microvessels of the lung. The resulting development of smooth muscle cells in these segments contributes to changes in vasoreactivity and increased pulmonary artery pressure. This in vivo study determines the temporal and spatial expression of endogenous endothelial nitric oxide synthase (NOS III) and inducible NOS (NOS II), important enzymes regulating vasoreactivity and inflammation, in the adult rat lung during the development of experimental pulmonary hypertension induced by oxidant injury. We analyzed the cellular distribution of these NOS isoforms, using specific antibodies, and assessed enzyme activity at baseline and after 1-28 days of hyperoxia (FIO2 0.87). The number of NOS III-immuno-positive endothelial cells increased early in hyperoxia and then remained high. By day 28, the relative number of these cells had increased from 40% in proximal vessels and 13-16% in distal alveolar vessels of the normal lung to 73-86% and 40-59%, respectively, in hyperoxia. Pulmonary alveolar macrophages (PAMs), normally few in number and only weakly immunopositive for NOS II or III in the normal lung, increased in number in hyperoxia and were strongly immunopositive for each isoform. These morphological data were supported by a temporal increase in total and calcium-independent NOS activity. Thus NOS expression and activity significantly increased in hyperoxia as pulmonary hypertension developed, and NOS III expression increased selectively in vascular endothelial cells, while both NOS isoforms were expressed by the PAM population. We conclude that this increase in expression of a potent vasodilator, an antiproliferative agent for smooth muscle cells, and an antioxidant molecule represents an adaptive response to protect the lung from oxidant-induced vascular and epithelial injury.

Programmed cell death (apoptosis) occurs during normal development of the central nervous system. However, the mechanisms that determine which neurons will succumb to apoptosis are poorly understood. Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors for only a few hours during late fetal or early neonatal life triggered widespread apoptotic neurodegeneration in the developing rat brain, suggesting that the excitatory neurotransmitter glutamate, acting at NMDA receptors, controls neuronal survival. These findings may have relevance to human neurodevelopmental disorders involving prenatal (drug-abusing mothers) or postnatal (pediatric anesthesia) exposure to drugs that block NMDA receptors.

We have developed a model for head trauma in infant rats in an attempt to study mechanisms of neurodegeneration in the developing brain and were able to morphologically characterize two distinct types of brain damage. The first type or primary damage evolved within 4 hrs after trauma and occurred by an excitotoxic mechanism. The second type or secondary damage evolved within 6-24 hrs and occurred by an apoptotic mechanism. Primary damage remained localized to the parietal cortex at the site of impact. Secondary damage affected distant sites such as the cingulate/retrosplenial cortex, subiculum, frontal cortex, thalamus, hippocampal dentate gyrus and striatum. Histological evidence of delayed cell death was preceded by decrease of bcl-2- in conjunction with increase of c-jun-mRNA-levels, already evident at 1 hr after trauma. Increase of CPP32-like activity and elevated concentrations of oligonucleosomes in affected brain regions represented additional findings to indicate that this secondary disseminated degenerative reaction is apoptotic in nature. At the age of 7 days, secondary apoptotic damage was more severe than primary excitotoxic damage, but its severity declined with increasing age. In 7-days-old rats, NMDA antagonists protected against primary excitotoxic damage but increased severity of secondary apoptotic damage whereas the free radical scavenger SPBN, the tumor necrosis factor (TNF) inhibitor pentoxifylline and the antioxidant N-acetylcystein mitigated apoptotic damage. These findings demonstrate that in the developing rat brain apoptosis and not excitotoxicity determines neuropathologic outcome following head trauma. Whereas radical scavengers and TNF-inhibitors may prove useful in treatment of pediatric head trauma, great caution should be applied in regards to the use of NMDA antagonists because of the inherent risk of apoptosis promotion.

The immunohistochemical profile of 23 pleomorphic adenomas and 7 normal salivary glands was studied. We used antisera to vimentin (V), desmin (D), epithelial membrane antigen (EMA), prostate specific antigen (PSA), pancytokeratin, carcinoembryonic antigen (CEA), glial fibrillary acidic protein (GFAP) and S-100 protein. In the ducts and myoepithelial cells of normal salivary glands immunopositivity to most of the cytoskeletal proteins, EMA and CEA was observed. GFAP was localized only in cells of striated ducts. Major differences in the expression of various antigens among tubular structures, solid sheets, the myxoid and chondroid in the pleomorphic adenoma were encountered. Appearance of GFAP as a sign of stromal transformation into myxoid and chondroid was detected. Judging from these comparative immunohistochemical characteristics between normal salivary glands and pleomorphic adenomas, we assume that tumour cells originate from the reserve cells of intercalated and striated ducts.

Intimal cells play an important role in the biology of the vascular wall. Variability in the metabolic activity of intimal smooth muscle cells (SMC), as well as the differential expression of cellular cytoskeletal proteins depend on factors such as degree of differentiation, aging, atherosclerosis, etc. Myosin ATPase activity and cytoskeletal proteins were studied in the intima of bovine femoral arteries and veins of mature animals. In some arteries the intima was thickened and two distinct layers--inner elastic hyperplastic (EHL) and outer, musculo-elastic (MEL) were observed. ATPase activity was well defined in endothelial cells (EC) as well as in SMC. However, differential enzymatic expression was observed in thickened intimas. SMC in the EHL were ATPase negative, while in the MEL they were ATPase positive. All EC and SMC in the "normal" intimas were vimentin positive, desmin and cytokeratin negative. In vessels with thickened intimas, the EHL showed intensive vimentin positivity; in the MEL desmin immunoreactive SMC were numerous as were as those in the media. Vimentin-positive SMC occupied their innermost part. Differences in the expression of ATPase activity and cytoskeletal proteins is discussed in terms of possible migration of medial SMC and/or morphological modulation observed in vessels with altered vascular walls.

The aim of the present study was the evaluation of CEA-radio-immunoassay and CEA-immunohistochemistry in the management of pleomorphic adenoma and mucoepidermoid carcinoma of salivary glands. 23 pleomorphic adenomas, 9 mucoepidermoid carcinomas, and 7 normal salivary glands were examined. CEA-concentration in serum and saliva were assayed before and after surgery. Polyclonal CEA antibody was used for immunohistochemical CEA detection in the tumor tissue and in the normal salivary glands. The mean CEA concentrations were found to be 14.94 ng/ml in the serum and 216.67 ng/ml in the saliva of patients with mucoepidermoid carcinoma. These values were considerably higher compared to healthy controls (188.64 ng/ml in saliva) and in patients with pleomorphic adenoma - 7.65 ng/ml in serum and 189.35 ng/ml in saliva (P < 0.001). A correlation was found between high CEA concentration in the saliva and the intensity of CEA expression in the tumour tissue. An increased synthesis and secretion of CEA was determined by the prevalence of tubular structures, a high proliferative activity in pleomorphic adenoma, and its malignant transformation.