Global alterations to the choroid plexus blood-CSF barrier in amyotrophic lateral sclerosis.

The choroid plexus (CP) is a highly vascularized structure located in the ventricles that forms the blood-CSF barrier (BCSFB) and separates the blood from the cerebrospinal fluid (CSF). In addition to its role as a physical barrier, the CP functions in CSF secretion, transport of nutrients into the central nervous system (CNS) and a gated point of entry of circulating immune cells into the CNS. Aging and neurodegeneration have been reported to affect CP morphology and function and increase protein leakage from blood to the CSF. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with both upper and lower motor neuron loss, as well as altered proteomic and metabolomic signatures in the CSF. The role of the BCSFB and the CP in ALS is unknown. Here we describe a transcriptomic and ultrastructural analysis of BCSFB and CP alterations in human postmortem tissues from ALS and non-neurologic disease controls. ALS-CP exhibited widespread disruptions in tight junctional components of the CP epithelial layer and vascular integrity. In addition, we detected loss of pericytes around ALS blood vessels, accompanied by activation of platelet aggregation markers vWF and Fibrinogen, reminiscent of vascular injury. To investigate the immune component of ALS-CP, we conducted a comprehensive analysis of cytokines and chemokine panels in CP lysates and found a significant down-regulation of M-CSF and V-CAM1 in ALS, as well as up-regulation of VEGF-A protein. This phenotype was accompanied by an infiltration of MERTK positive macrophages into the parenchyma of the ALS-CP when compared to controls. Taken together, we demonstrate widespread structural and functional disruptions of the BCSFB in human ALS increasing our understanding of the disease pathology and identifying potential new targets for ALS therapeutic development.

Creating virtual 3-dimensional models for teaching pre-clinical tooth preparation: Students' usages and perceptions.

PURPOSE: This research aimed to evaluate the students' usage and perceptions of using smartphones in their general dental education and learning tooth preparation with the individually designed virtual 3D instructional models in the pre-clinical removable partial denture course. MATERIALS AND METHODS: Second-year dental students were asked to voluntarily participate in a survey to investigate their demographic information, general usages of smartphones, perception of smartphones usage in dental education (construct 1) and perception of individually designed virtual 3D instructional models (construct 2). Students' responses of general usages of the smartphones were compared with their demographic and educational backgrounds using nonparametric Kruskal-Wallis test (for age) and Fisher's exact test (for sex, race and educational background). The sums of scores of the construct 1 and construct 2 were tested for associations with student's demographic and educational backgrounds using the Pearson product-moment correlation (for age), t test (for sex and educational background) or one-way ANOVA F test (for race) (α = .05). RESULTS: A 75% response rate (N = 90) was achieved in this study, and all 90 participants owned smartphones. Students' responses to general usages of the smartphones were not significantly influenced by their demographic background. For the construct 1, more than 73% of participants responded either agree or strongly agree to the usage of smartphones in general dental education and pre-clinical setting; however, only 49% of participants responded the same way in the clinical setting. For the construct 2, 48 of 90 participants viewed the 3D models, and more than 73% of these 48 participants responded either agree or strongly agree to the usage of the 3D models in the pre-clinical course. Student's demographic background did not have significant influence on the sums of scores of the construct 1 and construct 2. CONCLUSIONS: Within the limitations of this study, high usages and ownerships of smartphones were found amongst the students surveyed. The individually designed virtual 3D instructional models as supplemental teaching materials in the pre-clinical course were perceived positively by the students.

Comparison of digital scanning and polyvinyl siloxane impression techniques by dental students: instructional efficiency and attitudes towards technology.

INTRODUCTION: With increasing use of digital scanning with restorative procedures in the dental office, it becomes necessary that educational institutions adopt instructional methodology for introducing this technology together with conventional impression techniques. OBJECTIVE: To compare the time differences between instructing dental students on digital scanning (DS) (LAVA C.O.S. digital impression system) and a conventional impression technique (CI) (polyvinyl siloxane), and to compare students' attitudes and beliefs towards both techniques. MATERIALS AND METHODS: Volunteer sophomore dental students (n = 25) with no prior experience in clinical impressions were recruited and IRB consent obtained. Participants responded to a pre-and post-exposure questionnaire. Participants were instructed on the use of both DS and CI for a single tooth full coverage crown restoration using a consecutive sequence of video lecture, investigator-led demonstration and independent impression exercise. The time necessary for each step (minutes) was recorded. Statistical significance was calculated using dependent t-tests (time measurements) and 2-sample Mann-Whitney (questionnaire responses). RESULTS: The time spent teaching students was greater for DS than CI for video lecture (15.95 and 10.07 min, P = 0.0000), demonstration time (9.06 and 4.70 min, P = 0.0000) and impression time (18.17 and 8.59 min, P = 0.0000). Prior to the instruction and practice, students considered themselves more familiar with CI (3.96) than DS (1.96) (P = 0.0000). After the instruction and practice, participants reported CI technique proved significantly easier than expected (pre-instruction: 3.52 and post-instruction: 4.08, P = 0.002). However, overall participants' perception of ease of use for DS was not influenced by this instruction and practice experience (pre-instruction: 3.84 and post-instruction: 3.56, P = 0.106). Despite the results, 96% of participants expressed an expectation that DS will become their predominant impression technique during their careers. CONCLUSIONS: Dental students with no clinical experience have high expectations for digital scanning, and despite their initial difficulty, expect it to become their primary impression technique during their professional futures. The instructional time necessary for introducing DS into the curriculum is significantly greater than CI in both classroom (lecture) and clinical simulation settings (investigator-led demonstration).

p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53ß are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53ß, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53ß overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53ß expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.

Predictable Restorative Work Flow for Computer-Aided Design/Computer-Aided Manufacture-Fabricated Ceramic Veneers Utilizing a Virtual Smile Design Principle.

The purpose of this case report was to present the use of a contemporary digital photograph-assisted virtual smile design principle, an intraoral digital impression, and computer-aided design/computer-aided manufacture-fabricated lithium disilicate ceramic veneers to treat a patient with esthetic needs in the maxillary anterior region. By using the proposed digital restorative work flow, this case report demonstrated an effective communication pathway between the patient, clinician, and dental laboratory technician. Effective communication can help to achieve a more predictable and satisfactory esthetic outcome.

Inhibitor of DNA binding-4 promotes angiogenesis and growth of glioblastoma multiforme by elevating matrix GLA levels.

Inhibitor of differentiation-4 is highly expressed in glioblastoma multiforme (GBM). We report a novel pro-angiogenic function for inhibitor of differentiation-4 in the growth of glioblastoma xenografts. Tumor-derived cell cultures expressing elevated levels of ID4 produced enlarged xenografts in immunosuppressed mice that were better vascularized than corresponding control tumors and expressed elevated matrix GLA protein (MGP) that mediated enhanced tumor angiogenesis. Inhibition of MGP resulted in smaller and less vascularized xenografts. Our finding shows a novel function for ID4 in tumor angiogenesis, and identifies ID4 and MGP as possible therapeutic targets for GBM.

Tumor necrosis factor-alpha induces changes in mitochondrial cellular distribution in motor neurons.

Motor neuron (MN) mitochondrial abnormalities and elevation in spinal fluid levels of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The mechanism of neuron death in ALS remains unclear, along with the contributions of mitochondrial dysfunction and inflammation in the process. Cell cultures enriched for MN derived from embryonic rat spinal cords were established and directly exposed in vitro to recombinant TNF-alpha for varying lengths of time. Although cytokine exposure for up to 4 days failed to induce MN death, mitochondrial changes were observed shortly after initiating treatment. Our results demonstrate that TNF-alpha induced mitochondrial redistribution toward the soma in MN. We postulate that inflammation may precede, and in fact cause, the mitochondrial changes observed in ALS tissue.

Spontaneous murine neuroaxonal dystrophy: a model of infantile neuroaxonal dystrophy.

The neuroaxonal dystrophies (NADs) in human beings are fatal, inherited, neurodegenerative diseases with distinctive pathological features. This report describes a new mouse model of NAD that was identified as a spontaneous mutation in a BALB/c congenic mouse strain. The affected animals developed clinical signs of a sensory axonopathy consisting of hindlimb spasticity and ataxia as early as 3 weeks of age, with progression to paraparesis and severe morbidity by 6 months of age. Hallmark histological lesions consisted of spheroids (swollen axons), in the grey and white matter of the midbrain, brain stem, and all levels of the spinal cord. Ultrastructural analysis of the spheroids revealed accumulations of layered stacks of membranes and tubulovesicular elements, strongly resembling the ultrastructural changes seen in the axons of human patients with endogenous forms of NAD. Mouse NAD would therefore seem a potentially valuable model of human NADs.

Schwann cells and astrocytes induce synapse formation by spinal motor neurons in culture.

Glia constitute 90% of cells in the human nervous system, but relatively little is known about their functions. We have been focusing on the potential synaptic roles of glia in the CNS. We recently found that astrocytes increase the number of mature, functional synapses on retinal ganglion cells (RGCs) by sevenfold and are required for synaptic maintenance in vitro. These observations raised the question of whether glia similarly enhance synapse formation by other neuron types. Here we have investigated whether highly purified motor neurons isolated from developing rat spinal cords are able to form synapses in the absence of glia or whether glia similarly enhance synapse number. We show that spinal motor neurons (SMNs) form few synapses unless Schwann cells or astrocytes are present. Schwann cells increase the number of functional synapses by ninefold as measured by immunostaining, and increase spontaneous synaptic activity by several hundredfold. Surprisingly, the synapses formed between spinal motor neurons were primarily glutamatergic, as they could be blocked by CNQX. This synapse-promoting activity is not mediated by direct glial-neuronal cell contact but rather is mediated by secreted molecule(s) from the Schwann cells, as we previously found for astrocytes. Interestingly, the synapse-promoting activity from astrocytes and Schwann cells was functionally similar: Schwann cells also promoted synapse formation between retinal ganglion cells, and astrocytes promoted synapse formation between spinal motor neurons. These studies show that both astrocytes and Schwann cells strongly promote synapse formation between spinal motor neurons and demonstrate that glial regulation of synaptogenesis extends to other neuron types.

MRI study of immediate cell viability in focused ultrasound lesions in the rabbit brain.

The purpose of this study was to evaluate cell viability in MR imaged focused ultrasound (FUS) lesions using cell-viability staining with triphenyl tetrazolium chloride (TTC) and both light and electron microscopy. Ten paired ultrasonic lesions were created in 5 rabbit brains in vivo with an ultrasound beam of 1.5 MHz electrical power input to the transducer of 50 W and exposure duration of 15 seconds. T2-weighted fast spin-echo (FSE) MRI was performed to detect the FUS lesions in the brain 4 hours after treatment, after which the animals were immediately euthanized. Lesion sizes were measured on TTC-stained specimens, histological sections stained with hematoxylin and eosin (H&E), and T2-weighted MR images. The differences between the lesion diameters measured with the three methods were within the range of 0.1--0.7 mm. The lesion sizes measured from MRI correlated well with those seen from H&E sections. The measurements from MRI slightly overestimated lesion sizes on TTC-stained wet tissues by approximately one MRI pixel (0.31 mm). Electron microscopy demonstrated nuclear and cytoplasmic ultrastructural damage within the grey-white, non-TTC-stained lesion zone, whereas the TTC-stained normal tissue showed preservation of neuronal ultrastructure. Therefore, MR-imaged lesions represent a cell-death zone in rabbit brain 4 hours after FUS ablation, with slight overestimation by approximately one MRI pixel. J. Magn. Reson. Imaging 2001;13:23-30.

Spinal cord glioneuronal tumor with "rosetted" neuropil islands and meningeal dissemination: a case report.

Distinctive glioneuronal tumors arising within the cerebrum and displaying neuropil-like islands and tumor cells immunoreactive for neuronal and glial antigens have recently been described. We report a similar tumor in the cervico-thoracic region of the spinal cord in a 44-year-old woman that recurred 1 year later with dissemination to the lumbar dura and cauda equina. The tumor was composed of "rosetted" neuropil islands displaying immunoreactivity for synaptophysin, whereas the intervening tumor cells were more fibrillar and immunoreactive for GFAP. The tumor cell nuclei immediately surrounding these neuropil islands were immunoreactive to the newly characterized neuronal marker, anti-Hu. While several cases of neurocytomas have been described in the spinal cord, to the best of our knowledge, this is the first example of a glioneuronal tumor with "rosetted" neuropil islands to be reported in the spinal cord.

Acquired cerebral arteriovenous malformation induced by an anaplastic astrocytoma: an interesting case.

High grade gliomas foster an environment rich in angiogenic factors that promote neovascularity. We report a case of a cerebral arteriovenous malformation, which developed in the setting of a high grade astrocytoma. The patient presented with complaints of confusion and left hemiparesis. An initial cerebral angiogram was normal. Repeat angiography six weeks later demonstrated an extremely vascular lesion with arteriovenous shunting involving the right thalamus and occipital lobe. Histopathologic evaluation of open biopsy and autopsy specimens demonstrated a high grade astrocytoma in association with an arteriovenous malformation. Immunohistochemical staining with VEGF was diffusely positive. A possible role for the hyperangiogenic environment of a high grade astrocytoma resulting in the development of an arteriovenous malformation is discussed.

Melanotic craniopharyngioma: a report of two cases.

We report a 74-year-old woman and a 50-year-old woman with similar histories of headache and visual disturbance who were found to have adamantinomatous craniopharyngiomas which contained melanin pigment. This finding was confirmed by the Masson Fontana method and ultrastructural studies. These are only the second and third cases reported describing melanin pigment within a craniopharyngioma. The finding of melanin in craniopharyngiomas attests to their similarities with odontogenic tumors of the jaw, which can also contain melanin pigment and also supports the hypothesis that the histogenesis of these neoplasms derives from the vestiges of Rathke's pouch epithelium.

Differentially expressed GABAA-receptor subunits result in structurally and functionally receptor assemblies following excitatory afferent synaptic transmission.

Cerebellar granule cells isolated from postnatal day 7 rat pups are ideal for studying epigenetic events associated with the regulation of neuronal gene expression. These cultures contain from 90 to 95% glutamatergic granule cells and express mRNAs encoding a variety of ionotropic and metabotropic glutamate receptors as well as virtually all of the GABAA-receptor subunit mRNAs to different extents. A unique feature of this culture system is that the neurons undergo time-dependent maturation changes in vitro that mimic many of the characteristics of these receptors occurring in vivo. Granule cell cultures in vitro require depolarizing concentrations of KCl for long-term growth and survival. Both N-methyl-D-aspartate (NMDA) and GABA have been reported to exert trophic actions on these cells replacing the requirement for maintaining the cultures in high KCl. Cerebellar granule cells maintained under different conditions in vitro can be induced to alter their patterns of maturation, as indicated by the different temporal changes in gene expression of receptor subunit mRNAs and proteins. The focus of the current studies is the effect of NMDA afferent synaptic signaling on the changes in mRNA content and functional properties of GABAA receptors and how this may relate to comparable changes shown to occur in vivo.

NMDA-mediated modulation of gamma-aminobutyric acid type A receptor function in cerebellar granule neurons.

GABAA receptors are ligand-gated CI- ion channels with multiple clinically relevant drug-recognition sites. We have previously shown that stimulation of N-methyl-D-aspartic acid (NMDA)-specific glutamate receptors quantitatively alters selected GABAA receptor subunit mRNAs and proteins in primary cultures of rat cerebellar granule neurons. We used whole-cell recordings of GABA-elicited CI- currents and flunitrazepam binding experiments in granule cell cultures maintained in low K+ (12.5 mM), cells maintained in low K+ and treated with a single dose of NMDA (10 microM), and cell cultures maintained in depolarizing concentrations of K+ (25 mM). The EC50 obtained from the dose-response curves for GABA in eliciting a maximal response was comparable in neurons maintained in high K+ or in low K+ and treated with a single dose of NMDA, but that it increased significantly in cells maintained in low K+. The potentiation of GABA-gated CI- currents by flunitrazepam increased significantly, while the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was significantly more effective in cultures either maintained in high K+ or treated with NMDA. This was coincident with a twofold increase in the Bmax associated with flunitrazepam binding. To further characterize the receptor assemblies present in the depolarization and NMDA induced paradigms, the Zn(2+)-induced inhibition of GABA-gated CI- currents was reduced as was the inhibition mediated by furosemide. Our data indicate that GABAA receptor assemblies alter their composition in response to excitatory afferent receptor stimulation.

Exposure of neuronal cultures to K+ depolarization or to N-methyl-D-aspartate increases the transcription of genes encoding the alpha 1 and alpha 5 GABAA receptor subunits.

The transcription rates of the alpha 1, alpha 5, and alpha 6 gamma-aminobutyric acidA receptor subunit genes were analyzed in cultures maintained in low KCl (12.5 mM), in low KCl treated with NMDA (10 microM), and in high KCl (25 mM). alpha 1 and alpha 5 transcription rates were significantly increased in response to NMDA or high KCl treatment, while alpha 6 and cyclophilin transcription rates were not changed by either condition. These data suggest that following NMDA or high KCl treatment of granule cells, changes in alpha 1 and alpha 5 mRNA content are a consequence of a specific transcriptional rate increase of the corresponding subunit genes.