Virtual reality ultrasound imaging of the normal and abnormal fetal central nervous system

OBJECTIVES: In fetal ultrasound imaging, teaching and experience are of paramount importance to improve prenatal detection rates of fetal abnormalities. Yet both aspects depend on exposure to normal and, in particular, abnormal 'specimens'. We aimed to generate a number of simple virtual reality (VR) objects of the fetal central nervous system for use as educational tools. METHODS: We applied a recently proposed algorithm for the generation of fetal VR object movies to the normal and abnormal fetal brain and spine. Interactive VR object movies were generated from ultrasound volume data from normal fetuses and fetuses with typical brain or spine anomalies. Pathognomonic still images from all object movies were selected and annotated to enable recognition of these features in the object movies. RESULTS: Forty-six virtual reality object movies from 22 fetuses (two with normal and 20 with abnormal brains) were generated in an interactive display format (QuickTime) and key images were annotated. The resulting .mov files are available for download from the website of this journal. CONCLUSIONS: VR object movies can be generated from educational ultrasound volume datasets, and may prove useful for teaching and learning normal and abnormal fetal anatomy.

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.

Fetal ventral mesencephalon of human and rat origin maintained in vitro and transplanted to 6-hydroxydopamine-lesioned rats gives rise to grafts rich in dopaminergic neurons

Free-floating roller tube cultures of human fetal (embryonic age 6-10 weeks post-conception) and rat fetal (embryonic day 13) ventral mesencephalon were prepared. After 7-15 days in vitro, the mesencephalic tissue cultures were transplanted into the striatum of adult rats that had received unilateral injections of 6-hydroxydopamine into the nigrostriatal bundle 3-5 weeks prior to transplantation. Graft survival was assessed in tyrosine hydroxylase (TH)-immunostained serial sections of the grafted brains up to post-transplantation week 4 for the human fetal xenografts and post-transplantation week 11 for the rat fetal allografts. D-amphetamine-induced rotation was monitored up to 10 weeks after transplantation in the allografted animals and compared with that of lesioned-only control animals. All transplanted animals showed large, viable grafts containing TH-immunoreactive (ir) neurons. The density of TH-ir neurons in the human fetal xenografts and in rat fetal allografts was similar. A significant amelioration of the amphetamine-induced rotation was observed in the animals that received cultured tissue allografts. These results promote the feasibility of in vitro maintenance of fetal human and rat nigral tissue prior to transplantation using the free-floating roller tube technique.

A three-dimensional histological atlas of the human basal ganglia. II. Atlas deformation strategy and evaluation in deep brain stimulation for Parkinson disease

OBJECT: The localization of any given target in the brain has become a challenging issue because of the increased use of deep brain stimulation to treat Parkinson disease, dystonia, and nonmotor diseases (for example, Tourette syndrome, obsessive compulsive disorders, and depression). The aim of this study was to develop an automated method of adapting an atlas of the human basal ganglia to the brains of individual patients. METHODS: Magnetic resonance images of the brain specimen were obtained before extraction from the skull and histological processing. Adaptation of the atlas to individual patient anatomy was performed by reshaping the atlas MR images to the images obtained in the individual patient using a hierarchical registration applied to a region of interest centered on the basal ganglia, and then applying the reshaping matrix to the atlas surfaces. RESULTS: Results were evaluated by direct visual inspection of the structures visible on MR images and atlas anatomy, by comparison with electrophysiological intraoperative data, and with previous atlas studies in patients with Parkinson disease. The method was both robust and accurate, never failing to provide an anatomically reliable atlas to patient registration. The registration obtained did not exceed a 1-mm mismatch with the electrophysiological signatures in the region of the subthalamic nucleus. CONCLUSIONS: This registration method applied to the basal ganglia atlas forms a powerful and reliable method for determining deep brain stimulation targets within the basal ganglia of individual patients.

Volumetry and diffusion tensor imaging of hippocampal subregions in schizophrenia

Previous MRI-volumetric studies in schizophrenic psychoses have demonstrated more or less pronounced volume reductions of the hippocampus in patients. Correspondingly, neuropathological examinations on the brains of schizophrenics showed diverse structural changes of the hippocampus. Employing a high-resolution 3D-MPRAGE sequence, we found volume reductions in most hippocampal subregions of schizophrenic patients, which, however, did not reach significant levels. An analysis of co-registered diffusion tensor imaging (DTI) data revealed significant alterations of the inter-voxel coherences in single hippocampal subdivisions of these patients, supporting the assumption of characteristic microstructural tissue changes relevant for the pathogenesis of schizophrenic psychoses. Our results argue for the usage of additional MRI modalities like DTI in order to detect subtle regional alterations of hippocampal structure in schizophrenics.

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 degrees C, subsequently displayed a high thermotolerance, being able to grow at 42 degrees C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.

Brain CYP1A in seabream, Sparus aurata exposed to benzo(a)pyrene

This study compares basal and induced expression of cytochrome P4501A-CYP1A in the brain of gilthead seabream, Sparus aurata. Larval or adult seabream were exposed to benzo(a)pyrene -B(a)P- and the CYP1A response was assessed by analyzing CYP1A mRNA (RT-PCR), CYP1A protein (expression levels: ELISA, western blotting; cellular localization: immunohistochemistry), and CYP1A catalytic activity (7-ethoxyresorufin-O-deethylase-EROD). In the brain of adult S. aurata, CYP1A immunostaining was generally detected in the vasculature. It was present in the neuronal fibers and glial cells of the olfactory bulbs and the ventral telencephalon. ELISA and RT-PCR analyses confirmed CYP1A expression in the brains of non-exposed seabream. B(a)P exposure led to increased CYP1A staining mainly in neuronal fibers and glial cells of the olfactory bulbs, but also in the vascular endothelia. EROD activity, however, could not be detected in the brain of adult seabream, neither in control nor in exposed fish. In the developing brain of S. aurata larvae, immunohistochemical staining detected CYP1A protein exclusively in endothelia of the olfactory placode and in retina. Staining intensity of CYP1A slightly increases with larval development, especially in vascular brain endothelia. Exposing the larvae to 0.3 or 0.5 microg B(a)P/L from hatching until 15 days post hatching (dph) did not result in enhanced CYP1A immunostaining in the brain. In samples of whole seabream larvae, both from controls and BaP treatments, neither CYP1A mRNA, protein nor catalytic activity were detectable. The results demonstrate that CYP1A is expressed already and inducible in the larval brain, but that the regional and cellular expression differs partly between larval and adult brain. This may have implications for the toxicity of CYP1A-inducing xenobiotics on early and mature life stages of seabream.

Adult Neurogenesis in Drosophila

Adult neurogenesis has been linked to several cognitive functions and neurological disorders. Description of adult neurogenesis in a model organism like Drosophila could facilitate the genetic study of normal and abnormal neurogenesis in the adult brain. So far, formation of new neurons has not been detected in adult fly brains and hence has been thought to be absent in Drosophila. Here, we used an improved lineage-labeling method to show that, surprisingly, adult neurogenesis occurs in the medulla cortex of the Drosophila optic lobes. We also find that acute brain damage to this region stimulates adult neurogenesis. Finally, we identify a factor induced by acute damage, which is sufficient to specifically activate the proliferation of a cell type with adult neuroblast characteristics. Our results reveal unexpected plasticity in the adult Drosophila brain and describe a unique model for the genetic analysis of adult neurogenesis, plasticity, and brain regeneration.

Matrix metalloproteinase inhibition lowers mortality and brain injury in experimental pneumococcal meningitis

Pneumococcal meningitis (PM) results in high mortality rates and long-lasting neurological deficits. Hippocampal apoptosis and cortical necrosis are histopathological correlates of neurofunctional sequelae in rodent models and are frequently observed in autopsy studies of patients who die of PM. In experimental PM, inhibition of matrix metalloproteinases (MMPs) and/or tumor necrosis factor (TNF)-converting enzyme (TACE) has been shown to reduce brain injury and the associated impairment of neurocognitive function. However, none of the compounds evaluated in these studies entered clinical development. Here, we evaluated two second-generation MMP and TACE inhibitors with higher selectivity and improved oral availability. Ro 32-3555 (Trocade, cipemastat) preferentially inhibits collagenases (MMP-1, -8, and -13) and gelatinase B (MMP-9), while Ro 32-7315 is an efficient inhibitor of TACE. PM was induced in infant rats by the intracisternal injection of live Streptococcus pneumoniae. Ro 32-3555 and Ro 32-7315 were injected intraperitoneally, starting at 3 h postinfection. Antibiotic (ceftriaxone) therapy was initiated at 18 h postinfection, and clinical parameters (weight, clinical score, mortality rate) were recorded. Myeloperoxidase activities, concentrations of cytokines and chemokines, concentrations of MMP-2 and MMP-9, and collagen concentrations were measured in the cerebrospinal fluid. Animals were sacrificed at 42 h postinfection, and their brains were assessed by histomorphometry for hippocampal apoptosis and cortical necrosis. Both compounds, while exhibiting disparate MMP and TACE inhibitory profiles, decreased hippocampal apoptosis and cortical injury. Ro 32-3555 reduced mortality rates and cerebrospinal fluid TNF, interleukin-1β (IL-1β) and collagen levels, while Ro 32-7315 reduced weight loss and cerebrospinal fluid TNF and IL-6 levels.

Brain-derived neurotrophic factor protects against multiple forms of brain injury in bacterial meningitis

BACKGROUND Brain-derived neurotrophic factor (BDNF) blocks activation of caspase-3, reduces translocation of apoptosis-inducing factor (AIF), attenuates excitotoxicity of glutamate, and increases antioxidant enzyme activities. The mechanisms of neuroprotection suggest that BDNF may be beneficial in bacterial meningitis. METHODS To assess a potentially beneficial effect of adjuvant treatment with BDNF in bacterial meningitis, 11-day-old infant rats with experimental meningitis due to Streptococcus pneumoniae or group B streptococci (GBS) were randomly assigned to receive intracisternal injections with either BDNF (3 mg/kg) or equal volumes (10 mu L) of saline. Twenty-two hours after infection, brains were analyzed, by histomorphometrical examination, for the extent of cortical and hippocampal neuronal injury. RESULTS Compared with treatment with saline, treatment with BDNF significantly reduced the extent of 3 distinct forms of brain cell injury in this disease model: cortical necrosis in meningitis due to GBS (median, 0.0% [range, 0.0%-33.7%] vs. 21.3% [range, 0.0%-55.3%]; P<.03), caspase-3-dependent cell death in meningitis due to S. pneumoniae (median score, 0.33 [range, 0.0-1.0] vs. 1.10 [0.10-1.56]; P<.05), and caspase-3-independent hippocampal cell death in meningitis due to GBS (median score, 0 [range, 0-2] vs. 0.88 [range, 0-3.25]; P<.02). The last form of injury was associated with nuclear translocation of AIF. CONCLUSION BDNF efficiently reduces multiple forms of neuronal injury in bacterial meningitis and may hold promise as adjunctive therapy for this disease.

Intracisternal application of endotoxin enhances the susceptibility to subsequent hypoxic-ischemic brain damage in neonatal rats.

Perinatal brain damage is associated not only with hypoxic-ischemic insults but also with intrauterine inflammation. A combination of antenatal inflammation and asphyxia increases the risk of cerebral palsy >70 times. The aim of the present study was to determine the effect of intracisternal (i.c.) administration of endotoxin [lipopolysaccharides (LPS)] on subsequent hypoxic-ischemic brain damage in neonatal rats. Seven-day-old Wistar rats were subjected to i.c. application of NaCl or LPS (5 microg/pup). One hour later, the left common carotid artery was exposed through a midline neck incision and ligated with 6-0 surgical silk. After another hour of recovery, the pups were subjected to a hypoxic gas mixture (8% oxygen/92% nitrogen) for 60 min. The animals were randomized to four experimental groups: 1) sham control group, left common carotid artery exposed but not ligated (n = 5); 2) LPS group, subjected to i.c. application of LPS (n = 7); 3) hypoxic-ischemic study group, i.c. injection of NaCl and exposure to hypoxia after ligation of the left carotid artery (n = 17); or 4) hypoxic-ischemic/LPS study group, i.c. injection of LPS and exposure to hypoxia after ligation of the left carotid artery (n = 19). Seven days later, neonatal brains were assessed for neuronal cell damage. In a second set of experiments, rat pups received an i.c. injection of LPS (5 microg/pup) and were evaluated for tumor necrosis factor-alpha expression by immunohistochemistry. Neuronal cell damage could not be observed in the sham control or in the LPS group. In the hypoxic-ischemic/LPS group, neuronal injury in the cerebral cortex was significantly higher than in animals that were subjected to hypoxia/ischemia after i.c. application of NaCl. Injecting LPS intracisternally caused a marked expression of tumor necrosis factor-alpha in the leptomeninges. Applying LPS intracisternally sensitizes the immature rat brain to a subsequent hypoxic-ischemic insult.

The role of nitric oxide in Listeria encephalitis of ruminants and in rats intracisternally infected with Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular bacteria which infects a wide range of hosts. In ruminants, infection with L. monocytogenes frequently causes encephalitis, which is usually fatal in sheep and goat, while cattle often recover with antibiotic therapy. Since the role of NO in the control of Listeria is controversial, we have studied the expression of iNOS in the brains of cattle, sheep and goats which had succumbed to listeria encephalitis. iNOS was demonstrated in decreasing intensity in the M phi of microabscesses from cattle, sheep and goat. iNOS expression was accompanied by NT in the microabscesses of cattle, but was only present to a low degree in sheep and was absent in goats. This is indirect evidence for differences in the ability to produce NO in the three species. Presence of iNOS and NT were inversely correlated with the numbers of bacteria. While microabscesses of goats contained high amounts of L. monocytogenes they occurred only rarely in cattle. To corroborate our hypothesis that NO is involved in the control of listeria encephalitis a new animal model was developed. Eleven day old infant rats were infected intracisternally with a low dose of L. monocytogenes. This resulted in a transient meningoencephalitis with moderate clinical signs and low mortality. Listeria proliferated strongly in the inflammatory lesions during the first days of infection, reached a peak at day 4 and were eliminated until day 7. The presence of bacteria was closely accompanied by high numbers of iNOS-expressing M phi and the formation of NT. Administration of the iNOS inhibitor L-NIL or the radical scavenger PBN resulted in rapid death of the treated animals. However, the increase in bacterial numbers was one order of magnitude higher for animals treated with PBN compared with L-NIL administration. This shows that NO plays an important role in the control of a brain infection with Listeria, but suggests that reactive oxidants other than NO are also involved. In conclusion, our findings point to a possible involvement of the differences in the ability to express iNOS and subsequent NO production in the different clinical outcome of listeria encephalitis in cattle and small ruminants.

Erythrina abyssinica prevents meningoencephalitis in chronic Trypanosoma brucei brucei mouse model

Human African trypanosomiasis is prevalent in Sub-sahara African countries that lie between 14° North and 29° south of the equator. Sixty million people are at risk of infection. Trypanosoma brucei gambesience occurs in West and Central Africa while Trypanosoma brucei rhodesience occurs in East and Southern Africa. The neurological stage of the disease is characterized by neuroinflammation. About 10% of patients treated with the recommended drug, melarsoprol develop post treatment reactive encephalopathy, which is fatal in 50% of these patients, thus melarsoprol is fatal in 5% of all treated patients. This study was aimed at establishing the potential activity of Erythrina abyssinica in reducing neuroinflammation following infection with Trypanosoma brucei brucei. Swiss white mice were divided into ten groups, two control groups and eight infected groups. Infected mice received either methanol or water extract of Erythrina abyssinica at 12.5, 25, 50 or 100 mg/kg body weight. Parasite counts were monitored in peripheral circulation from the third day post infection up to the end of the study. Brains were processed for histology, immunohistochemistry scanning and transmission electron microscopy. Following infection, trypanosomes were observed in circulation 3 days post-infection, with the parasitaemia occurring in waves. In the cerebrum, typical brain pathology of chronic trypanosomiasis was reproduced. This was exhibited as astrocytosis, perivascular cuffing and infiltration of inflammatory cells into the neuropil. However, mice treated with Erythrina abyssinica water extract exhibited significant reduction in perivascular cuffing, lymphocytic infiltration and astrocytosis in the cerebrum. The methanol extract did not have a significant difference compared to the non-treated group. This study provides evidence of anti-inflammatory properties of Erythrina abyssinica and may support its wide use as a medicinal plant by various communities in Kenya.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aβ42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

White matter matters: a multimodal approach to investigate white matter microstructure and metabolism

White matter connects different brain areas and applies electrical insulation to the neuron’s axons with myelin sheaths in order to enable quick signal transmission. Due to its modulatory properties in signal conduction, white matter plays an essential role in learning, cognition and psychiatric disorders (Fields, 2008a). In respect thereof, the non-invasive investigation of white matter anatomy and function in vivo provides the unique opportunity to explore the most complex organ of our body. Thus, the present thesis aimed to apply a multimodal neuroimaging approach to investigate different white matter properties in psychiatric and healthy populations. On the one hand, white matter microstructural properties were investigated in a psychiatric population; on the other hand, white matter metabolic properties were assessed in healthy adults providing basic information about the brain’s wiring entity. As a result, three research papers are presented here. The first paper assessed the microstructural properties of white matter in relation to a frequent epidemiologic finding in schizophrenia. As a result, reduced white matter integrity was observed in patients born in summer and autumn compared to patients born in winter and spring. Despite the large genetic basis of schizophrenia, accumulating evidence indicates that environmental exposures may be implicated in the development of schizophrenia (A. S. Brown, 2011). Notably, epidemiologic studies have shown a 5–8% excess of births during winter and spring for patients with schizophrenia on the Northern Hemisphere at higher latitudes (Torrey, Miller, Rawlings, & Yolken, 1997). Although the underlying mechanisms are unclear, the seasonal birth effect may indicate fluctuating environmental risk factors for schizophrenia. Thus, exposure to harmful factors during foetal development may result in the activation of pathologic neural circuits during adolescence or young adulthood, increasing the risk of schizophrenia (Fatemi & Folsom, 2009). While white matter development starts during the foetal period and continues until adulthood, its major development is accomplished by the age of two years (Brody, Kinney, Kloman, & Gilles, 1987; Huang et al., 2009). This indicates a vulnerability period of white matter that may coincide with the fluctuating environmental risk factors for schizophrenia. Since microstructural alterations of white matter in schizophrenia are frequently observed, the current study provided evidence for the neurodevelopmental hypothesis of schizophrenia. In the second research paper, the perfusion of white matter showed a positive correlation between white matter microstructure and its perfusion with blood across healthy adults. This finding was in line with clinical studies indicating a tight coupling between cerebral perfusion and WM health across subjects (Amann et al., 2012; Chen, Rosas, & Salat, 2013; Kitagawa et al., 2009). Although relatively little is known about the metabolic properties of white matter, different microstructural properties, such as axon diameter and myelination, might be coupled with the metabolic demand of white matter. Furthermore, the ability to detect perfusion signal in white matter was in accordance with a recent study showing that technical improvements, such as pseudo-continuous arterial spin labeling, enabled the reliable detection of white matter perfusion signal (van Osch et al., 2009). The third paper involved a collaboration within the same department to assess the interrelation between functional connectivity networks and their underlying structural connectivity.