Comprehensive small animal imaging strategies on a clinical 3 T dedicated head MR-scanner; adapted methods and sequence protocols in CNS pathologies

Background

Small animal models of human diseases are an indispensable aspect of pre-clinical research. Being dynamic, most pathologies demand extensive longitudinal monitoring to understand disease mechanisms, drug efficacy and side effects. These considerations often demand the concomitant development of monitoring systems with sufficient temporal and spatial resolution.

Methodology and Results

This study attempts to configure and optimize a clinical 3 Tesla magnetic resonance scanner to facilitate imaging of small animal central nervous system pathologies. The hardware of the scanner was complemented by a custom-built, 4-channel phased array coil system. Extensive modification of standard sequence protocols was carried out based on tissue relaxometric calculations. Proton density differences between the gray and white matter of the rodent spinal cord along with transverse relaxation due to magnetic susceptibility differences at the cortex and striatum of both rats and mice demonstrated statistically significant differences. The employed parallel imaging reconstruction algorithms had distinct properties dependent on the sequence type and in the presence of the contrast agent. The attempt to morphologically phenotype a normal healthy rat brain in multiple planes delineated a number of anatomical regions, and all the clinically relevant sequels following acute cerebral ischemia could be adequately characterized. Changes in blood-brain-barrier permeability following ischemia-reperfusion were also apparent at a later time. Typical characteristics of intra-cerebral haemorrhage at acute and chronic stages were also visualized up to one month. Two models of rodent spinal cord injury were adequately characterized and closely mimicked the results of histological studies. In the employed rodent animal handling system a mouse model of glioblastoma was also studied with unequivocal results.

Conclusions

The implemented customizations including extensive sequence protocol modifications resulted in images of high diagnostic quality. These results prove that lack of dedicated animal scanners shouldn''t discourage conventional small animal imaging studies.     

Nonhematopoietic cells are key players in innate control of bacterial airway infection

Airborne pathogens encounter several hurdles during host invasion, including alveolar macrophages (AMs) and airway epithelial cells (AECs) and their products. Although growing evidence indicates pathogen-sensing capacities of epithelial cells, the relative contribution of hematopoietic versus nonhematopoietic cells in the induction of an inflammatory response and their possible interplay is still poorly defined in vivo in the context of infections with pathogenic microorganisms. In this study, we show that nonhematopoietic cells, including AECs, are critical players in the inflammatory process induced upon airway infection with Legionella pneumophila, and that they are essential for control of bacterial infections. Lung parenchymal cells, including AECs, are not infected themselves by L. pneumophila in vivo but rather act as sensors and amplifiers of inflammatory cues delivered by L. pneumophila-infected AM. We identified AM-derived IL-1β as the critical mediator to induce chemokine production in nonhematopoietic cells in the lung, resulting in swift and robust recruitment of infection-controlling neutrophils into the airways. These data add a new level of complexity to the coordination of the innate immune response to L. pneumophila and illustrate how the cross talk between leukocytes and nonhematopoietic cells contributes to efficient host protection.     

Nitrate reductase in needles,roots and trunk wood of spruce trees [Picea abies (L.) Karst.]

Summary Nitrate reductase (EC 1.6.6.2) activity (NRA), as measured by an in vivo assay, is present in needle leaves and mycorrhizal fine root tips of adult Norway spruce [Picea abies (L.) Karst.] in at least equal amounts on a fresh weight basis, in both adult and 5-year-old trees. NRA could also be demonstrated in trunk wood of deroted trees after fertilization with 5 mM , exhibiting a longitudinal profile in the trunk. Inducibility in needles can more efficiently be achieved by NO₂ (100 g·m^-3) than by 5 mM nitrate, which is effective only in root-amputated trees. A remarkably high level of needle-NRA in unfertilized trees, which are characterized by a very low level of nitrate in the xylem sap, suggests that NRA in spruce needles may in part be constitutive. Organic-N is a major nitrogen source for the needles even in root-amputated trees, indicating pronounced exchange processes between ray parenchyma and trunk xylem, which in turn are modified by the nitrogen source fed to the trunk stump. Intact trees exhibit a very similar amino acid composition of the xylem sap, regardless of whether or has been fed. The amino acid pattern of the needles is not thrown out of balance by flooding with and , which occurs in fertilized derooted trees. This indicates a distinct potential for homoeostasis of nitrogen entrance-metabolism (i.e. NRA and glutamine synthetase activity) in the needles. In the ectomycorrhiza/fine root-system (EMC), marked differences in NRA were observed depending on root-tip diameter and along the longitudinal profile of the fine roots. EMC-nitrate reductase is strongly enhanced by . Needle-NRA exhibits a circannual rhythm. An early summer maximum is followed by a December minimum. This activity pattern matches well the transitory increase of soluble nitrogen in spring and the total protein maximum in winter. In an indirect way assimilatory NRA may well contribute to nitrogen overfertilization (by consumption of NO_X) as one possible cause of the contemporary decline of spruce populations.     

Comparative genomic hybridisation and ultrafast pyrosequencing revealed remarkable differences between the Sinorhizobium meliloti genomes of the model strain Rm1021 and the field isolate SM11

Genomic variation between the Sinorhizobium meliloti model strain Rm1021 and the field isolate SM11 was assessed by using the genome-wide S. meliloti Rm1021 Sm6k-oligonucleotide microarray in a comparative genomic hybridisation experiment. Several gene clusters present in the Rm1021 genome are missing in the SM11 genome. In detail, three missing gene clusters were identified for the chromosome, five for megaplasmid pSymA and two for megaplasmid pSymB. To confirm these hybridisation results, the draft genome sequence of the S. meliloti field isolate SM11 was established by 454-pyrosequencing. Three sequencing runs on the ultrafast Genome Sequencer 20 System yielded 112.5 million bases. These could be assembled into 905 larger contigs resulting in a nearly 15-fold coverage of the 7.1Mb SM11 genome. The missing gene regions identified by comparative genomic hybridisation could be confirmed by the results of the 454-sequencing project. An in-depth analysis of these gene regions resulted in the following findings: (i) a complete type I restriction/modification system encoded by a composite transposon is absent in the chromosome of strain SM11. (ii) Most of the Rm1021 denitrification genes and the complete siderophore biosynthesis operon were found to be missing on SM11 megaplasmid pSymA. (iii) S. meliloti SM11 megaplasmid pSymB lacks a complete cell surface carbohydrate synthesis gene cluster. (iv) Several genes that are absent in the SM11 genome could be assigned to insertion sequences and transposons.     

The Role of Specific Tomato Volatiles in Tomato-Whitefly Interaction

Bemisia tabaci (whitefly) infestations and the subsequent transfer of viruses are the cause of severe losses in crop production and horticultural practice. To improve biological control of B. tabaci, we investigated repellent properties of plant-produced semiochemicals. The mix of headspace volatiles, collected from naturally repellent wild tomato accessions, influenced B. tabaci initial choice behavior, indicating a role for plant semiochemicals in locating host plants. A collection of wild tomato accessions and introgression lines (Solanum pennellii LA716 × Solanum lycopersicum ‘Moneyberg’) were extensively screened for attractiveness to B. tabaci, and their headspace profiles were determined by means of gas chromatography-mass spectrometry. Correlation analysis revealed that several terpenoids were putatively involved in tomato-whitefly interactions. Several of these candidate compounds conferred repellence to otherwise attractive tomato plants when applied to the plant''s branches on paper cards. The sesquiterpenes zingiberene and curcumene and the monoterpenes p-cymene, α-terpinene, and α-phellandrene had the strongest effects in free-choice bioassays. These terpenes also elicited a response of receptors on the insect''s antennae as determined by electroantennography. Conversely, the monoterpene β-myrcene showed no activity in both assays. B. tabaci apparently uses, besides visual cues, specific plant volatile cues for the initial selection of a host. Altering whitefly choice behavior by manipulation of the terpenoid composition of the host headspace may therefore be feasible.During the last decades, a worldwide spread of the pest insects Bemisia tabaci (Gennadius) and greenhouse whitefly (Trialeurodes vaporariorum) has led to local devastation of vegetable and ornamental crops, resulting in large economic losses. The damage whiteflies cause by their feeding behavior, such as affected biochemistry and development (for review, see Inbar and Gerling, 2008), is far exceeded by the secondary, indirect crop losses due to virus transmission. Specifically B. tabaci outbreaks are associated with the emergence of viruses for which they serve as vectors (Polston and Anderson, 1997). B. tabaci is capable of transmitting >100 different virus species of which the majority belong to the genus Begomovirus, such as Tomato yellow leaf curl virus, Tomato mottle virus (Jones, 2003), and African cassava mosaic virus (Maruthi et al., 2001). Damage caused by virus infection ranges from mild symptoms, such as leaf discolorations, to overall yield reduction, severe fruit necrosis, flower and fruit abortions, and plant death. Viral diseases are particularly severe since no chemical control is available and good sources of virus resistance for interspecific crossing are not always available (Maruthi et al., 2003). To date, only a limited number of virus resistance genes have been identified, and due to high mutation rates, viruses rapidly evolve (Drake and Holland, 1999; García-Andrés et al., 2006) and break monogenic resistances. Herbivores, such as whiteflies and thrips, can apparently benefit from transmitting viruses (Medeiros et al., 2004; Jiu et al., 2007; Belliure et al., 2008).B. tabaci was originally restricted to subtropical regions and greenhouses. However, the new and extremely invasive B and Q biotypes have the ability to rapidly adapt to more temperate zones and new host species (Jones, 2003; Wan et al., 2008). To date, the main control strategy for many crops is the application of insecticides, though effective spraying is complicated because of the insect''s preference for the abaxial side of the leaf (Simmons, 1994). Moreover, B. tabaci is difficult to control chemically due to emerging resistance to active ingredients (Horowitz et al., 2005). A new biological control agent, the phytoseiid predator Typhlodromips swirskii, has only been successful on plants without trichomes in closed greenhouses (Nomikou et al., 2002). The root-knot nematode resistance gene Mi1.2, which confers partial resistance to B. tabaci (Nombela et al., 2003), is widely used in modern tomato (Solanum spp.) varieties but is not sufficient to provide adequate protection against whitefly infestations.During insect host selection, orientation, and landing, both visual and olfactory cues play a predominant role (Visser, 1988). Color is an important factor in host-plant selection, and it was shown that B. tabaci reacts to blue-UV and yellow wavelengths (Van Lenteren and Noldus, 1990). The olfactory stimuli associated with the host plant initiate host targeting, whereas visual cues improve the accuracy of landing. In the initial phase of host targeting, olfaction may cause a positive chemotactic response, i.e. a flight up an odor gradient. Plant odor specificity might be achieved by a particular ratio of constituent volatiles (Bruce et al., 2005a). In the case of whiteflies, the role of olfaction in attraction or repellence has not received much prior attention. After host contact, B. tabaci evaluates host plant quality by labial dabbing and probing using piercing mouthparts. By probing, persistent viruses are transmitted via the insects'' salivary glands and mouthparts (Ghanim et al., 1998; Rosell et al., 1999). Therefore, to avoid virus transmission by B. tabaci, probing should be prevented.Volatile organic compounds released by plants can act as semiochemicals. They play an important role in enabling insects to recognize host plants from a distance (Schütz et al., 1997; Bruce et al., 2005a) or in attracting predators and parasitoids upon herbivory (De Moraes et al., 1998; Van Poecke and Dicke, 2002; Kappers et al., 2005). Moreover, they can play a role in the direct defense against herbivores and pathogens (Kessler and Baldwin, 2001; Shiojiri et al., 2006). A large number of different plant volatiles, with numerous ecological roles, have been identified so far (Sacchettini and Poulter, 1997; Pichersky et al., 2006). The largest class of plant volatiles is derived from the isoprenoid or terpenoid pathway. Solanaceous plants, like tomato, often make use of these terpenes for the defense against herbivores (Snyder et al., 1993; Kennedy, 2003). Some terpenes have been shown to exhibit repellent properties to insects (Peterson et al., 2002; Birkett et al., 2004; Terry et al., 2007). These plant-produced semiochemicals can potentially be used as insect repellents of natural origin, thus providing an alternative to the use of pesticides (Peterson and Coats, 2001). Engineering terpene emission to make crop plants more attractive to herbivore enemies has already been shown to be feasible (Degenhardt et al., 2003; Kappers et al., 2005; Schnee et al., 2006).The aim of this study is to identify the role of plant volatiles in the B. tabaci-tomato host interaction and to identify the terpenes that cause repellence of a selection of wild tomato accessions. The potential of several terpenes as repellent olfactory cues in B. tabaci host-preference behavior has been assessed in behavioral studies and through electroantennography (EAG).     

Phylogenetic Analysis of Bacterial Communities Associated with Leaves of the Seagrass Halophila stipulacea by a Culture-Independent Small-Subunit rRNA Gene Approach

Abstract The phylogenetic diversity of the bacterial community associated with leaves of the marine plant Halophila stipulacea in the northern Gulf of Elat was examined by 16S rRNA gene (rDNA) sequence analyses of a clone library. For 59 clones corresponding to 51 ARDRA (amplified rDNA restriction analysis) groups, the sequence of ∼1 kb was determined, and the fraction of the corresponding ARDRA groups of the leaf library was calculated. The class Proteobacteria was represented by 62.6% of the clone sequences. Most sequences originated from members of the γ-subclass (27.3%), affiliated with members of the genera Pseudomonas, Vibrio, Marinomonas, Oceanospirillum, and other marine groups. Affiliation to the α-subclass was determined for 24.2% of the sequences. They were related to the genera Hyphomonas, Roseobacter, Ruegeria, and Rhizobiaceae. Several α-proteobacterial sequences were distantly related to known sequences. Only 4% of the clone sequences were related to β-Proteobacteria. Additionally, 7.1% of the sequences possibly belonged to the class Proteobacteria, but branched deeply from known subclasses. Several sequences were affiliated to members of the orders Verrucomicrobiales and Planctomycetales, the Holophaga/Acidobacterium phylum, and chloroplasts of marine diatoms. Received: 20 March 1999; Accepted: 13 August 1999; Online Publication: 2 March 2000     

Neural stem cells for spinal cord repair

Spinal cord injury (SCI) causes the irreversible loss of spinal cord parenchyma including astroglia, oligodendroglia and neurons. In particular, severe injuries can lead to an almost complete neural cell loss at the lesion site and structural and functional recovery might only be accomplished by appropriate cell and tissue replacement. Stem cells have the capacity to differentiate into all relevant neural cell types necessary to replace degenerated spinal cord tissue and can now be obtained from virtually any stage of development. Within the last two decades, many in vivo studies in small animal models of SCI have demonstrated that stem cell transplantation can promote morphological and, in some cases, functional recovery via various mechanisms including remyelination, axon growth and regeneration, or neuronal replacement. However, only two well-documented neural-stem-cell-based transplantation strategies have moved to phase I clinical trials to date. This review aims to provide an overview about the current status of preclinical and clinical neural stem cell transplantation and discusses future perspectives in the field.     

Failure of Schwann cells as supporting cells for adult neural progenitor cell grafts in the acutely injured spinal cord

Adult neural progenitor cells (NPC) co-grafted with fibroblasts replace cystic lesion defects and promote cell-contact-mediated axonal regeneration in the acutely injured spinal cord. Fibroblasts are required as a platform to maintain NPC within the lesion; however, they are suspected to create an inhospitable milieu for regenerating central nervous system (CNS) axons. Therefore, we thought to replace fibroblasts by primary Schwann cells, which might serve as a superior scaffold to maintain NPC within the lesion and might further enhance axon regrowth and remyelination following spinal cord injury. Adult rats underwent a cervical dorsal column transection immediately followed by transplantation of either NPC/Schwann cell or NPC/Schwann cell/fibroblast co-grafts. Animals receiving Schwann cell or fibroblast grafts alone, or Schwann cell/fibroblast co-grafts served as controls. At 3 weeks after injury/transplantation, histological analysis revealed that only fibroblast-containing grafts were able to replace the cystic lesion defect. In both co-cultures and co-grafts, Schwann cells and NPC were segregated. Almost all NPC migrated out of the graft into the adjacent host spinal cord. As a consequence, only peripheral-type myelin, but no CNS-type myelin, was detected within co-grafts containing NPC/Schwann cells. Corticospinal axon regeneration into Schwann-cell-containing co-grafts was reduced. Taken together, Schwann cells within NPC grafts contribute to remyelination. However, Schwann cells fail as a supporting platform to maintain NPC within the graft and impair CNS axon regeneration; this makes them an unfavorable candidate to support/augment NPC grafts following spinal cord injury.This work was supported by the Institute International de Recherche en Paraplégie Geneva, on behalf of an anonymous donation, and ReForM-Program, University of Regensburg, School of Medicine.