N-myc controls proliferation, morphogenesis, and patterning of the inner ear.

Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no apparent defects, whereas inactivation of N-myc results in reduced growth caused by a lack of proliferation. Reciprocally, the misexpression of N-myc in the inner ear increases proliferation. Morphogenesis of the inner ear in N-myc mouse mutants is severely disturbed, including loss of the lateral canal, fusion of the cochlea with the sacculus and utriculus, and stunted outgrowth of the cochlea. Mutant cochleas are characterized by an increased number of cells exiting the cell cycle that express the cyclin-dependent kinase inhibitor p27(Kip1) and lack cyclin D1, both of which control the postmitotic state of hair cells. Analysis of different molecular markers in N-myc mutant ears reveals the development of a rudimentary organ of Corti containing hair cells and the underlying supporting cells. Differentiated cells, however, fail to form the highly ordered structure characteristic for the organ of Corti but appear as rows or clusters with an excess number of hair cells. The Kölliker's organ, a transient structure neighboring the organ of Corti and a potential source of ectopic hair cells, is absent in the mutant ears. Collectively, our data suggest that N-myc regulates growth, morphogenesis, and pattern formation during the development of the inner ear.

Spatial and temporal down-regulation of transgene expression using the TRSID-silencer in mice: application to Prnp.

Spatial and temporal control of ovine prion protein (Prnp) gene expression was achieved in mice using two transgenes: a Prnp minigene with tet-operator sequences inserted 5' to exon 1 and a mouse neurofilament genomic clone carrying the chimeric-repressor TRSID cDNA. In bi-transgenic mice, ovine PrP(C) expression could be reversibly controlled in neuronal cells by doxycycline treatment whereas it remains constant in other cell types. Overall, this model opens opportunities to assess the involvement of cell types in prion diseases and PrP physiological function. It demonstrates the potentiality of the TRSID-silencer to precisely control temporal and spatial gene expression in vivo.

Temporal Profile of MicroRNA Expression in Contused Cortex after Traumatic Brain Injury in Mice.

MicroRNAs (miRNAs) were recently identified as important regulators of gene expression under a wide range of physiological and pathophysiological conditions. Thus, they may represent a novel class of molecular targets for the management of traumatic brain injury (TBI). In this study, we investigated the temporal profile of miRNA expression during the development of secondary brain damage after experimental TBI. For this purpose, we used a controlled cortical impact model in C57Bl/6 mice (n = 6) to induce a cortical contusion and analyzed miRNA expression in the traumatized cortex by microarray analysis during the development of secondary contusion expansion-i.e., at 1, 6, and 12 h after TBI. Of a total 780 mature miRNA sequences analyzed, 410 were detected in all experimental groups. Of these, 158 miRNAs were significantly upregulated or downregulated in TBI compared with sham-operated animals, and 52 miRNAs increased more than twofold. We validated the upregulation of five of the most differentially expressed miRNAs (miR-21*, miR-144, miR-184, miR-451, miR-2137) and the downregulation of four of the most differentially expressed miRNAs (miR-107, miR-137, miR-190, miR-541) by quantitative polymerase chain reaction (qPCR). miR-2137, the most differentially expressed miRNA after TBI, was further investigated by in situ hybridization and was found to be upregulated in neurons within the traumatic penumbra. This study gives a comprehensive picture of miRNA expression levels during secondary contusion expansion after TBI and may pave the way for the identification of novel targets for the management of brain trauma.

Identification of the Vascular Source of Vasogenic Brain Edema following Traumatic Brain Injury Using In Vivo 2-Photon Microscopy in Mice.

Vasogenic brain edema due to vascular leakage is one of the most important factors determining the clinical outcome of patients following acute brain injury. To date, performing a detailed in vivo quantification of vascular leakage has not been possible. Here, we used in vivo 2-photon microscopy (2-PM) to determine the spatial (3D) and temporal development of vasogenic brain edema following traumatic brain injury (TBI) in mice; in addition, we identified the vessel types involved in vascular leakage. Thirteen male Tie2-GFP mice (6-8 weeks old) were subjected to controlled cortical impact (CCI) or a sham operation; subsequently, a cranial window was prepared adjacent to the injury site, and tetramethylrhodamine-dextran (TMRM, 40 mg/kg, MW 40,000) was injected intravenously to visualize blood plasma leakage. Parenchymal fluorescence intensity was monitored in three regions for 2-4 h post-CCI, reaching from the surface of the brain to a depth of 300 µm, and TMRM leakage was measured as an increase in TMRM fluorescence intensity outside the vessel lumen and in the parenchyma. In the CCI group, vascular leakage was detected in all investigated regions as early as 2.5 h post-injury. This leakage increased over time and was more pronounced proximal to the primary contusion. Both arterioles and venules contributed similarly to brain edema formation and their contribution was independent of vessel size; however, capillaries were the major contributor to leakage. In summary, using 2-PM to perform in vivo 3D deep-brain imaging, we found that TBI induces vascular leakage from capillaries, venules, and arterioles. Thus, all three vessel types are involved in trauma-induced brain edema and should be considered when developing novel therapies for preventing vasogenic brain edema.

Endothelial nitric oxide synthase mediates arteriolar vasodilatation after traumatic brain injury in mice.

Brain edema and increased cerebral blood volume (CBV) contribute to intracranial hypertension and hence to unfavorable outcome after traumatic brain injury (TBI). The increased post-traumatic CBV may be caused in part by arterial vasodilatation. The aim of the current study was to uncover the largely unknown mechanisms of post-traumatic arteriolar vasodilatation. The diameter of pial arterioles and venules was monitored by intravital fluorescence microscopy before (baseline) and for 30 min after controlled cortical impact in C57BL/6 and endothelial nitric oxide synthase (eNOS)-/- mice (n=5-6/group) and in C57BL/6 mice (n=6/group) receiving vehicle (phosphate-buffered saline [PBS]) or 4-amino-tetrahydro-L-biopterine (VAS203), a NOS inhibitor previously shown to reduce post-traumatic intracranial hypertension. Temperature, end-tidal partial pressure of carbon dioxide (pCO2), and mean arterial blood pressure were kept within the physiological range throughout the experiments. Arteriolar diameters were stable during baseline monitoring but increased significantly in C57BL/6 mice after controlled cortical impact (136±7% of baseline; p<0.001 vs. baseline). This response was reduced by 78% in eNOS-/- mice (108±3% of baseline; p<0.005 vs. wild-type). Application of VAS203, a NOS inhibitor, or PBS did not affect vessels diameter before TBI. After trauma, however, administration of VAS203 reduced arteriolar diameter to 92±2% of baseline (p<0.05). The diameter of pial veins was not affected. Our results suggest that arteriolar vasodilatation after TBI is largely mediated by excess production of endothelial nitric oxide. Accordingly, our data may explain the beneficial effects of the NOS inhibitor VAS203 in the early phase after TBI and suggest that inhibition of excess endothelial nitric oxide production may represent a novel therapeutic strategy following TBI.

Prnp knockdown in transgenic mice using RNA interference.

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.