Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons.

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q<0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting ß-cells and neurons and underline the existence of trans­nosology pathways in diabetes and its co-morbidities.

Long-term distribution of biodegradable microparticles in rat muscle quantified noninvasively by MRI.

PURPOSE: To demonstrate the feasibility of MRI to monitor longitudinally the fate of PLGA microparticles in muscle tissue after intramuscular injection in rats using standard equipment. METHODS: MRI was performed at different time points and until day 28 after intramuscular administration of microparticles. Image segmentation was used to quantify the MRI signals. Histology was performed at selected time points to validate the in vivo observations. The SOM230-long acting release formulation was used as test compound. RESULTS: Microparticles were detected in vivo until 28 days following their administration. Imaging and histology data indicated that the MRI signals followed three phases: in an early phase (≤ 48 h after injection), vehicle, edema and hydration of microparticles contributed to the signals. In the second (days 3-17) and third phases (day 17 onward), microparticle hydration was the main contributor. SOM230 in blood displayed peaks at days 2 and 17. CONCLUSION: MRI was suitable to follow longitudinally the presence of PLGA microparticles in the rat muscle without labeling them. This is advantageous, because labeling could potentially alter the properties and pharmacokinetics of the microparticles. Data were consistent with an initial compound release followed by diffusion and microparticle erosion as main mechanisms of SOM230 release.

Hyaluronidase modulates bleomycin-induced lung injury detected noninvasively in small rodents by radial proton MRI.

PURPOSE: To assess whether hyaluronic acid, a building block of proteoglycans and extracellular matrix with hydrophilic characteristics, might contribute to magnetic resonance imaging (MRI)-detected proton signals elicited by bleomycin in the lung. To this end, hyaluronidase, which degrades hyaluronic acid, was administered to bleomycin-challenged animals. MATERIALS AND METHODS: Fibrosis was induced by oropharyngeal aspiration (OA) of bleomycin. Mice received bleomycin once daily (0.1 mg/kg) on 6 consecutive days, while rats were given a single dose (2 or 4 mg/kg). Hyaluronidase, budesonide, and the respective vehicles were also administered via OA. Animals were examined using a radial ultrashort echo time sequence. Histology of picrosirius reflecting collagen and tissue gene analysis were performed postmortem. RESULTS: In mice, hyaluronidase induced an increase of high intensity signals by 34 ± 12 µL (means ± SD, P = 0.007), consistent with the ability of the degradation products of hyaluronic acid to provoke acute inflammation. Budesonide was able to resolve hyaluronidase-induced signals or to prevent their formation. Combined administration of budesonide and hyaluronidase to bleomycin-treated rats resulted in an overall decrease (-17.1 ± 7%, P = 0.02) of the MRI-detected bleomycin-induced signals. Moreover, the relative gene expression of hyaluronidase was reduced (-61.8 ± 10.2%, P < 0.001) in fibrotic lungs. CONCLUSION: The present data indicate that hyaluronic acid contributes to the bleomycin-induced responses detected by MRI in the lung.

Adriamycin-induced nephropathy in rats: functional and cellular effects characterized by MRI.

PURPOSE: To assess with magnetic resonance imaging (MRI) adriamycin-induced nephropathy in living rats, an established model for proteinuric renal disease was used. MATERIALS AND METHODS: Functional information of contrast agent clearance was obtained with dynamic contrast-enhanced (DCE) imaging following intravenous Gd-DOTA administration. Perfusion data were obtained with a bolus tracking technique comprising intravenous injection of superparamagnetic iron oxide (SPIO) nanoparticles. Cellular information was derived from anatomical images acquired 24 hours after SPIO. Treatment with the transforming growth factor-ß123 (TGF-ß1,2,3 ) antibody, 1D11, started 1 week after adriamycin. Histology was performed at week 6 post-adriamycin. RESULTS: Tracer washout rates derived by DCE-MRI decreased by 65.5% with respect to baseline at week 6 post-adriamycin. The impaired kidney function agreed with glomerulopathy, nephropathy and fibrosis revealed histologically (picrosirius collagen staining in adriamycin-treated rats increased by 125.8% [P = 0.005] with respect to controls). Perfusion was reduced by 16.1%. Images acquired 24 hours after SPIO presented contrast changes that correlated inversely with the histologically determined iron content (R = -0.74, P = 2.6 × 10(-4) ). In adriamycin-challenged animals, iron was found in macrophages and in sclerotic tubuli, only in areas where macrophages were present. Treatment with 1D11 did not improve the adriamycin-induced renal injury. CONCLUSION: MRI provides longitudinal functional and cellular (macrophage infiltration) information that correlates with nephropathy development in adriamycin-challenged rats.

Lung volume quantified by MRI reflects extracellular-matrix deposition and altered pulmonary function in bleomycin models of fibrosis: effects of SOM230.

Idiopathic pulmonary fibrosis is a progressive and lethal disease, characterized by loss of lung elasticity and alveolar surface area, secondary to alveolar epithelial cell injury, reactive inflammation, proliferation of fibroblasts, and deposition of extracellular matrix. The effects of oropharyngeal aspiration of bleomycin in Sprague-Dawley rats and C57BL/6 mice, as well as of intratracheal administration of ovalbumin to actively sensitized Brown Norway rats on total lung volume as assessed noninvasively by magnetic resonance imaging (MRI) were investigated here. Lung injury and volume were quantified by using nongated or respiratory-gated MRI acquisitions [ultrashort echo time (UTE) or gradient-echo techniques]. Lung function of bleomycin-challenged rats was examined additionally using a flexiVent system. Postmortem analyses included histology of collagen and hydroxyproline assays. Bleomycin induced an increase of MRI-assessed total lung volume, lung dry and wet weights, and hydroxyproline content as well as collagen amount. In bleomycin-treated rats, gated MRI showed an increased volume of the lung in the inspiratory and expiratory phases of the respiratory cycle and a temporary decrease of tidal volume. Decreased dynamic lung compliance was found in bleomycin-challenged rats. Bleomycin-induced increase of MRI-detected lung volume was consistent with tissue deposition during fibrotic processes resulting in decreased lung elasticity, whereas influences by edema or emphysema could be excluded. In ovalbumin-challenged rats, total lung volume quantified by MRI remained unchanged. The somatostatin analog, SOM230, was shown to have therapeutic effects on established bleomycin-induced fibrosis in rats. This work suggests MRI-detected total lung volume as readout for tissue-deposition in small rodent bleomycin models of pulmonary fibrosis.

Noninvasive magnetic resonance imaging detection of cerebral amyloid angiopathy-related microvascular alterations using superparamagnetic iron oxide particles in APP transgenic mouse models of Alzheimer's disease: application to passive Abeta immunotherapy.

Cerebral amyloid angiopathy (CAA) is a common feature of Alzheimer's disease (AD). More advanced stages are accompanied by microhemorrhages and vasculitis. Peripheral blood-borne macrophages are intimately linked to cerebrovascular pathology coincident with AD. Magnetic resonance imaging (MRI) was used to noninvasively study microvascular lesions in amyloid precursor protein transgenic mouse AD models. Foci of signal attenuation were detected in cortical and thalamic brain regions of aged APP23 mice. Their strength and number was considerably enhanced by intravenous administration of iron oxide nanoparticles, which are taken up by macrophages through absorptive endocytosis, 24 h before image acquisition. The number of cortical sites displaying signal attenuation increased with age. Histology at these sites demonstrated the presence of iron-containing macrophages in the vicinity of CAA-affected blood vessels. A fraction of the sites additionally showed thickened vessel walls and vasculitis. Consistent with the visualization of CAA-associated lesions, MRI detected a much smaller number of attenuated signal sites in APP23xPS45 mice, for which a strong presenilin mutation caused a shift toward amyloid ß(42), thus reducing vascular amyloid. Similar results were obtained with APP24 and APP51 mice, which develop significantly less CAA and microvascular pathology than APP23. In a longitudinal study, we noninvasively demonstrated the reinforced formation of microvascular pathology during passive amyloid ß immunotherapy of APP23 mice. Histology confirmed that foci of signal attenuation reflected an increase in CAA-related lesions. Our data demonstrate that MRI has the sensitivity to noninvasively monitor the development of vascular pathology and its possible enhancement by amyloid ß immunotherapy in transgenic mice modeling AD.

Bleomycin-induced lung injury in mice investigated by MRI: model assessment for target analysis.

Magnetic resonance imaging (MRI) has been used to follow the course of bleomycin-induced lung injury in mice and to investigate two knockout mouse lines with the aim of providing potential therapeutic targets. Bleomycin (0.25 mg/kg) was administered intranasally six times, once a day. MRI was carried out on spontaneously breathing animals up to day 70 after bleomycin. Neither cardiac nor respiratory gating was applied during image acquisition. A long lasting response following bleomycin has been detected by MRI in the lungs of male C57BL/6 mice. Histology showed that, from day 14-70 after bleomycin, fibrosis was the predominant component of the injury. Female C57BL/6 mice displayed a smaller response than males. Bleomycin-induced injury was significantly more pronounced in C57BL/6 than in Balb/C mice. MRI and histology demonstrated a protection against bleomycin insult in female heterozygous and male homozygous cancer Osaka thyroid kinase knockout animals. In contrast, no protection was seen in cadherin-11 knockout animals. In summary, MRI can quantify, in spontaneously breathing mice, bleomycin-induced lung injury. With the ability for repetitive measurements in the same animal, the technique is attractive for in vivo target analysis and compound profiling in this murine model.

Noninvasive assessment of bleomycin-induced lung injury and the effects of short-term glucocorticosteroid treatment in rats using MRI.

PURPOSE: To demonstrate the feasibility of proton MRI to noninvasively quantify bleomycin-induced injury and the effects of glucocorticosteroids in a rat model of lung fibrosis. MATERIALS AND METHODS: Sprague-Dawley rats received bleomycin intra-tracheally and underwent MRI up to day 70 following injury onset. A subgroup of animals was treated with budesonide. RESULTS: The response in the first 2 weeks post-bleomycin, characterized by diffuse MRI signals, was related primarily to inflammation as confirmed by histology. Later, increased signals reflected principally tissue remodeling involved in fibrosis development, as suggested by histological analysis revealing collagen deposition in the same areas where MRI signals had been detected. Budesonide administration at days 6 and 13 after bleomycin resulted in decreased MRI signals 24 h after each corticosteroid application. However, no complete signal resolution was observed. Histology showed that budesonide affected inflammation but not fibrosis. CONCLUSION: The ability of MRI to noninvasively quantify lung injury in bleomycin-treated rats will facilitate in vivo pharmacological studies in this model of pulmonary fibrosis. Repetitive measurements open new avenues in testing compounds as the responses at several time points during the course of treatment can be easily compared. Specifically, studies at the chronic phase, when fibrosis is already established, become amenable.

Effects of the fibroblast activation protein inhibitor, PT100, in a murine model of pulmonary fibrosis.

Bleomycin (BLM) induced lung injury is detectable in C57BL/6 mice using magnetic resonance imaging (MRI). We investigated the effects of the fibroblast activation protein (FAP) inhibitor, PT100, in this model. BLM (0.5mg/kg/day) was administered on days -7, -6, -5, -2, -1, 0 in the nostrils of male mice. PT100 (40µg/mouse) or vehicle (0.9%NaCl) was dosed per os twice daily from day 1-14. MRI was performed before BLM and at days 0, 7 and 14. After the last MRI acquisition, animals were euthanised and the lungs harvested for histological and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. As evidenced longitudinally by MRI, the BLM-elicited lesions in the lungs of vehicle-treated mice progressed over time. In contrast, responses elicited by BLM did not progress in animals receiving PT100. Histology demonstrated significant less fibrosis in PT100- than in vehicle-treated, BLM-challenged mice. Significant correlation (R=0.91, P<0.001, N=24) was found between the volumes of BLM-induced lesions detected in vivo by MRI and the collagen content determined histologically (picrosirius staining). FAP was overexpressed in the lungs of BLM-challenged mice. Upon PT100 treatment, FAP expression was reduced. Significant differences in the MMP-12, MIP-1α, and MCP-3 mRNA expression levels in the lungs of PT100- compared to vehicle-treated mice were also revealed by qRT-PCR. The IBA-1 level determined histologically was higher in the lungs of PT100- compared to vehicle-treated mice. Taken together, these observations suggest that treatment with PT100 in this murine model of pulmonary fibrosis had an anti-fibro-proliferative effect and increased macrophage activation.

[Late abdominal compartment syndrome secondary to traumatic gallbladder rupture]. / Syndrome du compartiment abdominal tardif secondaire à une rupture traumatique de la vésicule biliaire.

A case of abdominal compartment syndrome following hepatic rupture with gallbladder tear is reported. We discuss the physiology, diagnosis criteria and treatment of this potentially life-threatening complication.

Administration of bleomycin via the oropharyngeal aspiration route leads to sustained lung fibrosis in mice and rats as quantified by UTE-MRI and histology.

Pulmonary fibrosis can be experimentally induced in small rodents by bleomycin. The antibiotic is usually administered via the intratracheal or intranasal routes. In the present study, we investigated the oropharyngeal aspiration of bleomycin as an alternative route for the induction of lung fibrosis in rats and mice. The development of lung injury was followed in vivo by ultrashort echo time magnetic resonance imaging (UTE-MRI) and by post-mortem analyses (histology of collagen, hydroxyproline determination, and qRT-PCR). In C57BL/6 mice, oropharyngeal aspiration of bleomycin led to more prominent lung fibrosis as compared to intranasal administration. Consequently, the oropharyngeal aspiration route allowed a dose reduction of bleomycin and, therewith, a model refinement. Moreover, the distribution of collagen after oropharyngeal aspiration of bleomycin was more homogenous than after intranasal administration: for the oropharyngeal aspiration route, fibrotic areas appeared all over the lung lobes, while for the intranasal route fibrotic lesions appeared mainly around the largest superior airways. Thus, oropharyngeal aspiration of bleomycin induced morphological changes that were more comparable to the human disease than the intranasal administration route did. Oropharyngeal aspiration of bleomycin led to a homogeneous fibrotic injury also in rat lungs. The present data suggest oropharyngeal aspiration of bleomycin as a less invasive means to induce homogeneous and sustained fibrosis in the lungs of mice and rats.

Temporally controlled site-specific mutagenesis in the germ cell lineage of the mouse testis.

We have obtained a PrP-Cre-ER(T) transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ER(T) recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ER(T) is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ER(T) line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis.

Germ cell expression of the transcriptional co-repressor TIF1beta is required for the maintenance of spermatogenesis in the mouse.

The gene for transcriptional intermediary factor 1beta (TIF1beta) encodes a transcriptional co-repressor known to play essential roles in chromatin remodeling as well as in early embryonic development. During spermatogenesis, TIF1beta is preferentially associated with heterochromatin structures of Sertoli cells and round spermatids, as well as with meiotic chromosomes. Its expression is tightly regulated within spermatocyte and spermatid populations, and it is undetectable in spermatogonia. Spatiotemporally controlled ablation of TIF1beta by using a germ cell lineage-specific CreER(T)/loxP system leads to testicular degeneration. This degeneration is not due to impairment of chromatin remodeling processes during meiosis and spermiogenesis, as TIF1beta-deficient spermatocytes are able to complete their differentiation into spermatozoa. It rather occurs as a consequence of shedding of immature germ cells (spermatocytes and spermatids), and disappearance of stem spermatogonia. These results indicate that TIF1beta has important functions in the homeostasis of the seminiferous epithelium, and probably plays a crucial role in the network of paracrine interactions between germ cell subpopulations and/or Sertoli cells.

Physiological and retinoid-induced proliferations of epidermis basal keratinocytes are differently controlled.

To investigate the roles of retinoic acid (RA) receptors (RARs) in the physiology of epidermis that does not express RAR beta, conditional spatio-temporally controlled somatic mutagenesis was used to selectively ablate RAR alpha in keratinocytes of RAR gamma-null mice. Keratinocyte proliferation was maintained in adult mouse epidermis lacking both RAR alpha and RAR gamma, as well as in RAR beta-null mice. All RAR-mediated signalling pathways are therefore dispensable in epidermis for homeostatic keratinocyte renewal. However, topical treatment of mouse skin with selective retinoids indicated that RXR/RAR gamma heterodimers, in which RXR transcriptional activity was subordinated to that of its RAR gamma partner, were required for retinoid-induced epidermal hyperplasia, whereas RXR homodimers and RXR/RAR alpha heterodimers were not involved. RA-induced keratinocyte proliferation was studied in mutant mice in which RXR alpha, RXR alpha and RAR alpha, RAR gamma, or RXR alpha and RAR gamma genes were specifically disrupted in either basal or suprabasal keratinocytes. We demonstrate that the topical retinoid signal is transduced by RXR alpha/RAR gamma heterodimers in suprabasal keratinocytes, which, in turn, stimulate proliferation of basal keratinocytes via a paracrine signal that may be heparin-binding EGF-like growth factor.