Induction of apoptosis by mercury compounds depends on maturation and is not associated with microglial activation

The earliest sign of neurotoxicity observed after exposure of three-dimensional brain cell cultures to low concentrations of mercury compounds is a microglial reaction. We hypothesized that an induction of apoptosis by mercury compounds could be an activating signal of the microglial reaction. Aggregating brain cell cultures of fetal rat telencephalon were treated for 10 days with either mercury chloride or monomethylmercury chloride at noncytotoxic concentrations during two developmental periods: from day 5 to 15, corresponding to an immature stage, and from day 25 to 35 corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. It was found that both mercury compounds caused a significant increase in the number of apoptotic cells, but exclusively in immature cultures exhibiting also spontaneous apoptosis. Double staining by the TUNEL technique combined with either neuronal or astroglial markers revealed that the proportion of cells undergoing apoptosis was highest for astrocytes. Furthermore neither an association nor a colocalization was found between apoptotic cells and microglial cells. In conclusion, it appears that the induction of apoptosis by mercury compounds in immature cells is only an acceleration of a spontaneously occurring process, and that it is not a directly related to the early microglial reaction.

The neurochemical profile quantified by in vivo 1H NMR spectroscopy.

Proton NMR spectroscopy is emerging from translational and preclinical neuroscience research as an important tool for evidence based diagnosis and therapy monitoring. It provides biomarkers that offer fingerprints of neurological disorders even in cases where a lesion is not yet observed in MR images. The collection of molecules used as cerebral biomarkers that are detectable by (1)H NMR spectroscopy define the so-called "neurochemical profile". The non-invasive quality of this technique makes it suitable not only for diagnostic purposes but also for therapy monitoring paralleling an eventual neuroprotection. The application of (1)H NMR spectroscopy in basic and translational neuroscience research is discussed here.

An angiotensin II- and NF-kappaB-dependent mechanism increases connexin 43 in murine arteries targeted by renin-dependent hypertension.

AIMS: Connexins (Cxs) play a role in the contractility of the aorta wall. We investigated how connexins of the endothelial cells (ECs; Cx37, Cx40) and smooth muscle cells (SMCs; Cx43, Cx45) of the aorta change during renin-dependent and -independent hypertension. METHODS AND RESULTS: We subjected both wild-type (WT) mice and mice lacking Cx40 (Cx40(-/-)), to either a two-kidney, one-clip procedure or to N-nitro-l-arginine-methyl-ester treatment, which induce renin-dependent and -independent hypertension, respectively. All hypertensive mice featured a thickened aortic wall, increased levels of Cx37 and Cx45 in SMC, and of Cx40 in EC (except in Cx40(-/-) mice). Cx43 was up-regulated, with no effect on its S368 phosphorylation, only in the SMCs of renin-dependent models of hypertension. Blockade of the renin-angiotensin system of Cx40(-/-) mice normalized blood pressure and prevented both aortic thickening and Cx alterations. Ex vivo exposure of WT aortas, carotids, and mesenteric arteries to physiologically relevant levels of angiotensin II (AngII) increased the levels of Cx43, but not of other Cx. In the aortic SMC line of A7r5 cells, AngII activated kinase-dependent pathways and induced binding of the nuclear factor-kappa B (NF-kappaB) to the Cx43 gene promoter, increasing Cx43 expression. CONCLUSION: In both large and small arteries, hypertension differently regulates Cx expression in SMC and EC layers. Cx43 is selectively increased in renin-dependent hypertension via an AngII activation of the extracellular signal-regulated kinase and NF-kappaB pathways.

Probing the interaction between feline immunodeficiency virus and CD134 by using the novel monoclonal antibody 7D6 and the CD134 (Ox40) ligand.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+ T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+ T cells, with weaker expression on CD8+ T cells, concordant with the expansion of FIV into CD8+ T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.

Differential regulation of Na-K-ATPase isoform gene expression by T3 during rat brain development.

A fetal rat telencephalon organotypic cell culture system was found to reproduce the developmental pattern of Na-K-adenosinetriphosphatase (ATPase) gene expression observed in vivo [Am. J. Physiol. 258 (Cell Physiol. 27): C1062-C1069, 1990]. We have used this culture system to study the effects of triiodothyronine (T3; 0.003-30 nM) on mRNA abundance and basal transcription rates of Na-K-ATPase isoforms. Steady-state mRNA levels were low at culture day 6 (corresponding to the day of birth) but distinct for each isoform alpha 3 much greater than beta 1 = beta 2 greater than alpha 2 greater than alpha 1. At culture day 6, T3 did not modify mRNA abundance of any isoform. At culture day 12 (corresponding to day 7 postnatal), T3 increased the mRNA level of alpha 2 (4- to 7-fold), beta 2 (4- to 5-fold), alpha 1 (3- to 6-fold), and beta 1 (1.5-fold), whereas alpha 3 mRNA levels remained unchanged. Interestingly, the basal transcription rate for each isoform differed strikingly (alpha 2 greater than alpha 1 much greater than beta 1 = beta 2 greater than alpha 3) but remained stable throughout 12 days of culture and was not regulated by T3. Thus we observed an inverse relationship between rate of transcription and rate of mRNA accumulation for each alpha-isoform, suggesting that alpha 1- and alpha 2-mRNA are turning over rapidly whereas alpha 3-mRNA is turning over slowly. Our data indicate that one of the mechanisms by which T3 selectively controls Na-K-ATPase gene expression during brain development in vitro occurs at the posttranscriptional level.

Insulin and IGF-1 enhance the expression of the neuronal monocarboxylate transporter MCT2 by translational activation via stimulation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin pathway.

MCT2 is the main neuronal monocarboxylate transporter essential for facilitating lactate and ketone body utilization as energy substrates. Our study reveals that treatment of cultured cortical neurons with insulin and IGF-1 led to a striking enhancement of MCT2 immunoreactivity in a time- and concentration-dependent manner. Surprisingly, neither insulin nor IGF-1 affected MCT2 mRNA expression, suggesting that regulation of MCT2 protein expression occurs at the translational rather than the transcriptional level. Investigation of the putative signalling pathways leading to translation activation revealed that insulin and IGF-1 induced p44- and p42 MAPK, Akt and mTOR phosphorylation. S6 ribosomal protein, a component of the translational machinery, was also strongly activated by insulin and IGF-1. Phosphorylation of p44- and p42 MAPK was blocked by the MEK inhibitor PD98058, while Akt phosphorylation was abolished by the PI3K inhibitor LY294002. Phosphorylation of mTOR and S6 was blocked by the mTOR inhibitor rapamycin. In parallel, it was observed that LY294002 and rapamycin almost completely blocked the effects of insulin and IGF-1 on MCT2 protein expression, whereas PD98059 and SB202190 (a p38K inhibitor) had no effect on insulin-induced MCT2 expression and only a slight effect on IGF-1-induced MCT2 expression. At the subcellular level, a significant increase in MCT2 protein expression within an intracellular pool was observed while no change at the cell surface was apparent. As insulin and IGF-1 are involved in synaptic plasticity, their effect on MCT2 protein expression via an activation of the PI3K-Akt-mTOR-S6K pathway might contribute to the preparation of neurons for enhanced use of nonglucose energy substrates following altered synaptic efficacy.

Influence of environmental factors on the expression of phenotypic characters by chicken dorsal root ganglion cells in culture.

Primary sensory neurons were grown under four conditions of culture. The influence of nonneuronal cells, horse serum or both was studied on the phenotypic expression of certain neuronal subpopulations. The number of neurons expressing acetylcholinesterase, alpha-bungarotoxin-binding sites or a high uptake capacity for glutamine was enhanced by nonneuronal cells. The horse serum increases the neuronal subpopulation exhibiting a carbonic anhydrase activity. Certain phenotypic changes fit conditions consistent with an epigenetic induction rather than a cell selection.

Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model.

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.

A new perspective on the evolutionary history of western European Sorex araneus group revealed by paternal and maternal molecular markers.

The species of the common shrew (Sorex araneus) group are morphologically very similar, but have undergone a spectacular chromosomal evolution. We investigate here the evolutionary history of the Sorex araneus group distributed in western Europe. In particular, we clarify the position of a difficult species, S. granarius, using sex-specific (mtDNA and Y-chromosome) markers. The karyotype of S. granarius is generally considered similar to the common ancestor of the restricted group considered here. The mtDNA data (1.4 kb) confirms the close relationship between S. granarius and S. araneus sensu stricto (hereafter S. araneus s.s.), but the Y-chromosome (3.4 kb) produces a quite different picture: S. granarius is closely related to another species, S. coronatus. Comparison of mtDNA and Y-chromosome phylogenies suggests that the genetic and chromosomal evolution in this group are disconnected processes. The evolutionary history of the south-western European populations of the S. araneus group can only be understood considering secondary contacts between taxa after their divergence, implying genetic exchanges by means of hybridization and/or introgression.

Selective in vivo visualization of immune-cell infiltration in a mouse model of autoimmune myocarditis by fluorine-19 cardiac magnetic resonance.

BACKGROUND: The goal of this study was to characterize the performance of fluorine-19 ((19)F) cardiac magnetic resonance (CMR) for the specific detection of inflammatory cells in a mouse model of myocarditis. Intravenously administered perfluorocarbons are taken up by infiltrating inflammatory cells and can be detected by (19)F-CMR. (19)F-labeled cells should, therefore, generate an exclusive signal at the inflamed regions within the myocardium. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in BALB/c mice. After intravenous injection of 2×200 µL of a perfluorocarbon on day 19 and 20 (n=9) after immunization, in vivo (19)F-CMR was performed at the peak of myocardial inflammation (day 21). In 5 additional animals, perfluorocarbon combined with FITC (fluorescein isothiocyanate) was administered for postmortem immunofluorescence and flow-cytometry analyses. Control experiments were performed in 9 animals. In vivo (19)F-CMR detected myocardial inflammation in all experimental autoimmune myocarditis-positive animals. Its resolution was sufficient to identify even small inflammatory foci, that is, at the surface of the right ventricle. Postmortem immunohistochemistry and flow cytometry confirmed the presence of perfluorocarbon in macrophages, dendritic cells, and granulocytes, but not in lymphocytes. The myocardial volume of elevated (19)F signal (rs=0.96; P<0.001), the (19)F signal-to-noise ratio (rs=0.92; P<0.001), and the (19)F signal integral (rs=0.96; P<0.001) at day 21 correlated with the histological myocarditis severity score. CONCLUSIONS: In vivo (19)F-CMR was successfully used to visualize the inflammation specifically and robustly in experimental autoimmune myocarditis, and thus allowed for an unprecedented insight into the involvement of inflammatory cells in the disease process.

Photodynamic therapy enhances lipodoxorubcin distribution in sarcoma lung metastasis by lowering tumor interstitial fluid pressure in a rodent model

Objective: The management of sarcoma metastasis by systemic chemotherapy is often unsatisfactory. This has paradoxally been attributed to the leakiness of tumor neovessels which induce high intratumor interstitial fluid pressure (IFP) and limit convection forces that are important for drug distribution. In a rodent model, we have recently shown that photodynamic (PDT) pre treatment of lung metastasis could enhance their uptake of chemotherapy. We hypothesized that PDT transiently decreases tumor IFP which enhances convection and promotes drug distribution.Methods: Sarcoma tumors were generated sub-pleurally in the lungs of 12 rats. Animals were randomized at 10 days into i. no pre-treatment (control) and ii. low dose PDT pre-treatment (0・0625 mg/kg Visudyne, 10J/cm2 and 35 mW/cm2) followed by intravenous Liposomal doxorubicin (LiporubicinTM) administration. Using the wick-in-needle technique, we determined tumor and normal tissue IFP before, during and after PDT. In parallel, the uptake of LiporubicinTM was determined by high performance liquid chromatography in tumor and lung tissues.Results: Tumor IFP was significantly higher than normal tissue IFP in all animals. PDT pre-treatment did not affect normal tissue IFP but caused a significant decrease in tumor IFP (mean decrease by 2+/− 1mmHg) which lasted an average of 30 minutes before reaching baseline values. Tumor but not normal lung tissue LiporubicinTM uptake was significantly increased by 67% with PDT pre-treatment when liporubicin was allowed to circulate for one hour.Conclusion: Photodynamic therapy pre-treatment enhances LiporubicinTM uptake in sarcoma lung metastasis by transiently decreasing tumor IFP. These PDT conditions seem to specifically modulate tumor neovessels but not normal lung vessels.

c-Myc controls the development of CD8alphaalpha TCRalphabeta intestinal intraepithelial lymphocytes from thymic precursors by regulating IL-15-dependent survival.

The murine gut epithelium contains a large population of thymus-derived intraepithelial lymphocytes (IELs), including both conventional CD4(+) and CD8alphabeta(+) T cells (expressing T-cell receptor alphabeta [TCRalphabeta]) and unconventional CD8alphaalpha(+) T cells (expressing either TCRalphabeta or TCRgammadelta). Whereas conventional IELs are widely accepted to arise from recirculation of activated CD4(+) and CD8alphabeta(+) T cells from the secondary lymphoid organs to the gut, the origin and developmental pathway of unconventional CD8alphaalpha IELs remain controversial. We show here that CD4-Cre-mediated inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biologic activities, selectively impairs the development of CD8alphaalpha TCRalphabeta IELs. In the absence of c-Myc, CD4(-) CD8(-) TCRalphabeta(+) thymic precursors of CD8alphaalpha TCRalphabeta IELs are present but fail to develop on adoptive transfer in immunoincompetent hosts. Residual c-Myc-deficient CD8alphaalpha TCRalphabeta IEL display reduced proliferation and increased apoptosis, which correlate with significantly decreased expression of interleukin-15 receptor subunits and lower levels of the antiapoptotic protein Bcl-2. Transgenic overexpression of human BCL-2 resulted in a pronounced rescue of CD8alphaalpha TCRalphabeta IEL in c-Myc-deficient mice. Taken together, our data support a model in which c-Myc controls the development of CD8alphaalpha TCRalphabeta IELs from thymic precursors by regulating interleukin-15 receptor expression and consequently Bcl-2-dependent survival.

Mouse fetal trisomy 13 and hypotrophy of the spinal cord: effect on calbindin-D28k and calretinin expressed by neurons of the spinal cord and dorsal root ganglia.

Trisomy 13 was detected in 10% of mouse embryos obtained from pregnant females which were doubly heterozygous for Robertsonian chromosomes involving chromosome 13. The developing dorsal root ganglia and spinal cords were examined in trisomy 13 and littermate control mice between days 12 and 18 of gestation (E12-18). The overall size of the dorsal root ganglia and number of ganglion cells within a given ganglion were not altered, but the number of neurons immunoreactive for calbindin and calretinin was reduced. The trisomic spinal cord was reduced in size with neurons lying in a tightly compact distribution in the gray matter. In trisomic fetuses, the extent of the neuropil of the spinal cord was reduced, and may represent a diminished field of interneuronal connectivity, due to reduced arborization of dendritic processes of the neurons present, particularly of calbindin-immunostained neurons. Furthermore, the subpopulation of calretinin-immunoreactive neurons and axons was also reduced in developing trisomic gray and white matter, respectively. Thus, overexpression of genes on mouse chromosome 13 exerts a deleterious effect on the development of neuropil, affecting both dendritic and axonal arborization in the trisomy 13 mouse. The defect of calbindin or calretinin expression by subsets of dorsal root ganglion or spinal cord neurons may result from deficient cell-to-cell interactions with targets which are hypoplastic.

Critical single proximal left arterial descending coronary artery stenosis to mimic chronic myocardial ischemia: a new model induced by minimal invasive technology.

BACKGROUND/AIMS: The present report examines a new pig model for progressive induction of high-grade stenosis, for the study of chronic myocardial ischemia and the dynamics of collateral vessel growth. METHODS: Thirty-nine Landrace pigs were instrumented with a novel experimental stent (GVD stent) in the left anterior descending coronary artery. Eight animals underwent transthoracic echocardiography at rest and under low-dose dobutamine. Seven animals were examined by nuclear PET and SPECT analysis. Epi-, mid- and endocardial fibrosis and the numbers of arterial vessels were examined by histology. RESULTS: Functional analysis showed a significant decrease in global left ventricular ejection fraction (24.5 +/- 1.6%) 3 weeks after implantation. There was a trend to increased left ventricular ejection fraction after low-dose dobutamine stress (36.0 +/- 6.6%) and a significant improvement of the impaired regional anterior wall motion. PET and SPECT imaging documented chronic hibernation. Myocardial fibrosis increased significantly in the ischemic area with a gradient from epi- to endocardial. The number of arterial vessels in the ischemic area increased and coronary angiography showed abundant collateral vessels of Rentrop class 1. CONCLUSION: The presented experimental model mimics the clinical situation of chronic myocardial ischemia secondary to 1-vessel coronary disease.

Rôle de la bradykinine dans l'hypotension induite par le fragment actif dérivé du facteur XII [Role of bradykinin in hypotension induced by the active factor derived from factor XII]

The active fragment derived from factor XII (factor XIIf) was purified from human plasma and administered intravenously to normotensive conscious rats. Factor XIIf-mediated hypotension was dose-dependent and augmented by pretreatment with captopril, an inhibitor of the angiotensin I- and bradykinin-processing enzyme. In contrast, factor XIIf-induced hypotension was not enhanced by blockade of the renin-angiotensin system by saralasin, a competitive antagonist of angiotensin II at the vascular receptor level. These results suggest that factor XIIf-mediated hypotension is due to the formation of bradykinin.