Tissue-Like 3D Standard and Protocols for Microscope Quality Management.

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation, instrument-specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 µm thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include the following: 1) microscope resolution with automated analysis of the point-spread function (PSF), 2) automated signal-to-noise ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth, but the relative intensities of different objects at similar depths are maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g., magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved scientific rigor and reproducibility.

Impact of Cosmetic Lotions on Nanoparticle Penetration through ex vivo C57BL/6 Hairless Mouse and Human Skin: A Comparison Study.

Understanding the interactions of nanoparticles (NPs) with skin is important from a consumer and occupational health and safety perspective, as well as for the design of effective NP-based transdermal therapeutics. Despite intense efforts to elucidate the conditions that permit NP penetration, there remains a lack of translatable results from animal models to human skin. The objectives of this study are to investigate the impact of common skin lotions on NP penetration and to quantify penetration differences of quantum dot (QD) NPs between freshly excised human and mouse skin. QDs were mixed in 7 different vehicles, including 5 commercial skin lotions. These were topically applied to skin using two exposure methods; a petri dish protocol and a Franz diffusion cell protocol. QD presence in the skin was quantified using Confocal Laser Scanning Microscopy. Results show that the commercial vehicles can significantly impact QD penetration in both mouse and human skin. Lotions that contain alpha hydroxyl acids (AHA) facilitated NP penetration. Lower QD signal was observed in skin studied using a Franz cell. Freshly excised human skin was also studied immediately after the sub-cutaneous fat removal process, then after 24 hours rest ex vivo. Resting human skin 24 hours prior to QD exposure significantly reduced epidermal presence. This study exemplifies how application vehicles, skin processing and the exposure protocol can affect QD penetration results and the conclusions that maybe drawn between skin models.

Development and characterization of antibody reagents for detecting nanoparticles.

The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in solution. We present a proof-of-concept for the generation of NProbes and their use for detecting quantum dots and titanium dioxide NPs in vitro and in an ex vivo human skin model. Continued development and refinement of NProbes to detect NPs that vary in composition, shape, size, and surface coating will comprise a powerful tool kit that can be used to advance nanotechnology research particularly in the nanotoxicology and nanotherapeutics fields.

Differences in amygdala cell proliferation between adolescent and young adult rats.

Adolescence is characterized by changes in both behavior and neural organization. During this period, the amygdala, a structure that mediates social and emotional behaviors, is changing in terms of neural and glia density. We examined cell proliferation within the amygdala of adolescent (post natal day (PND) 31) and adult (PND 70) male Sprague-Dawley rats using BrdU (bromodeoxyuridine) to label dividing cells. BrdU-labeled cells were distributed throughout the amygdala, often found in fibers surrounding major nuclei. Using two independent cell counting strategies under light and confocal microcopy, respectively, we found significantly more labeled cells in the amygdala in adolescent compared to adult animals (239.3 ± 87.18 vs. 44.75 ± 13.68; n=4/group; p<.05). BrdU/doublecortin (DCX) positive cells constitute approximately 30% of all dividing cells in the amygdala in both adolescents and adults. These data suggest that compared to young adulthood, adolescence is a relatively active period of cell proliferation in the amygdala. Moreover, the normal decline in dividing cells with age does not preferentially affect cells co-containing DCX-immunoreactivity.

The ligand-mediated nuclear mobility and interaction with estrogen-responsive elements of estrogen receptors are subtype specific.

17ß-Estradiol (E(2)) plays important roles in functions of many tissues. E(2) effects are mediated by estrogen receptor (ER) α and ß. ERs regulate transcriptions through estrogen-responsive element (ERE)-dependent and ERE-independent modes of action. ER binding to ERE constitutes the basis of the ERE-dependent pathway. Direct/indirect ER interactions with transcription complexes define ERE-independent signaling. ERs share functional features. Ligand-bound ERs nevertheless induce distinct transcription profiles. Live cell imaging indicates a dynamic nature of gene expressions by highly mobile ERs. However, the relative contribution of ER mobility at the ERE-independent pathway to the overall kinetics of ER mobility remains undefined. We used fluorescent recovery after a photo-bleaching approach to assess the ligand-mediated mobilities of ERE binding-defective ERs, ER(EBD). The decrease in ERα mobility with E(2) or the selective ER modulator 4-hydroxyl-tamoxifen (4HT) was largely due to the interaction of the receptor with ERE. Thus, ERα bound to E(2) or 4HT mediates transcriptions from the ERE-independent pathway with remarkably fast kinetics that contributes fractionally to the overall motility of the receptor. The antagonist Imperial Chemical Industries 182 780 immobilized ERαs. The mobilities of ERß and ERß(EBD) in the presence of ligands were indistinguishable kinetically. Thus, ERß mobility is independent of the nature of ligands and the mode of interaction with target sites. Chimeric ERs indicated that the carboxyl-termini are critical regions for subtype-specific mobility. Therefore, while ERs are highly mobile molecules interacting with target sites with fast kinetics, an indication of the hit-and-run model of transcription, they differ mechanistically to modulate transcriptions.

A neurotoxic phosphoform of Elk-1 associates with inclusions from multiple neurodegenerative diseases.

Neurodegenerative diseases are characterized by a number of features including the formation of inclusions, early synaptic degeneration and the selective loss of neurons. Molecules serving as links between these shared features have yet to be identified. Identifying candidates within the diseased microenvironment will open up novel avenues for therapeutic intervention. The transcription factor Elk-1 resides within multiple brain areas both in nuclear and extranuclear neuronal compartments. Interestingly, its de novo expression within a single dendrite initiates neuronal death. Given this novel regionalized function, we assessed whether extranuclear Elk-1 and/or phospho-Elk-1 (pElk-1) protein might be associated with a spectrum of human neurodegenerative disease cases including Lewy body Disease (e.g. Parkinson's), Alzheimer's disease, and Huntington's Disease. We first determined the importance of Elk-1 post-translational modifications on its ability to initiate regionalized cell death. We next screened human cases from three major neurodegenerative diseases to look for remarkable levels of Elk-1 and/or pElk-1 protein as well as their association with inclusions characteristic of these diseases. We compared our findings to age-matched control cases. We find that the ability of Elk-1 to initiate regionalized neuronal death depends on a specific phosphosite, T417. Furthermore, we find that T417(+) Elk-1 uniquely associates with several types of inclusions present in cases of human Lewy body Disease, Alzheimer's disease, and Huntington's Disease. These results suggest a molecular link between the presence of inclusions and neuronal loss that is shared across a spectrum of neurodegenerative disease.

Epithelial ablation of Bcl-XL increases sensitivity to oxygen without disrupting lung development.

Recent studies indicate that the antiapoptotic Bcl-X(L), one of five isoforms expressed by the Bcl-X gene, protects a variety of cell lines exposed to hyperoxia. However, its role in lung development and protection against oxidative stress in vivo is not known. Here, we show Bcl-X(L) is the predominant isoform expressed in the lung, and the only isoform detected in respiratory epithelium. Because loss of Bcl-X(L) is embryonically lethal, Bcl-X(L) was ablated throughout the respiratory epithelium by mating mice with a floxed exon II of the Bcl-X gene with mice expressing Cre under control of the surfactant protein-C promoter. Interestingly, the loss of Bcl-X(L) in respiratory epithelium was perinatally lethal in approximately 50% of the expected offspring. However, some adult mice lacking the gene were obtained. The epithelial-specific ablation of Bcl-X(L) did not disrupt pulmonary function, the expression of epithelial cell-specific markers, or lung development. However, it shifted the lung toward a proapoptotic state, defined by a reduction in antiapoptotic Mcl-1, an increase in proapoptotic Bak, and increased sensitivity of the respiratory epithelium to hyperoxia. Intriguingly, increased 8-oxoguanine lesions seen during hyperoxia were also evident as lungs transitioned to room air at birth, a time when perinatal lethality in some mice lacking Bcl-X(L) was observed. These findings reveal that the epithelial-specific expression of Bcl-X(L) is not required for proper lung development, but functions to protect respiratory epithelial cells against oxygen-induced toxicity, such as during hyperoxia and the lung's first exposure to ambient air.

Chronic neuron-specific tumor necrosis factor-alpha expression enhances the local inflammatory environment ultimately leading to neuronal death in 3xTg-AD mice.

Inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta, appear integral in initiating and/or propagating Alzheimer's disease (AD)-associated pathogenesis. We have previously observed a significant increase in the number of mRNA transcripts encoding the pro-inflammatory cytokine TNF-alpha, which correlated to regionally enhanced microglial activation in the brains of triple transgenic mice (3xTg-AD) before the onset of overt amyloid pathology. In this study, we reveal that neurons serve as significant sources of TNF-alpha in 3xTg-AD mice. To further define the role of neuronally derived TNF-alpha during early AD-like pathology, a recombinant adeno-associated virus vector expressing TNF-alpha was stereotactically delivered to 2-month-old 3xTg-AD mice and non-transgenic control mice to produce sustained focal cytokine expression. At 6 months of age, 3xTg-AD mice exhibited evidence of enhanced intracellular levels of amyloid-beta and hyperphosphorylated tau, as well as microglial activation. At 12 months of age, both TNF receptor II and Jun-related mRNA levels were significantly enhanced, and peripheral cell infiltration and neuronal death were observed in 3xTg-AD mice, but not in non-transgenic mice. These data indicate that a pathological interaction exists between TNF-alpha and the AD-related transgene products in the brains of 3xTg-AD mice. Results presented here suggest that chronic neuronal TNF-alpha expression promotes inflammation and, ultimately, neuronal cell death in this AD mouse model, advocating the development of TNF-alpha-specific agents to subvert AD.

VIP is a transcriptional target of Nurr1 in dopaminergic cells.

The orphan nuclear receptor Nurr1 is required for the development of the ventral mesencephalic dopaminergic neurons. These are the same neurons that are invariantly lost in patients with Parkinson's disease. Nurr1 mRNA expression is not confined to the developing midbrain, and yet Nurr1 appears to be essential for either the maturation of progenitors into fully post-mitotic dopaminergic neurons and/or once formed, their survival. The function of Nurr1 in the transactivation of gene(s) important for neuronal development and/or maintenance is uncharacterized. To characterize potential downstream target genes of Nurr1, we sought to identify mRNAs that are differentially affected by Nurr1 expression. Using a dopaminergic cell line in which Nurr1 content was tightly regulated, differential display analysis identified transcripts altered by Nurr1 expression, including the mRNA encoding vasoactive intestinal peptide (VIP). Herein, we demonstrate that Nurr1 regulates VIP mRNA and protein levels, and transactivates the VIP promoter through Nurr1-responsive cis elements. In addition, dopaminergic cells release and utilize VIP to mediate survival when challenged with paraquat. Nurr1 regulation of VIP is also demonstrated in vivo as loss of Nurr1 function results in diminished VIP mRNA levels within the developing midbrain.

Dysregulation of gene expression in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse substantia nigra.

Parkinson's disease pathogenesis proceeds through several phases, culminating in the loss of dopaminergic neurons of the substantia nigra (SN). Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of oxidative SN injury is frequently used to study degeneration of dopaminergic neurons in mice and non-human primates, an understanding of the temporal sequence of molecular events from inhibition of mitochondrial complex 1 to neuronal cell death is limited. Here, microarray analysis and integrative data mining were used to uncover pathways implicated in the progression of changes in dopaminergic neurons after MPTP administration. This approach enabled the identification of small, yet consistently significant, changes in gene expression within the SN of MPTP-treated animals. Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity, and cell cycle and apoptosis. The discovery and validation of these alterations provide molecular evidence for an evolving cascade of injury, dysfunction, and cell death.

Progressive reduction of synaptophysin message in single neurons in Alzheimer disease.

The data presented here examine 2 hypotheses: 1) that viable but vulnerable single neurons remaining in the Alzheimer brain lose synaptic markers, and 2) that the extent of this loss is related to the disease state of these single neurons when disease state is defined by immunoreactivity. We used double immunohistochemistry (IHC) to define neurofibrillary tangle (NFT) and phosphorylation status of tau at selected defined epitopes. This double IHC was combined with quantitative in situ hybridization for message for the synaptic marker, synaptophysin, in 1,127 single hippocampal CA1 pyramidal neurons from 15 Alzheimer disease (AD) and 4 control cases. We found that there is a graded, progressive, decrease of synaptophysin message expressed by single neurons related to immunohistochemical markers of tau status, and that neurons in similar immunohistochemically defined classes show similar losses of synaptophysin message regardless of whether they were sampled from clinical control brains or advanced AD. The resulting conclusions are consistent with a suggestion that differences among clinically defined AD and control status are defined by the numbers of neurons in various disease states.