Browsing by Subject "in vivo"

Now showing 1 - 7 of 7
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    Acridine Orange as a Novel Photosensitizer for Photodynamic Therapy in Glioblastoma
    (Elsevier, 2018) Osman, Hany; Elsahy, Deena; Saadatzadeh, M. Reza; Pollok, Karen E.; Yocom, Steven; Hattab, Eyas; Georges, Joseph; Cohen-Gadol, Aaron A.; Neurological Surgery, School of Medicine
    Object Photodynamic therapy is an exciting treatment modality that combines the effects of a chemical agent with the physical energy from light or radiation to result in lysis of cells of interest. Acridine orange is a molecule with fluorescence properties that was demonstrated to possess photosensitizing properties. The objective of this study was to investigate the photodynamic effect of acridine orange on glioblastoma cell viability and growth. Methods Glioblastoma cells (n = 8000 cells/well at 0 hours) were exposed to acridine orange followed by white unfiltered light-emitting diodes (LED) light. Cultures were exposed to either 10 or 30 minutes of light. The cell number per well was determined at 0, 24, 48, and 72 hours after exposure. Results A dramatic cytocidal effect of acridine orange after exposure to as little as 10 minutes of white light was observed. There was almost complete eradication of the glioblastoma cells over a 72-hour period. Although acridine orange or light alone exhibited some effect on cell growth, it was not as pronounced as the combination of acridine orange and light. Conclusions This is the first study to demonstrate the photodynamic effect of acridine orange in glioblastoma cells. This data supports the need for further studies to characterize and evaluate whether this striking cytotoxic effect can be achieved in vivo. The combination of acridine orange and exposure to white unfiltered LED light may have potential future applications in management of glioblastoma.
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    Assessing neuronal ciliary localization of Melanin Concentrating Hormone Receptor 1 in vivo
    (2021-08) Kamba, Tisianna K.; Berbari, Nicolas F.; Mastracci, Teresa; Dai, Guoli
    Obesity is a growing pandemic that claims close to three hundred thousand lives per year in the United States alone. Despite strong interest and investment in potential treatments, obesity remains a complex and challenging disorder. In the study of obesity, mouse models have been excellent tools that help in understanding the function of different genes that contribute to this disease of energy homeostasis. However, it was surprising when disfunction in primary cilia was found to be linked to syndromic obesity. To understand the role of primary cilia in obesity, a growing subset of GPCRs have been identified to selectively localize to the organelle. Several of which have known roles in energy homeostasis. In some examples, ciliary GPCRs appear to dynamically localize to the organelle; such is the case of GPR161 and smoothened in the hedgehog signaling pathway. Thus, we were interested to see if other GPCRs dynamically localize to the primary cilia as part of their regulation of energy homeostasis. For example, the GPCR MCHR1 selectively localizes to the cilia and is involved in energy homeostasis. Although much is known about the expression of the receptor in the brain, how its ciliary subcellular localization impacts its roles in energy homeostasis is unknown. Observing neuronal cilia in vivo remains a difficult task as some of the available tools such as tagged alleles rely on overexpression of ciliary protein which may impact function. Additionally, most of the work is done in vitro, leaving much to be discovered about neuronal cilia in vivo. In this thesis, we show that using a newly constructed reporter allele mCherryMCHR1, we can see ciliary expression of MCHR1 in the brain of developing and adult mice; more specifically in the ARC and PVN. Subsequently, using a novel Artificial intelligence analysis approach, we measured the length and composition of MCHR1 positive cilia under physiological conditions associated with MCHR1 function. Although in this work we are reporting no changes in dynamic localization of MCHR1 in the hypothalamus specifically, we are not excluding the potential for changes in other regions of the brain or under other conditions; and we are suggesting that pharmacological approaches may help highlight potential ciliary GPCR dynamic localization.
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    In Vivo Microscopy in Neurosurgical Oncology
    (Elsevier, 2018) Osman, Hany; Georges, Joseph; Elsahy, Deena; Hattab, Eyas; Yocom, Steven; Cohen-Gadol, Aaron A.; Neurological Surgery, School of Medicine
    Intraoperative neurosurgical histopathologic diagnoses rely on evaluation of rapid tissue preparations such as frozen sections and smears with conventional light microscopy. Though useful, these techniques are time consuming and therefore unable to provide real-time intraoperative feedback. In vivo molecular imaging techniques are emerging as novel methods for generating real-time diagnostic histopathologic images of tumors and their surrounding tissues. These imaging techniques rely on contrast generated by exogenous fluorescent dyes, autofluorescence of endogenous molecules, fluorescence decay of excited molecules, or light scattering. Large molecular imaging instruments are being miniaturized for clinical in vivo use. This review discusses pertinent imaging systems that have been developed for neurosurgical use and imaging techniques currently under NADPH development for neurosurgical molecular imaging.
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    Loss of inversin contributes to renal cystic disease through altered cellular processes and decreased sodium transport in renal epithelial cells
    (2017-05) Kulkarni, Nalini H.; Blazer-Yost, Bonnie L.
    Type II nephronophthisis (NPHP2) is an autosomal recessive renal cystic disorder characterized by mutations in the inversin gene. Humans and mice with mutations in inversin have enlarged cystic kidneys. Increased kidney size in NPHP2 may involve altered cell growth, apoptosis, electrolyte transport and fluid accumulation in the cysts. To test this hypothesis, histology and transcriptome analysis were performed on one-day old wild-type and inv/inv mice to uncover molecular pathways altered in the mutant mice. Histology of inv/inv mice kidneys showed dilated cystic tubules compared to wild type. Pathway analysis of transcriptome data showed that inversin exerts its effects on kidneys, at least in part, through the transcriptional regulation of genes implicated in inflammation, immune response, cellular metabolism, cell cycle and ion transport. Genes involved in inflammation or immune response were upregulated whereas the genes involved in cell cycle progression and ion transport were downregulated. To validate the array findings from inv/inv mice kidneys, functional consequence of inversin loss on transepithelial ion transport was measured by electrophysiological techniques in shRNA mediated inversin-depleted renal cell type isolated from mouse cortical collecting duct (mCCD). Depletion of inversin decreased vasopressin-induced Na+ absorption, but did not alter Cl- secretion in mCCD cells. Addition of amiloride, a specific blocker of the epithelial sodium channel (ENaC), abolished basal ion transport in both inversin knockdown and control cells indicating ENaC involvement. Loss of inversin decreased Na+ absorption and this effect, in part, was mediated by transcriptional and post-translational regulation of ENaC mediators. To better understand inversin function in renal cells, transcriptome analysis was performed in control and inversin-depleted mCCD cells. Pathway analysis showed that inversin-depletion altered the genes represented in cell cycle, cellular assembly and organization, DNA replication, cell proliferation and ion transport in this isolated renal cell type. In concordance with the array data from inv/inv mice kidneys, a decrease in the expression of cell cycle, ion transport and apoptotic genes were observed accompanied by an upregulation of genes implicated in inflammatory or immune response indicating a direct effect of inversin on renal cells. Together, this study utilized a combination of transcriptome and functional analyses to unravel the role of inversin in renal cells. These data demonstrate that loss of inversin can cause a delay in cell cycle progression with a decrease in cell proliferation and apoptosis which in turn can perturb the development of the renal tubule. Also, a decrease in Na+ reabsorption together with differential regulation of other ion transporters can result in altered electrolyte transport contributing to cystogenesis, cyst growth, fluid accumulation and cyst expansion in NPHP2.
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    Neurosurgical Flexible Probe Microscopy with Enhanced Architectural and Cytological Detail
    (Elsevier, 2019-08) Osman, Hany; Elsahy, Deena; Slivova, Veronika; Thompson, Corey; Georges, Joseph; Yocom, Steven; Cohen-Gadol, Aaron A.; Neurological Surgery, School of Medicine
    Background Microscopic delineation and clearance of tumor cells at neurosurgical excision margins potentially reduce tumor recurrence and increase patient survival. Probe-based in vivo fluorescence microscopy technologies are promising for neurosurgical in vivo microscopy. Objective We sought to demonstrate a flexible fiberoptic epifluorescence microscope capable of enhanced architectural and cytological imaging for in vivo microscopy during neurosurgical procedures. Methods Eighteen specimens were procured from neurosurgical procedures. These specimens were stained with acridine orange and imaged with a 3-dimensional (3D)-printed epifluorescent microscope that incorporates a flexible fiberoptic probe. Still images and video sequence frames were processed using frame alignment, signal projection, and pseudo-coloring, resulting in resolution enhancement and an increased field of view. Results Images produced displayed good nuclear contrast and architectural detail. Grade 1 meningiomas demonstrated 3D chords and whorls. Low-grade meningothelial nuclei showed streaming and displayed regularity in size, shape, and distribution. Oligodendrogliomas showed regular round nuclei and a variably staining background. Glioblastomas showed high degrees of nuclear pleomorphism and disarray. Mitoses, vascular proliferation, and necrosis were evident. Conclusions We demonstrate the utility of a 3D-printed, flexible probe microscope for high-resolution microscopic imaging with increased architectural detail. Enhanced in vivo imaging using this device may improve our ability to detect and decrease microscopic tumor burden at excision margins during neurosurgical procedures.
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    Pharmacologic inhibition of PI3K p110δ in mutant Shp2E76K-expressing mice
    (Impact Journals, 2017-10-03) Deng, Lisa; Virts, Elizabeth L.; Kapur, Reuben; Chan, Rebecca J.; Pediatrics, School of Medicine
    Juvenile myelomonocytic leukemia is a childhood malignancy that lacks effective chemotherapies and thus has poor patient outcomes. PI3K p110δ has been found to promote hyperproliferation of cells expressing mutant Shp2. In this study, we tested the efficacy of a PI3Kδ inhibitor in mice expressing the Shp2 gain-of-function mutation, E76K. We found that in vivo treatment of mice led to significantly decreased splenomegaly, reduced frequency of bone marrow progenitor cells, and increased terminally differentiated peripheral blood myeloid cells. The survival of drug-treated mice was significantly prolonged compared to vehicle-treated controls, although mice from both groups ultimately succumbed to a similar myeloid cell expansion. PI3Kδ inhibitors are currently used to treat patients with relapsed lymphoid malignancies, such as chronic lymphocytic leukemia. The current findings provide evidence for using PI3Kδ inhibitors as a treatment strategy for JMML and potentially other myeloid diseases.
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    Refractory testicular germ cell tumors are highly sensitive to the second generation DNA methylation inhibitor guadecitabine
    (Impact Journals, 2017-01-10) Albany, Costantine; Hever-Jardine, Mary P.; von Herrmann, Katherine M.; Yim, Christina Y.; Tam, Janice; Warzecha, Joshua M.; Shin, Leah; Bock, Sarah E.; Curran, Brian S.; Chaudhry, Aneeq S.; Kim, Fred; Sandusky, George E.; Taverna, Pietro; Freemantle, Sarah J.; Christensen, Brock C.; Einhorn, Lawrence H.; Spinella, Michael J.; Medicine, School of Medicine
    Testicular germ cell tumors (TGCTs) are the most common cancers of young males. A substantial portion of TGCT patients are refractory to cisplatin. There are no effective therapies for these patients, many of whom die from progressive disease. Embryonal carcinoma (EC) are the stem cells of TGCTs. In prior in vitro studies we found that EC cells were highly sensitive to the DNA methyltransferase inhibitor, 5-aza deoxycytidine (5-aza). Here, as an initial step in bringing demethylation therapy to the clinic for TGCT patients, we evaluated the effects of the clinically optimized, second generation demethylating agent guadecitabine (SGI-110) on EC cells in an animal model of cisplatin refractory testicular cancer. EC cells were exquisitely sensitive to guadecitabine and the hypersensitivity was dependent on high levels of DNA methyltransferase 3B. Guadecitabine mediated transcriptional reprogramming of EC cells included induction of p53 targets and repression of pluripotency genes. As a single agent, guadecitabine completely abolished progression and induced complete regression of cisplatin resistant EC xenografts even at doses well below those required to impact somatic solid tumors. Low dose guadecitabine also sensitized refractory EC cells to cisplatin in vivo. Genome-wide analysis indicated that in vivo antitumor activity was associated with activation of p53 and immune-related pathways and the antitumor effects of guadecitabine were dependent on p53, a gene rarely mutated in TGCTs. These preclinical findings suggest that guadecitabine alone or in combination with cisplatin is a promising strategy to treat refractory TGCT patients.