scholarly journals Nanoparticle-induced inflammation and fibrosis in ex vivo murine precision-cut liver slices and effects of nanoparticle exposure conditions

2021 ◽  
Vol 95 (4) ◽  
pp. 1267-1285 ◽  
Author(s):  
Roberta Bartucci ◽  
Alex Z. van der Meer ◽  
Ykelien L. Boersma ◽  
Peter Olinga ◽  
Anna Salvati
Keyword(s):  
Ex Vivo ◽  
Long Term Effects ◽  
Tissue Slices ◽  
Nanoparticle Exposure ◽  
Liver Slices ◽  
Nanoparticle Dispersions ◽  
Corona Formation ◽  
Modified Polystyrene ◽  
Exposure Conditions

AbstractChronic exposure and accumulation of persistent nanomaterials by cells have led to safety concerns on potential long-term effects induced by nanoparticles, including chronic inflammation and fibrosis. With this in mind, we used murine precision-cut liver tissue slices to test potential induction of inflammation and onset of fibrosis upon 72 h exposure to different nanomaterials (0–200 µg/ml). Tissue slices were chosen as an advanced ex vivo 3D model to better resemble the complexity of the in vivo tissue environment, with a focus on the liver where most nanomaterials accumulate. Effects on the onset of fibrosis and inflammation were investigated, with particular care in optimizing nanoparticle exposure conditions to tissue. Thus, we compared the effects induced on slices exposed to nanoparticles in the presence of excess free proteins (in situ), or after corona isolation. Slices exposed to daily-refreshed nanoparticle dispersions were used to test additional effects due to ageing of the dispersions. Exposure to amino-modified polystyrene nanoparticles in serum-free conditions led to strong inflammation, with stronger effects with daily-refreshed dispersions. Instead, no inflammation was observed when slices were exposed to the same nanoparticles in medium supplemented with serum to allow corona formation. Similarly, no clear signs of inflammation nor of onset of fibrosis were detected after exposure to silica, titania or carboxylated polystyrene in all conditions tested. Overall, these results show that liver slices can be used to test nanoparticle-induced inflammation in real tissue, and that the exposure conditions and ageing of the dispersions can strongly affect tissue responses to nanoparticles.

scholarly journals TMOD-31. ADAPTING ENGINEERED CELL THERAPIES TO UNDERSTAND AND OVERCOME GLIOBLASTOMA RESISTANCE USING INTEGRATED IN VIVO AND EX VIVO MODELS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi269-vi269
Author(s):  
Andrew Satterlee ◽  
Denise Dunn ◽  
Scott Floyd ◽  
Shawn Hingtgen
Keyword(s):  
Primary Tumor ◽  
Ex Vivo ◽  
Tumor Burden ◽  
In Vivo Studies ◽  
Pcr Analysis ◽  
Tissue Slices ◽  
Live Animal ◽  
Invasive Tumor ◽  
Recurrent Tumors

Abstract Genetically engineered neural stem cells (NSCs) are a promising therapy for the highly aggressive brain cancer glioblastoma (GBM), yet treatment durability remains a major challenge. We sought to define the events that contribute to dynamic adaption of GBM during NSC treatment and develop strategies to convert initial tumor kill into sustained GBM suppression. Using a unique hybrid tumor model treated with human skin-derived induced NSCs (iNSCs) releasing the pro-apoptotic agent TRAIL, we investigated how spatial distribution of tumor and iNSCs affects GBM adaption throughout recurrence. Serial bioluminescent imaging (BLI) was used to track tumor volumes in vivo, while a subset of mice were sacrificed 6, 13, and 20 days post-treatment to harvest brains and generate living ex vivo tissue slices. Live animal imaging showed iNSC-TRAIL treatment rapidly decreased tumor volumes when delivered into the primary tumor mass; however, minimal impact on tumor growth was observed when cells were delivered into distal regions of the brain. In contrast, high-resolution imaging of living brain sections showed extensive impacts of iNSC-TRAIL therapy that could not be visualized with BLI. The living slices showed iNSC-TRAIL treatment into the primary tumor decreased the solid, but not the invasive, tumor burden. Treatment into the lateral ventricles did impact tumor kill and was more effective at treating the invasive tumor burden and maintaining inhibition than treatment into the contralateral parenchyma. We next utilized the living tissue slices to explore the sensitivity of the recurrent tumors to TRAIL. When therapy was applied to slices harboring recurrent tumor, treatment again significantly reduced tumor volumes, suggesting that tumors had not acquired TRAIL resistance. These results informed an additional in vivo survival study and subsequent PCR analysis of untreated and recurrent tumors, and combine the fidelity of in vivo studies with the speed and spatial resolution of living brain slice technology.

scholarly journals Melanopsin+RGCs Are fully Resistant to NMDA-Induced Excitotoxicity

2019 ◽  
Vol 20 (12) ◽  
pp. 3012 ◽  
Author(s):  
Beatriz Vidal-Villegas ◽  
Johnny Di Pierdomenico ◽  
Juan A Miralles de Imperial-Ollero ◽  
Arturo Ortín-Martínez ◽  
Francisco M Nadal-Nicolás ◽  
...  
Keyword(s):  
Intravitreal Injection ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Retinal Ganglion ◽  
Long Term Effects ◽  
Pou Domain ◽  
Short And Long Term ◽  
Sd Oct

We studied short- and long-term effects of intravitreal injection of N-methyl-d-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using Spectral Domain-Optical Coherence Tomography (SD-OCT). Ex vivo analyses were done at 3, 7, or 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for brain-specific homeobox/POU domain protein 3A (Brn3a) and melanopsin (m), the total number of Brn3a+RGCs and m+RGCs were quantified, and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903 ± 3572 and 2358 ± 144 (mean ± SD; n = 10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 49%, 28%, 24%, and 19%, at 3, 7, 14 days, and 15 months, respectively. There was no further loss between 7 days and 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3 and 7 days, and were back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induced within a week a rapid loss of 72% of Brn3a+RGCs, a transient downregulation of melanopsin expression (but not m+RGC death), and a thinning of the inner retinal layers.

scholarly journals A practical method for multimodal registration and assessment of whole-brain disease burden using PET, MRI, and optical imaging

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Matthew L. Scarpelli ◽  
Debbie R. Healey ◽  
Shwetal Mehta ◽  
Vikram D. Kodibagkar ◽  
Christopher C. Quarles
Keyword(s):  
Optical Imaging ◽  
Imaging System ◽  
Ex Vivo ◽  
Imaging Techniques ◽  
Neurological Diseases ◽  
Spatial Scales ◽  
Phenotypic Heterogeneity ◽  
Tissue Slices ◽  
Rodent Brain

Abstract Many neurological diseases present with substantial genetic and phenotypic heterogeneity, making assessment of these diseases challenging. This has led to ineffective treatments, significant morbidity, and high mortality rates for patients with neurological diseases, including brain cancers and neurodegenerative disorders. Improved understanding of this heterogeneity is necessary if more effective treatments are to be developed. We describe a new method to measure phenotypic heterogeneity across the whole rodent brain at multiple spatial scales. The method involves co-registration and localized comparison of in vivo radiologic images (e.g. MRI, PET) with ex vivo optical reporter images (e.g. labeled cells, molecular targets, microvasculature) of optically cleared tissue slices. Ex vivo fluorescent images of optically cleared pathology slices are acquired with a preclinical in vivo optical imaging system across the entire rodent brain in under five minutes, making this methodology practical and feasible for most preclinical imaging labs. The methodology is applied in various examples demonstrating how it might be used to cross-validate and compare in vivo radiologic imaging with ex vivo optical imaging techniques for assessing hypoxia, microvasculature, and tumor growth.

scholarly journals SCIDOT-20. ADAPTING ENGINEERED CELL THERAPIES TO UNDERSTAND AND OVERCOME GLIOBLASTOMA RESISTANCE USING INTEGRATED IN VIVO AND EX VIVO MODELS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi275-vi275
Author(s):  
Andrew Satterlee ◽  
Denise Dunn ◽  
Scott Floyd ◽  
Shawn Hingtgen
Keyword(s):  
Primary Tumor ◽  
Ex Vivo ◽  
Tumor Burden ◽  
In Vivo Studies ◽  
Pcr Analysis ◽  
Tissue Slices ◽  
Live Animal ◽  
Invasive Tumor ◽  
Recurrent Tumors

Abstract Genetically engineered neural stem cells (NSCs) are a promising therapy for the highly aggressive brain cancer glioblastoma (GBM), yet treatment durability remains a major challenge. We sought to define the events that contribute to dynamic adaption of GBM during NSC treatment and develop strategies to convert initial tumor kill into sustained GBM suppression. Using a unique hybrid tumor model treated with human skin-derived induced NSCs (iNSCs) releasing the pro-apoptotic agent TRAIL, we investigated how spatial distribution of tumor and iNSCs affects GBM adaption throughout recurrence. Serial bioluminescent imaging (BLI) was used to track tumor volumes in vivo, while a subset of mice were sacrificed 6, 13, and 20 days post-treatment to harvest brains and generate living ex vivo tissue slices. Live animal imaging showed iNSC-TRAIL treatment rapidly decreased tumor volumes when delivered into the primary tumor mass; however, minimal impact on tumor growth was observed when cells were delivered into distal regions of the brain. In contrast, high-resolution imaging of living brain sections showed extensive impacts of iNSC-TRAIL therapy that could not be visualized with BLI. The living slices showed iNSC-TRAIL treatment into the primary tumor decreased the solid, but not the invasive, tumor burden. Treatment into the lateral ventricles did impact tumor kill and was more effective at treating the invasive tumor burden and maintaining inhibition than treatment into the contralateral parenchyma. We next utilized the living tissue slices to explore the sensitivity of the recurrent tumors to TRAIL. When therapy was applied to slices harboring recurrent tumor, treatment again significantly reduced tumor volumes, suggesting tumors had not acquired TRAIL resistance. These results informed an additional in vivo survival study and subsequent PCR analysis of untreated and recurrent tumors, and combine the fidelity of in vivo studies with the speed and spatial resolution of living brain slice technology.

scholarly journals Subchronic N-acetylcysteine Treatment Decreases Brain Kynurenic Acid Levels and Improves Cognitive Performance in Mice

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 147
Author(s):  
Tonali Blanco Ayala ◽  
Daniela Ramírez Ortega ◽  
Paulina Ovalle Rodríguez ◽  
Benjamín Pineda ◽  
Gonzalo Pérez de la Cruz ◽  
...  
Keyword(s):  
Cognitive Performance ◽  
Kynurenic Acid ◽  
Ex Vivo ◽  
Cognitive Impairments ◽  
Prolonged Treatment ◽  
Biosynthetic Enzyme ◽  
Tissue Slices ◽  
Aspartate Receptor ◽  
The Brain

The tryptophan (Trp) metabolite kynurenic acid (KYNA) is an α7-nicotinic and N-methyl-d-aspartate receptor antagonist. Elevated brain KYNA levels are commonly seen in psychiatric disorders and neurodegenerative diseases and may be related to cognitive impairments. Recently, we showed that N-acetylcysteine (NAC) inhibits kynurenine aminotransferase II (KAT II), KYNA’s key biosynthetic enzyme, and reduces KYNA neosynthesis in rats in vivo. In this study, we examined if repeated systemic administration of NAC influences brain KYNA and cognitive performance in mice. Animals received NAC (100 mg/kg, i.p.) daily for 7 days. Redox markers, KYNA levels, and KAT II activity were determined in the brain. We also assessed the effect of repeated NAC treatment on Trp catabolism using brain tissue slices ex vivo. Finally, learning and memory was evaluated with and without an acute challenge with KYNA’s bioprecursor L-kynurenine (Kyn; 100 mg/kg). Subchronic NAC administration protected against an acute pro-oxidant challenge, decreased KYNA levels, and lowered KAT II activity and improved memory both under basal conditions and after acute Kyn treatment. In tissue slices from these mice, KYNA neosynthesis from Trp or Kyn was reduced. Together, our data indicate that prolonged treatment with NAC may enhance memory at least in part by reducing brain KYNA levels.

scholarly journals Investigating fibrosis and inflammation in an ex vivo NASH murine model

2020 ◽  
Vol 318 (2) ◽  
pp. G336-G351
Author(s):  
Emilia Gore ◽  
Emilia Bigaeva ◽  
Anouk Oldenburger ◽  
Yvette J. M. Jansen ◽  
Detlef Schuppan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lipid Metabolism ◽  
Liver Disease ◽  
Nonalcoholic Steatohepatitis ◽  
Ex Vivo ◽  
Ex Vivo Model ◽  
Nonalcoholic Fatty Liver ◽  
Liver Slices ◽  
Expression Of Genes

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, characterized by excess fat accumulation (steatosis). Nonalcoholic steatohepatitis (NASH) develops in 15–20% of NAFLD patients and frequently progresses to liver fibrosis and cirrhosis. We aimed to develop an ex vivo model of inflammation and fibrosis in steatotic murine precision-cut liver slices (PCLS). NASH was induced in C57Bl/6 mice on an amylin and choline-deficient l-amino acid-defined (CDAA) diet. PCLS were prepared from steatohepatitic (sPCLS) and control (cPCLS) livers and cultured for 48 h with LPS, TGFβ1, or elafibranor. Additionally, C57Bl/6 mice were placed on CDAA diet for 12 wk to receive elafibranor or vehicle from weeks 7 to 12. Effects were assessed by transcriptome analysis and procollagen Iα1 protein production. The diets induced features of human NASH. Upon culture, all PCLS showed an increased gene expression of fibrosis- and inflammation-related markers but decreased lipid metabolism markers. LPS and TGFβ1 affected sPCLS more pronouncedly than cPCLS. TGFβ1 increased procollagen Iα1 solely in cPCLS. Elafibranor ameliorated fibrosis and inflammation in vivo but not ex vivo, where it only increased the expression of genes modulated by PPARα. sPCLS culture induced inflammation-, fibrosis-, and lipid metabolism-related transcripts, explained by spontaneous activation. sPCLS remained responsive to proinflammatory and profibrotic stimuli on gene expression. We consider that PCLS represent a useful tool to reproducibly study NASH progression. sPCLS can be used to evaluate potential treatments for NASH, as demonstrated in our elafibranor study, and serves as a model to bridge results from rodent studies to the human system. NEW & NOTEWORTHY This study showed that nonalcoholic steatohepatitis can be studied ex vivo in precision-cut liver slices obtained from murine diet-induced fatty livers. Liver slices develop a spontaneous inflammatory and fibrogenic response during culture that can be augmented with specific modulators. Additionally, the model can be used to test the efficacy of pharmaceutical compounds (as shown in this investigation with elafibranor) and could be a tool for preclinical assessment of potential therapies.

scholarly journals Type I IFN is siloed in endosomes

2020 ◽  
Vol 117 (30) ◽  
pp. 17510-17512 ◽  
Author(s):  
Jennie B. Altman ◽  
Justin Taft ◽  
Tim Wedeking ◽  
Conor N. Gruber ◽  
Michael Holtmannspötter ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cell Types ◽  
Specific Cell ◽  
Type I ◽  
Type I Ifn ◽  
Long Term Effects ◽  
Negative Regulators ◽  
Downstream Signaling

Type I IFN (IFN-I) is thought to be rapidly internalized and degraded following binding to its receptor and initiation of signaling. However, many studies report the persistent effects mediated by IFN-I for days or even weeks, both ex vivo and in vivo. These long-lasting effects are attributed to downstream signaling molecules or induced effectors having a long half-life, particularly in specific cell types. Here, we describe a mechanism explaining the long-term effects of IFN-I. Following receptor binding, IFN-I is siloed into endosomal compartments. These intracellular “IFN silos” persist for days and can be visualized by fluorescence and electron microscopy. However, they are largely dormant functionally, due to IFN-I−induced negative regulators. By contrast, in individuals lacking these negative regulators, such as ISG15 or USP18, this siloed IFN-I can continue to signal from within the endosome. This mechanism may underlie the long-term effects of IFN-I therapy and may contribute to the pathophysiology of type I interferonopathies.

scholarly journals Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance

2016 ◽  
Vol 37 (6) ◽  
pp. 2013-2024 ◽  
Author(s):  
Ain A Neuhaus ◽  
Yvonne Couch ◽  
Brad A Sutherland ◽  
Alastair M Buchan
Keyword(s):  
Electrical Impedance ◽  
Ex Vivo ◽  
Capillary Endothelium ◽  
Tissue Slices ◽  
In Vitro Techniques ◽  
Endothelin 1 ◽  
Mural Cells ◽  
Novel Method

Pericytes are contractile vascular mural cells overlying capillary endothelium, and they have been implicated in a variety of functions including regulation of cerebral blood flow. Recent work has suggested that both in vivo and ex vivo, ischaemia causes pericytes to constrict and die, which has implications for microvascular reperfusion. Assessing pericyte contractility in tissue slices and in vivo is technically challenging, while in vitro techniques remain unreliable. Here, we used isolated cultures of human brain vascular pericytes to examine their contractile potential in vitro using the iCelligence electrical impedance system. Contraction was induced using the vasoactive peptide endothelin-1, and relaxation was demonstrated using adenosine and sodium nitroprusside. Endothelin-1 treatment also resulted in increased proliferation, which we were able to monitor in the same cell population from which we recorded contractile responses. Finally, the observation of pericyte contraction in stroke was reproduced using chemical ischaemia, which caused a profound and irreversible contraction clearly preceding cell death. These data demonstrate that isolated pericytes retain a contractile phenotype in vitro, and that it is possible to quantify this contraction using real-time electrical impedance recordings, providing a significant new platform for assessing the effects of vasoactive and vasculoprotective compounds on pericyte contractility.

scholarly journals Preclinical Safety Evaluation of Intranasally Delivered Human Mesenchymal Stem Cells in Juvenile Mice

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1169
Author(s):  
Yolanda Aguilera ◽  
Nuria Mellado-Damas ◽  
Laura Olmedo-Moreno ◽  
Víctor López ◽  
Concepción Panadero-Morón ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Ex Vivo ◽  
Tumor Formation ◽  
Human Mscs ◽  
Long Term Effects ◽  
Serious Adverse Events ◽  
Immunodeficient Mice ◽  
Non Invasive ◽  
Promising Therapeutic Approach

Mesenchymal stem cell (MSC)-based therapy is a promising therapeutic approach in the management of several pathologies, including central nervous system diseases. Previously, we demonstrated the therapeutic potential of human adipose-derived MSCs for neurological sequelae of oncological radiotherapy using the intranasal route as a non-invasive delivery method. However, a comprehensive investigation of the safety of intranasal MSC treatment should be performed before clinical applications. Here, we cultured human MSCs in compliance with quality control standards and administrated repeated doses of cells into the nostrils of juvenile immunodeficient mice, mimicking the design of a subsequent clinical trial. Short- and long-term effects of cell administration were evaluated by in vivo and ex vivo studies. No serious adverse events were reported on mouse welfare, behavioral performances, and blood plasma analysis. Magnetic resonance study and histological analysis did not reveal tumor formation or other abnormalities in the examined organs of mice receiving MSCs. Biodistribution study reveals a progressive disappearance of transplanted cells that was further supported by an absent expression of human GAPDH gene in the major organs of transplanted mice. Our data indicate that the intranasal application of MSCs is a safe, simple and non-invasive strategy and encourage its use in future clinical trials.

scholarly journals Melanopsin+RGCs Are Fully Resistant to NMDA-Induced Excitotoxicity

2019 ◽  
Author(s):  
Beatriz Vidal-Villegas ◽  
Johnny Di Pierdomenico ◽  
Juan Antonio Miralles de Imperial-Ollero ◽  
Arturo Ortín-Martínez ◽  
Francisco Manuel Nadal-Nicolás ◽  
...  
Keyword(s):  
Intravitreal Injection ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Retinal Ganglion ◽  
Long Term Effects ◽  
Short And Long Term ◽  
The Mean ◽  
Sd Oct

We studied short- and long-term effects of intravitreal injection of N-methyl-D-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received  a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using SD-OCT.  Ex vivo analyses were done at 3, 7, 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for Brn3a and melanopsin, the total number of Brn3a+RGCs and m+RGCs were quantified and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903±3,572 and 2,358±144 (mean ± SD; n=10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 50% and 25%, at 3 and 14 days, respectively, but there was no further loss up to 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3-7 days and was back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induce a rapid loss of 75% of Brn3a+RGCs, a transient downregulation of melanopsin expression but not m+RGC death, and a thinning of the inner retinal layers.

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