scholarly journals Imaging of C-fos Activity in Neurons of the Mouse Parietal Association Cortex during Acquisition and Retrieval of Associative Fear Memory

2021 ◽  
Vol 22 (15) ◽  
pp. 8244
Author(s):  
Olga I. Ivashkina ◽  
Anna M. Gruzdeva ◽  
Marina A. Roshchina ◽  
Ksenia A. Toropova ◽  
Konstantin V. Anokhin
Keyword(s):  
Ex Vivo ◽  
Fear Memory ◽  
Experimental Models ◽  
Movement Planning ◽  
Fear Learning ◽  
Association Cortex ◽  
Transgenic Mouse Line ◽  
Two Photon ◽  
Parietal Association Cortex

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.

scholarly journals Ex vivo and in vivo imaging of mouse parietal association cortex activity in episodes of cued fear memory formation and retrieval

2019 ◽  
Author(s):  
Olga I. Ivashkina ◽  
Anna M. Gruzdeva ◽  
Marina A. Roshchina ◽  
Ksenia A. Toropova ◽  
Konstantin V. Anokhin
Keyword(s):  
Parietal Cortex ◽  
Ex Vivo ◽  
Anterior Part ◽  
Fear Memory ◽  
Memory Formation ◽  
Movement Planning ◽  
Association Cortex ◽  
Memory Processing ◽  
Two Photon

AbstractThe parietal cortex in rodents has an integrative function and participates in sensory and spatial processing, movement planning and decision-making. However, much less is known about its functions in associative memory processing. Here using Fos immunohistochemical mapping of neuronal activity and two-photon imaging in Fos-eGFP mice we show an involvement of anterior part of the parietal cortex (PtA) in the formation and retrieval of recent fear memory in mice. Using ex vivo c-fos imaging we demonstrate the specific activation of the PtA during recent memory retrieval. In vivo two-photon c-fos imaging confirms these results as well as establishes the activation of the PtA neurons during fear memory formation. Additionally, we describe a design of Fos-Cre-GCaMP transgenic mice to investigate long-term changes of calcium dynamics in neurons captured with Fos-TRAP technique during fear conditioning training.

scholarly journals Ocular Toxoplasmosis: Mechanisms of Retinal Infection and Experimental Models

Parasitologia ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 50-60
Author(s):  
Veronica Rodriguez Fernandez ◽  
Giovanni Casini ◽  
Fabrizio Bruschi
Keyword(s):  
Ex Vivo ◽  
Cell Damage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Treatment Strategies ◽  
Experimental Models ◽  
Ocular Toxoplasmosis ◽  
Cell Infection

Ocular toxoplasmosis (OT) is caused by the parasite Toxoplasma gondii and affects many individuals throughout the world. Infection may occur through congenital or acquired routes. The parasites enter the blood circulation and reach both the retina and the retinal pigment epithelium, where they may cause cell damage and cell death. Different routes of access are used by T. gondii to reach the retina through the retinal endothelium: by transmission inside leukocytes, as free parasites through a paracellular route, or after endothelial cell infection. A main feature of OT is the induction of an important inflammatory state, and the course of infection has been shown to be influenced by the host immunogenetics. On the other hand, there is evidence that the T. gondii phenotype also has an impact on the distribution of the pathology in different areas. Although considerable knowledge has been acquired on OT, a deeper knowledge of its mechanisms is necessary to provide new, more targeted treatment strategies. In particular, in addition to in vitro and in vivo experimental models, organotypic, ex vivo retinal explants may be useful in this direction.

Endothelial glycocalyx thickness and platelet-vessel wall interactions during atherogenesis

2011 ◽  
Vol 106 (11) ◽  
pp. 939-946 ◽  
Author(s):  
Mirjam oude Egbrink ◽  
Viviane Heijnen ◽  
Remco Megens ◽  
Wim Engels ◽  
Hans Vink ◽  
...  
Keyword(s):  
Vessel Wall ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Endothelial Glycocalyx ◽  
Knock Out ◽  
Two Photon ◽  
Common Carotid Arteries ◽  
Wall Interactions

SummaryThe endothelial glycocalyx (EG), the luminal cover of endothelial cells, is considered to be atheroprotective. During atherogenesis, platelets adhere to the vessel wall, possibly triggered by simultaneous EG modulation. It was the objective of this study to investigate both EG thickness and platelet-vessel wall interactions during atherogenesis in the same experimental model. Intravital fluorescence microscopy was used to study platelet-vessel wall interactions in vivo in common carotid arteries and bifurcations of C57bl6/J (B6) and apolipoprotein E knock-out (ApoE-/-) mice (age 7 – 31 weeks). At the same locations, EG thickness was determined ex vivo using two-photon laser scanning microscopy. In ApoE-/- bifurcations the overall median level of adhesion was 48 platelets/mm2 (interquartile range: 16 – 80), which was significantly higher than in B6 bifurcations (0 (0 – 16), p = 0.001). This difference appeared to result from a significant age-dependent increase in ApoE-/- mice, while no such change was observed in B6 mice. At the same time, the EG in ApoE-/- bifurcations was significantly thinner than in B6 bifurcations (2.2 vs. 2.5 μm, respectively; p < 0.05). This resulted from the fact that in B6 bifurcations EG thickness increased with age (from 2.4 μm in young mice to 3.0 μm in aged ones), while in bifurcations of ApoE-/- mice this growth appeared to be absent (2.2 μm at all ages). During atherogenesis, platelet adhesion to the wall of the carotid artery bifurcation increases significantly. At the same location, EG growth with age is hampered. Therefore, glycocalyx-reinforcing strategies could possibly ameliorate atherosclerosis.

scholarly journals p-Coumaric Acid as An Active Ingredient in Cosmetics: A Review Focusing on its Antimelanogenic Effects

Antioxidants ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 275 ◽  
Author(s):  
Yong Chool Boo
Keyword(s):  
Active Ingredient ◽  
Antioxidant Activities ◽  
Ex Vivo ◽  
Skin Permeation ◽  
Skin Pigmentation ◽  
Experimental Models ◽  
Coumaric Acid ◽  
Inflammatory Reactions ◽  
Skin Lightening

Controlling unwanted hyperpigmentation is a major challenge in dermatology and cosmetology, and safe and efficacious antimelanogenic agents are deemed useful for this purpose. p-Coumaric acid is a natural metabolite contained in many edible plants, and its antioxidant activities in reducing oxidative stress and inflammatory reactions have been demonstrated in various experimental models. p-Coumaric acid has the optimal structure to be a competitive inhibitor of tyrosinase that catalyzes key reactions in the melanin biosynthetic pathway. Experimental evidence supports this notion as it was found to be a more potent inhibitor of tyrosinase, especially toward human enzymes, than other well-known tyrosinase inhibitors such as arbutin and kojic acid. p-Coumaric acid inhibited melanin synthesis in murine melanoma cells, human epidermal melanocytes, and reconstituted three-dimensional human skin models. Ex-vivo skin permeation experiments and in-vivo efficacy tests for p-coumaric acid confirmed its efficient transdermal delivery and functional efficacy in reducing erythema development and skin pigmentation due to ultraviolet radiation exposure. Human studies further supported its effectiveness in hypopigmentation and depigmentation. These findings suggest that p-coumaric acid has good potential to be used as a skin-lightening active ingredient in cosmetics. Future studies are needed to extensively examine its safety and efficacy and to develop an optimized cosmetic formulation for the best performance in skin lightening.

scholarly journals Study of Melatonin as Preventive Agent of Gastrointestinal Damage Induced by Sodium Diclofenac

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 180 ◽  
Author(s):  
Aroha B. Sánchez ◽  
Beatriz Clares ◽  
María J. Rodríguez-Lagunas ◽  
María J. Fábrega ◽  
Ana C. Calpena
Keyword(s):  
Side Effects ◽  
Redox State ◽  
Ex Vivo ◽  
Inhibitory Effect ◽  
Experimental Models ◽  
In Vivo Studies ◽  
Histological Evaluation ◽  
Sodium Diclofenac ◽  
Cox 2

Safety profile of nonsteroidal anti-inflammatory drugs (NSAIDs) has been widely studied and both therapeutic and side effects at the gastric and cardiovascular level have been generally associated with the inhibitory effect of isoform 1 (COX-1) and 2 (COX-2) cyclooxygenase enzymes. Now there are evidences of the involvement of multiple cellular pathways in the NSAIDs-mediated-gastrointestinal (GI) damage related to enterocyte redox state. In a previous review we summarized the key role of melatonin (MLT), as an antioxidant, in the inhibition of inflammation pathways mediated by oxidative stress in several diseases, which makes us wonder if MLT could minimize GI NSAIDs side effects. So, the aim of this work is to study the effect of MLT as preventive agent of GI injury caused by NSAIDs. With this objective sodium diclofenac (SD) was administered alone and together with MLT in two experimental models, ex vivo studies in pig intestine, using Franz cells, and in vivo studies in mice where stomach and intestine were studied. The histological evaluation of pig intestine samples showed that SD induced the villi alteration, which was prevented by MLT. In vivo experiments showed that SD altered the mice stomach mucosa and induced tissue damage that was prevented by MLT. The evaluation by quantitative reverse transcription PCR (RT-qPCR) of two biochemical markers, COX-2 and iNOS, showed an increase of both molecules in less injured tissues, suggesting that MLT promotes tissue healing by improving redox state and by increasing iNOS/NO that under non-oxidative condition is responsible for the maintenance of GI-epithelium integrity, increasing blood flow and promoting angiogenesis and that in presence of MLT, COX-2 may be responsible for wound healing in enterocyte. Therefore, we found that MLT may be a preventive agent of GI damages induced by NSAIDs.

scholarly journals Ultrasound-assisted gatifloxacin delivery in mouse cornea, in vivo

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Uk Jegal ◽  
Jun Ho Lee ◽  
Jungbin Lee ◽  
Hyerin Jeong ◽  
Myoung Joon Kim ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Ophthalmic Solution ◽  
Ultrasound Treatment ◽  
Ocular Infection ◽  
Therapeutic Effects ◽  
Corneal Surface ◽  
Ultrasound Waves ◽  
Two Photon Microscopy ◽  
Two Photon

Abstract Gatifloxacin is a 4th generation fluoroquinolone antibiotic used in the clinic to treat ocular infection. One limitation of gatifloxacin is its relatively poor corneal penetration, and the increase of its trans-corneal delivery would be beneficial to reduce the amount or frequency of daily dose. In this study, ultrasound treatment was applied to enhance the trans-corneal delivery of gatifloxacin without damage. Experiments were conducted on mouse eyes in ex vivo and in vivo conditions. Ultrasound waves with 1 MHz in frequency, 1.3 W/cm2 in intensity were applied onto the mouse cornea for 5 minutes, and then gatifloxacin ophthalmic solution was instilled and left there for 10 minutes. 3D gatifloxacin distribution in the cornea was measured by two-photon microscopy (TPM) imaging based on its intrinsic fluorescence. Longitudinal TPM imaging of ultrasound treated mouse corneas showed the increase of initial gatifloxacin intensities on the corneal surface compared to untreated mouse corneas by 67%, and then the increased gatifloxacin delivery into the cornea from the surface at later time. The delivered gatifloxacin in the corneal epithelium stayed longer in the ultrasound treated corneas than in the untreated corneas. The enhanced trans-corneal delivery and extended stay of gatifloxacin in the mouse cornea by ultrasound treatment could be beneficial for therapeutic effects. This study demonstrated the detail process of enhanced trans-corneal gatifloxacin delivery by ultrasound treatment.

scholarly journals Two-Photon Microscopy Allows Imaging and Characterization of Cochlear Microvasculature In Vivo

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Friedrich Ihler ◽  
Mattis Bertlich ◽  
Bernhard Weiss ◽  
Steffen Dietzel ◽  
Martin Canis
Keyword(s):  
Fluorescence Microscopy ◽  
Inner Ear ◽  
Ex Vivo ◽  
Small Scale ◽  
Published Data ◽  
Cochlear Blood Flow ◽  
Two Photon ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence

Impairment of cochlear blood flow has been discussed as factor in the pathophysiology of various inner ear disorders. However, the microscopic study of cochlear microcirculation is limited due to small scale and anatomical constraints. Here, two-photon fluorescence microscopy is applied to visualize cochlear microvessels. Guinea pigs were injected with Fluorescein isothiocyanate- or Texas red-dextrane as plasma marker. Intravital microscopy was performed in four animals and explanted cochleae from four animals were studied. The vascular architecture of the cochlea was visualized up to a depth of90.0±22.7 μm. Imaging yielded a mean contrast-to-noise ratio (CNR) of3.3±1.7. Mean diameter in vivo was16.5±6.0 μm for arterioles and8.0±2.4 μm for capillaries. In explanted cochleae, the diameter of radiating arterioles and capillaries was measured with12.2±1.6 μm and6.6±1.0 μm, respectively. The difference between capillaries and arterioles was statistically significant in both experimental setups (P<0.001andP=0.022, two-way ANOVA). Measured vessel diameters in vivo and ex vivo were in agreement with published data. We conclude that two-photon fluorescence microscopy allows the investigation of cochlear microvessels and is potentially a valuable tool for inner ear research.

Ex vivo development of functional human lymph node and bronchus-associated lymphoid tissue

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2483-2489 ◽  
Author(s):  
Rabindra Tirouvanziam ◽  
Ibrahim Khazaal ◽  
Victoire N'Sondé ◽  
Marie-Alix Peyrat ◽  
Annick Lim ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
Mast Cells ◽  
Lymphoid Tissue ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Experimental Models ◽  
In Vivo Model ◽  
Functional Relations

We introduce a novel in vivo model of human mucosal immunity, based on the implantation of human fetal bronchial mucosa and autologous peribronchial lymph node (PLN) in the severe combined immunodeficiency (SCID) mouse. In the SCID host, human fetal bronchi implanted alone retain macrophages and mast cells but lose T cells. In contrast, fetal bronchi co-implanted with PLN contain, in addition to macrophages and mast cells, numerous T cells and B cells, often clustered in intramucosal bronchus-associated lymphoid tissue (BALT). Functionally, bronchus–PLN cografts are able to mount robust αβ and γδ T-cell–mediated immune responses to Pseudomonas aeruginosa and 3,4-epoxy-3-methyl-1-butyl-diphosphate challenges. No other autologous lymphoid organ (bone marrow, thymus, liver) allows for BALT development in co-implanted bronchi, which suggests special ontogenetic and functional relations between extramucosal PLN and intramucosal BALT. Overall, the bronchus–PLN cograft appears as a promising model for human bronchial immune development and function. Our study is the first to document long-term ex vivo maintenance of functional human lymph nodes as native appendices to mucosal tissue. Our results, therefore, suggest a simple strategy for developing similar experimental models of human immune function in other mucosae.

scholarly journals Imbalance of flight-freeze responses and their cellular correlates in the Nlgn3-/y rat model of autism

2020 ◽  
Author(s):  
Natasha J Anstey ◽  
Vijayakumar Kapgal ◽  
Shashank Tiwari ◽  
Thomas C Watson ◽  
Anna KH Toft ◽  
...  
Keyword(s):  
Emotional Disorders ◽  
Rat Model ◽  
Ex Vivo ◽  
Transmembrane Protein ◽  
Autism Spectrum ◽  
Fear Learning ◽  
Flight Behaviour ◽  
Cellular Excitability ◽  
Lower Magnitude

SummaryMutations in the postsynaptic transmembrane protein neuroligin-3 are highly correlative with autism spectrum disorders (ASDs) and intellectual disabilities (IDs). Fear learning is well studied in models of these disorders, however differences in fear response behaviours are often overlooked. Whilst examining fear in a rat model of ASD/ID lacking Nlgn3, we observed that they display a greater propensity to exhibit flight responses in contrast to classic freezing seen in wildtypes during fearful situations. Consequently, we examined the physiological underpinnings of this in neurons of the periaqueductal grey (PAG), a midbrain area involved in flight-or-freeze responses. In ex vivo slices from Nlgn3-/y, rats, dorsal PAG (dPAG) neurons showed intrinsic hyperexcitability. Further analysis of this revealed lower magnitude in vivo dPAG stimulation evoked flight behaviour in Nlgn3-/y, rats, indicating the functional impact of the increased cellular excitability. This study provides new insight into potential pathophysiologies leading to emotional disorders in individuals with ASD.

scholarly journals Short-Term Ex Vivo Culture of CTCs From Advance Breast Cancer Patients: Clinical Implications

2021 ◽  
Author(s):  
Nuria Carmona-Ule ◽  
Miriam González-Conde ◽  
Carmen Abuín ◽  
Juan F Cueva ◽  
Patricia Palacios ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Ex Vivo ◽  
Metastatic Breast ◽  
Experimental Models ◽  
Breast Cancer Patients ◽  
Phenotypic Analysis ◽  
Advance Breast Cancer ◽  
Ex Vivo Culture

Background: Circulating tumor cells (CTC) have relevance as prognostic markers in breast cancer. However, the functional properties of CTCs or their molecular characterization have not been well-studied. Experimental models indicate that only a few cells can survive in the circulation and eventually metastasize. Thus, it is essential to identify these surviving cells capable of forming such metastases. Methods: We isolated viable CTCs from 50 peripheral blood samples obtained from 35 patients with advanced metastatic breast cancer using RosetteSepTM for ex vivo culture. The CTCs were seeded and monitored on plates under low adherence conditions and with media supplemented with growth factors and Nanoemulsions. Phenotypic analysis was performed by immunofluorescence and gene expression analysis using RT-PCR and CTCs counting by Cellsearch&reg; system. Results: We found that in 75% of samples the CTC cultures lasted more than 23 days, predicting a shorter Progression-Free Survival in these patients, independently of having &ge; 5 CTC by Cellsearch&reg;. We also observed that CTCs before and after culture showed a different gene expression profile. Conclusions: the cultivability of CTCs is a predictive factor. Furthermore, the subset of cells capable of growing ex vivo show stem or mesenchymal features and may represent the CTC population with metastatic potential in vivo.

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