scholarly journals 3dSpAn: An interactive software for 3D segmentation and analysis of dendritic spines

2019 ◽  
Author(s):  
Nirmal Das ◽  
Ewa Baczynska ◽  
Monika Bijata ◽  
Blazej Ruszczycki ◽  
Andre Zeug ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Interactive Software ◽  
Seed Selection ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Observation Methods

AbstractThree dimensional segmentation and analysis of dendritic spines involve two major challenges: 1) how to segment individual spines from the dendrites and 2) how to quantitatively assess the morphology of individual spines. We developed a software named 3dSpAn to address these two issues by implementing our previously published 3D multiscale opening algorithm in shared intensity space and using effective morphological features for individual dendritic spine plasticity analysis. 3dSpAn consists of four modules: Preprocessing and ROI selection, Intensity thresholding and seed selection, Multiscale segmentation and Quantitative morphological feature extraction. We show the results of segmentation and morphological analysis for different observation methods, including in vitro and ex vivo imaging with confocal microscopy, and in vivo samples, using high-resolution two-photon microscopy. The software is freely available, the source code, windows installer, the software manual and video tutorial can be obtained from: https://sites.google.com/view/3dSpAn/.

scholarly journals Bidirectional in vivo structural dendritic spine plasticity revealed by two-photon glutamate uncaging in the mouse neocortex

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jun Noguchi ◽  
Akira Nagaoka ◽  
Tatsuya Hayama ◽  
Hasan Ucar ◽  
Sho Yagishita ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Time Course ◽  
Hippocampal Slices ◽  
Low Frequency ◽  
Structural Plasticity ◽  
Experimental Conditions ◽  
Two Photon ◽  
Spine Plasticity

Abstract Most excitatory synapses in the brain form on dendritic spines. Two-photon uncaging of glutamate is widely utilized to characterize the structural plasticity of dendritic spines in brain slice preparations in vitro. In the present study, glutamate uncaging was used to investigate spine plasticity, for the first time, in vivo. A caged glutamate compound was applied to the surface of the mouse visual cortex in vivo, revealing the successful induction of spine enlargement by repetitive two-photon uncaging in a magnesium free solution. Notably, this induction occurred in a smaller fraction of spines in the neocortex in vivo (22%) than in hippocampal slices (95%). Once induced, the time course and mean long-term enlargement amplitudes were similar to those found in hippocampal slices. However, low-frequency (1–2 Hz) glutamate uncaging in the presence of magnesium caused spine shrinkage in a similar fraction (35%) of spines as in hippocampal slices, though spread to neighboring spines occurred less frequently than it did in hippocampal slices. Thus, the structural plasticity may occur similarly in the neocortex in vivo as in hippocampal slices, although it happened less frequently in our experimental conditions.

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.

Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 942-952 ◽  
Author(s):  
Björn Petri ◽  
Jaswinder Kaur ◽  
Elizabeth M. Long ◽  
Hang Li ◽  
Sean A. Parsons ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Neutrophil Migration ◽  
Endothelial Activation ◽  
Specific Protein ◽  
Actin Binding ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Factor Α

Abstract The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin–binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1β [IL-1β]). Endothelial domes were significantly reduced in Lsp1−/− compared with wild-type (WT) mice. Lsp1−/− animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.

Robot-assisted endoscopic exploration of the spinal cord

2010 ◽  
Vol 224 (7) ◽  
pp. 1515-1529 ◽  
Author(s):  
L Ascari ◽  
C Stefanini ◽  
U Bertocchi ◽  
P Dario
Keyword(s):  
Spinal Cord ◽  
Subarachnoid Space ◽  
Ex Vivo ◽  
Hierarchical Control ◽  
Three Dimensional ◽  
Robot Assisted ◽  
Actuation System ◽  
Spinal Subarachnoid Space

This work presents the design and development of an integrated image-guided robot-assisted endoscopic system for the safe navigation within the spinal subarachnoid space, providing the surgeon with the direct vision of the structures (i.e. spinal cord, roots, vessels) and the possibility of performing some particularly useful operations, like local electrostimulation of nerve roots. The modelling, micro-fabrication, fluidic sustentation, and cable-based actuation system of a steerable tip for a multilumen flexible catheter is described; the hierarchical control system shared between the surgeon and the computer, and based on machine vision techniques and a simple but effective three-dimensional reconstruction is detailed. The Blind Expected Perception sensory-motor scheme is proposed in robot-assited endoscopy. Results from in vitro, ex vivo, and in vivo experiments show that the described model can accurately predict the shape of the catheter given the tension distribution on the cables, that the proposed actuation system can assure smooth and precise control of the catheter tip, that the fluidic sustentation of the catheter is essential in in vivo navigation, and that the proposed rear view mirror interface to show non-visible obstacles is appropriate; in conclusion, the results proved the validity of the proposed solution to develop an intrinsically safe robotic system for navigation and intervention in a narrow and challenging environment such as the spinal subarachnoid space.

scholarly journals Increased Mesenchymal Stem Cell Functionalization in Three-Dimensional Manufacturing Settings for Enhanced Therapeutic Applications

2021 ◽  
Vol 9 ◽  
Author(s):  
Dimitrios Kouroupis ◽  
Diego Correa
Keyword(s):  
Therapeutic Potential ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Therapeutic Applications ◽  
Cell Based Therapy ◽  
Donor Variability ◽  
Healing Bone ◽  
Preclinical Animal Models

Mesenchymal stem/stromal cell (MSC) exist within their in vivo niches as part of heterogeneous cell populations, exhibiting variable stemness potential and supportive functionalities. Conventional extensive 2D in vitro MSC expansion, aimed at obtaining clinically relevant therapeutic cell numbers, results in detrimental effects on both cellular characteristics (e.g., phenotypic changes and senescence) and functions (e.g., differentiation capacity and immunomodulatory effects). These deleterious effects, added to the inherent inter-donor variability, negatively affect the standardization and reproducibility of MSC therapeutic potential. The resulting manufacturing challenges that drive the qualitative variability of MSC-based products is evident in various clinical trials where MSC therapeutic efficacy is moderate or, in some cases, totally insufficient. To circumvent these limitations, various in vitro/ex vivo techniques have been applied to manufacturing protocols to induce specific features, attributes, and functions in expanding cells. Exposure to inflammatory cues (cell priming) is one of them, however, with untoward effects such as transient expression of HLA-DR preventing allogeneic therapeutic schemes. MSC functionalization can be also achieved by in vitro 3D culturing techniques, in an effort to more closely recapitulate the in vivo MSC niche. The resulting spheroid structures provide spatial cell organization with increased cell–cell interactions, stable, or even enhanced phenotypic profiles, and increased trophic and immunomodulatory functionalities. In that context, MSC 3D spheroids have shown enhanced “medicinal signaling” activities and increased homing and survival capacities upon transplantation in vivo. Importantly, MSC spheroids have been applied in various preclinical animal models including wound healing, bone and osteochondral defects, and cardiovascular diseases showing safety and efficacy in vivo. Therefore, the incorporation of 3D MSC culturing approach into cell-based therapy would significantly impact the field, as more reproducible clinical outcomes may be achieved without requiring ex vivo stimulatory regimes. In the present review, we discuss the MSC functionalization in 3D settings and how this strategy can contribute to an improved MSC-based product for safer and more effective therapeutic applications.

Organoids as ex vivo culture system to investigate infection-host interaction in gastric pre-carcinogenesis.

2022 ◽  
Vol 16 ◽  
Author(s):  
Cristina Di Giorgio ◽  
Rosalinda Roselli ◽  
Michele Biagioli ◽  
Silvia Marchianò ◽  
Eleonora Distrutti ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Cell Lines ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Mucosal Injury ◽  
In Vitro Models ◽  
World Population ◽  
Barr Virus

Abstract: Advancements in stem cell research have enabled the establishment of three-dimensional (3D) primary cell cultures, known as organoids. These culture systems follow the organization of an in vivo organ, as they enclose the different epithelial cell lines of which it is normally composed. Generation of these 3D cultures has bridged the gap between in vitro models, made up by two-dimensional (2D) cancer cell lines cultures, and in vivo animal models, that have major differences with human diseases. Organoids are increasingly used as a model to study colonization of gastric mucosa by infectious agents and to better understand host-microbe interactions and the molecular events that lead to infection, pathogen-epithelial cells interactions and mechanisms of gastric mucosal injury. In this review we will focus on the role of organoids as a tool to investigate molecular interactions of Helicobacter (H.) pylori and Epstein Barr Virus (EBV) and gastric mucosa and how these infections, that affect ≈ 45% of the world population, might progress to gastric cancer, a highly prevalent cancer and the third leading cause of cancer death.

scholarly journals Chronic imaging of mitochondria in the murine cerebral vasculature using in vivo two-photon microscopy

2020 ◽  
Vol 318 (6) ◽  
pp. H1379-H1386
Author(s):  
Ibolya Rutkai ◽  
Wesley R. Evans ◽  
Nikita Bess ◽  
Tomas Salter-Cid ◽  
Siniša Čikić ◽  
...  
Keyword(s):  
Cerebral Circulation ◽  
Three Dimensional ◽  
Cell Types ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Dimensional Reconstruction ◽  
Different Cell Types

We introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of long-term imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation.

Magnetically actuated microrobots as a platform for stem cell transplantation

2019 ◽  
Vol 4 (30) ◽  
pp. eaav4317 ◽  
Author(s):  
Sungwoong Jeon ◽  
Sangwon Kim ◽  
Shinwon Ha ◽  
Seungmin Lee ◽  
Eunhee Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Brain Slice ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Magnetically Actuated ◽  
Magnetic Microrobots ◽  
Hippocampal Neural Stem Cells ◽  
Human Nose

Magnetic microrobots were developed for three-dimensional culture and the precise delivery of stem cells in vitro, ex vivo, and in vivo. Hippocampal neural stem cells attached to the microrobots proliferated and differentiated into astrocytes, oligodendrocytes, and neurons. Moreover, microrobots were used to transport colorectal carcinoma cancer cells to tumor microtissue in a body-on-a-chip, which comprised an in vitro liver-tumor microorgan network. The microrobots were also controlled in a mouse brain slice and rat brain blood vessel. Last, microrobots carrying mesenchymal stem cells derived from human nose were manipulated inside the intraperitoneal cavity of a nude mouse. The results indicate the potential of microrobots for the culture and delivery of stem cells.

scholarly journals Calcium Imaging of Living Astrocytes in the Mouse Spinal Cord following Sensory Stimulation

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Giovanni Cirillo ◽  
Daniele De Luca ◽  
Michele Papa
Keyword(s):  
Spinal Cord ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Sensory Stimulation ◽  
Widespread Application ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Heart Beating ◽  
Sensory Activity

Astrocytic Ca2+dynamics have been extensively studied inex vivomodels; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca2+signaling in living central nervous system. Ca2+waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic activation during sensory activity is lacking. There are currently few methods to image living spinal cord: breathing and heart-beating artifacts have impeded the widespread application of this technique. We here imaged the living spinal cord by two-photon microscopy in C57BL6/J mice. Through pressurized injection, we specifically loaded spinal astrocytes using the red fluorescent dye sulforhodamine 101 (SR101) and imaged astrocytic Ca2+levels with Oregon-Green BAPTA-1 (OGB). Then, we studied astrocytic Ca2+levels at rest and after right electrical hind paw stimulation. Sensory stimulation significantly increased astrocytic Ca2+levels within the superficial dorsal horn of the spinal cord compared to rest. In conclusion,in vivomorphofunctional imaging of living astrocytes in spinal cord revealed that astrocytes actively participate to sensory stimulation.

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