scholarly journals Liver Sinusoidal Endothelial Cell (Lsec) Isolation Following a Liver Perfusion Technique

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Shahida Saharudin ◽  
Norlelawati A. Talib ◽  
Nor Zamzila Abdullah ◽  
Jamalludin Ab. Rahman ◽  
Zunariah Buyong
Keyword(s):  
Liver Perfusion ◽  
Light And Electron Microscopy ◽  
Average Concentration ◽  
Liver Sinusoidal Endothelial Cell ◽  
Sprague Dawley ◽  
Perfusion Technique ◽  
Liver Sinusoidal Endothelial Cells ◽  
Isolation Technique ◽  
And Function

Introduction: Liver perfusion has been the standard method to digest and isolate liver cells including liver sinusoidal endothelial cells (LSEC). Poor cannulating skills through portal vein results in a waste of animal resource. Familiarization of both liver perfusion technique and adhering strictly to aseptic technique during cell handling ensure high cell yield, minimum morphology disruption and cell contamination. We aimed to present a method of liver perfusion procedure followed by the isolation of LSEC. Materials and method: The study was conducted with the approval of IACUC committee. Seven Sprague Dawley rats underwent these procedures under anaesthesia. Liver perfusion was done as previously described. Briefly, LSEC were isolated by liberase enzyme perfusion of the liver, isopycnic sedimentation in a two- step Percoll gradient and selective adherence. The purification and cultivation of LSEC was evaluated by light and electron microscopy. Results: Purity and viability of LSEC after selective adherence was 80.5 ± 3.5% and ≥ 95 %, respectively. The average concentration of the cells ranged from 32 - 75 x 106 per 400 gm rat. After 8 hours of culture, LSEC monolayers were contaminated with less than 5% of other cells. Conclusion: This method is reliable and reproducible for the isolation of LSEC to enable the study of structure and function of these cells in vitro. However, improvement on the perfusion skills and isolation technique are vital to ensure better cell purity.

Effect of basolateral acidification on the frog oxynticopeptic cell

1990 ◽  
Vol 259 (4) ◽  
pp. G564-G570 ◽  
Author(s):  
S. Arvidsson ◽  
K. Carter ◽  
A. Yanaka ◽  
S. Ito ◽  
W. Silen
Keyword(s):  
Electron Microscopy ◽  
Gastric Mucosa ◽  
Light And Electron Microscopy ◽  
Structure And Function ◽  
Intracellular Acidification ◽  
Intracellular Acidosis ◽  
Normal Morphology ◽  
And Function ◽  
Oxynticopeptic Cells

The effects of intracellular acidosis induced by acidification of the basolateral (nutrient) perfusate on the structure and function of the oxynticopeptic cell were studied in in vitro frog gastric mucosa. Changing the pH of the unbuffered nutrient perfusate (UNB) from 7.2 to 3.5 acidified the oxynticopeptic cell with no change in potential difference (PD) or resistance (R). Intracellular pH (pHi), PD, and R were 7.05 +/- 0.01, 16 +/- 1 mV, 165 +/- 7 omega.cm2 before and 6.44 +/- 0.01, 16 +/- 2 mV, 170 +/- 9 omega.cm2 after nutrient acidification. Acid secretion (H+) increased from 0.86 +/- 0.07 to 1.88 +/- 0.18 mu eq.cm-2.h-1. Addition of forskolin to tissues perfused with nutrient pH (pHn) 3.5 decreased PD to 2 +/- 2 mV and further increased H+ to 3.07 +/- 0.19 mu eq.cm-2.h-1. By light and electron microscopy oxynticopeptic cells perfused with UNB, pHn 3.5, appeared normal. Oxynticopeptic cells in tissues pretreated with omeprazole and then exposed to UNB, pHn 3.5, had extensive morphological damage. On increasing the pH of the nutrient perfusate from 3.5 to 7.2 there was prompt recovery of pHi in untreated and forskolin-stimulated mucosae (pHi 6.87 +/- 0.06 and 6.85 +/- 0.04) but no recovery of pHi in tissues pretreated with omeprazole or cimetidine (pHi 6.26 +/- 0.04 and 6.44 +/- 0.06, n = 6, 30 min after reexposure to UNB, pHn 7.2). We conclude that in a secreting mucosa intracellular acidification of the oxynticopeptic cell to pHi 6.4 is associated with normal morphology, PD, R, and increased H+, and that intracellular acidosis is not de facto deleterious.

scholarly journals Protective role of microRNA-9-5p in oxygen glucose deprivation/reperfusion-induced injury in liver sinusoidal endothelial cell

2020 ◽  
Author(s):  
Yi Duan ◽  
Zhifeng Gao ◽  
Xiaoyu Wang ◽  
Yuanyuan Meng ◽  
Huan Zhang
Keyword(s):  
Inflammatory Response ◽  
Ischemia Reperfusion ◽  
Critical Role ◽  
Glucose Deprivation ◽  
Protective Role ◽  
Liver Sinusoidal Endothelial Cell ◽  
Liver Sinusoidal Endothelial Cells ◽  
Hepatic Ischemia

Abstract Background: Maintenance of the function and survival of liver sinusoidal endothelial cells (LSECs) play a crucial role in hepatic ischemia/reperfusion (I/R) injury, a major cause of liver impairment during surgical treatment. Emerging evidence indicate a critical role of microRNAs in I/R injury. This study aims to investigate whether miR-9-5p exert a protective effect on LSECs in vitro .Methods: We transfected LSECs with miR-9-5p mimic or mimic NC. LSECs were treated with oxygen and glucose deprivation (OGD, 5% CO2 and 95% N2), followed by glucose-free DMEM medium for 6 h, and high-glucose (HG, 30 mmol/L glucose) DMEM medium for 12 h. The biological role of miR-9-5p in I/R-induced LSEC injury was determined. Results: In the in vitro model of OGD/HG injury in LSECs, the expression levels of miR-9-5p were significantly downregulated and those of CXC chemokine receptor-4 (CXCR4) upregulated. LSEC I/R injury led to deteriorated cell death, enhanced oxidative stress and excessive inflammatory response. Mechanistically, we showed that miR-9-5p overexpression significantly upregulated both mRNA and protein levels of CXCR4, followed by rescue of LSECs, ameliorated inflammatory response, and deactivation of pro-apoptotic signaling pathways.Conclusion: miR-9-5p promotes LSEC survival and inhibits apoptosis and inflammatory response in LSECs following OGD/HG injury via downregulation of CXCR4.

scholarly journals FORMATION OF BONE TISSUE IN CULTURE FROM ISOLATED BONE CELLS

1974 ◽  
Vol 61 (2) ◽  
pp. 427-439 ◽  
Author(s):  
Itzhak Binderman ◽  
Dan Duksin ◽  
Arieh Harell ◽  
Ephraim Katzir (Katchalski) ◽  
Leo Sachs
Keyword(s):  
Electron Microscopy ◽  
Bone Tissue ◽  
Bone Cells ◽  
Light And Electron Microscopy ◽  
Bone Collagen ◽  
The Matrix ◽  
Three Stages ◽  
And Function

A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.

Alveolar macrophage depletion is associated with increased surfactant pool sizes in adult rats

2007 ◽  
Vol 103 (2) ◽  
pp. 637-645 ◽  
Author(s):  
Amy Forbes ◽  
Mike Pickell ◽  
Mehry Foroughian ◽  
Li-Juan Yao ◽  
James Lewis ◽  
...  
Keyword(s):  
Protein A ◽  
Surfactant Protein ◽  
Normal Lung ◽  
Sprague Dawley ◽  
Adult Rats ◽  
Cell Counts ◽  
And Function ◽  
The Impact ◽  
Pool Sizes

Pulmonary surfactant is a lipid-protein material that is essential for normal lung function. Maintaining normal and consistent alveolar amounts of surfactant is in part dependent on clearance of surfactant by alveolar macrophages (AM). The present study utilized a rat model of AM depletion to determine the impact on surfactant pool sizes and function over time. Male Sprague-Dawley rats were anesthetized and intratracheally instilled with PBS-liposomes (PBS-L) or dichloromethylene diphosphonic acid (DMDP) containing liposomes (DMDP-L) and were killed at various time points up to 21 days for compliance measurements, AM cell counts, and surfactant analysis. AM numbers were significantly decreased 1, 2, and 3 days after instillation in DMDP-L vs. PBS-L, with 72% depletion at 3 days. AM numbers returned to normal levels by 5 days. In DMDP-L rats, there was a rapid increase in surfactant-phospholipid pools, showing a ninefold increase in the amount of surfactant in the lavage 3 days after liposome instillation. Surfactant accumulation progressed up to 7 days, with pools normalizing by 21 days. The increase in surfactant was due to increases in both subfractions of surfactant, the large aggregates (LA) and small aggregates. Surfactant protein A levels, relative to LA phospholipids, were not increased. There was a decreased extent of surfactant conversion in vitro for LA from DMDP-L rats compared with controls. It is concluded that the procedure of AM depletion significantly affects surfactant metabolism. The increased endogenous surfactant must be considered when utilizing the AM depletion model to study the role of these cells during lung insults.

scholarly journals microRNA-9-5p protects liver sinusoidal endothelial cell against oxygen glucose deprivation/reperfusion injury

2021 ◽  
Vol 16 (1) ◽  
pp. 375-383
Author(s):  
Yi Duan ◽  
Yuanyuan Meng ◽  
Zhifeng Gao ◽  
Xiaoyu Wang ◽  
Huan Zhang
Keyword(s):  
Inflammatory Response ◽  
Ischemia Reperfusion ◽  
Critical Role ◽  
Glucose Deprivation ◽  
Liver Sinusoidal Endothelial Cell ◽  
Liver Sinusoidal Endothelial Cells ◽  
Hepatic Ischemia ◽  
Protein Levels

Abstract Background Maintenance of the function and survival of liver sinusoidal endothelial cells (LSECs) play a crucial role in hepatic ischemia/reperfusion (I/R) injury, a major cause of liver impairment during the surgical treatment. Emerging evidence indicates a critical role of microRNAs in I/R injury. This study aims to investigate whether miR-9-5p exerts a protective effect on LSECs. Methods We transfected LSECs with miR-9-5p mimic or mimic NC. LSECs were treated with oxygen and glucose deprivation (OGD, 5% CO2, and 95% N2), followed by glucose-free Dulbecco’s modified Eagle’s medium (DMEM) medium for 6 h and high glucose (HG, 30 mmol/L glucose) DMEM medium for 12 h. The biological role of miR-9-5p in I/R-induced LSEC injury was determined. Results In the in vitro model of OGD/HG injury in LSECs, the expression levels of miR-9-5p were significantly downregulated, and those of CXC chemokine receptor-4 (CXCR4) upregulated. LSEC I/R injury led to deteriorated cell death, enhanced oxidative stress, and excessive inflammatory response. Mechanistically, we showed that miR-9-5p overexpression significantly downregulated both mRNA and protein levels of CXCR4, followed by the rescue of LSECs, ameliorated inflammatory response, and deactivation of pro-apoptotic signaling pathways. Conclusions miR-9-5p promotes LSEC survival and inhibits apoptosis and inflammatory response in LSECs following OGD/HG injury via downregulation of CXCR4.

scholarly journals Chronic Low Dose Organic Arsenic Induced Liver Structural Damage

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Shahida S ◽  
Nor Zamzila A ◽  
Norlelawati AT ◽  
Jamalludin AR ◽  
Azliana AF ◽  
...  
Keyword(s):  
Structural Damage ◽  
Low Dose ◽  
Liver Perfusion ◽  
Arsenic Exposure ◽  
Inorganic Arsenic ◽  
Control Group ◽  
Sprague Dawley ◽  
Liver Sinusoidal Endothelial Cells ◽  
Organic Arsenic ◽  
And Control

Introduction: Over the decades, organic arsenic has been thought to be less toxic than inorganic arsenic. Monosodium methylarsonate (MSMA) is a potent organoarsenical herbicide that is still being used in most Asian countries. Reported studies on the effects of organic arsenic are mainly to the gastrointestinal system, however there are limited research on its impacts to the liver. Therefore, this study aimed to investigate the effect of MSMA exposure on hepatocytes and liver sinusoidal endothelial cells (LSEC). Materials and Methods: Fourteen Sprague Dawley rats (n=14) were divided equally into arsenic-exposed (n=7) and control (n=7) groups. The rats in arsenic-exposed group were given MSMA at 63.20 mg/kg daily for 6 months through oral gavage. While the rats in control group were given distilled water ad libitum. At the end of the duration, they were euthanized and underwent liver perfusion for tissue preservation. Liver tissues were harvested and processed for light microscopy, scanning and transmission electron microscopy. The findings were analysed descriptively. Results: MSMA had caused necrotic and apoptotic changes to the liver. Normal organelles morphology were loss in the hepatocytes while LSEC revealed defenestration. Conclusion: In this study, chronic low dose organic arsenic exposure showed evidence of toxicity to hepatocytes. Interestingly, LSEC demonstrated capillarization changes.

The liver sinusoidal endothelial cell: a cell type of controversial and confusing identity

2008 ◽  
Vol 294 (2) ◽  
pp. G391-G400 ◽  
Author(s):  
Kjetil Elvevold ◽  
Bård Smedsrød ◽  
Inigo Martinez
Keyword(s):  
Physiological Role ◽  
Structure And Function ◽  
Liver Sinusoidal Endothelial Cell ◽  
Cell Type ◽  
Liver Sinusoidal Endothelial Cells ◽  
Distinct Cell Type ◽  
Sound Communication ◽  
Distinct Cell ◽  
A Cell ◽  
And Function

A look through the literature on liver sinusoidal endothelial cells (LSECs) reveals that there are several conflicts among different authors of what this cell type is and does. Major controversies that will be highlighted in this review include aspects of the physiological role, the characterization, and the protocols of isolation and cultivation of these cells. Many of these conflicts may be ascribed to the fact that the cell was only recently established as a distinct cell type and that researchers from different disciplines tend to define their structure and function differently. This field is in need of a common platform to obtain a sound communication and a unified understanding of how to interpret novel research results. The aim of this review is to encourage scientists not to ignore the fact that there are, indeed, different opinions in the literature on LSECs. We also hope that this review will point out to the reader that some issues that may seem well established regarding our knowledge about the LSECs, in reality, are still unresolved and, indeed, controversial.

Transplantation of metanephroi after preservation in vitro

2001 ◽  
Vol 281 (2) ◽  
pp. R661-R665 ◽  
Author(s):  
Sharon A. Rogers ◽  
Marc R. Hammerman
Keyword(s):  
Growth Factors ◽  
Normal Structure ◽  
Sprague Dawley ◽  
Adult Rats ◽  
Rat Embryos ◽  
University Of Wisconsin ◽  
Distal Tubules ◽  
And Function ◽  
First Time

To determine whether transplanted metanephroi grow, differentiate, and function in hosts after preservation in vitro, we implanted metanephroi from embryonic day 15 ( E15) Sprague-Dawley rat embryos into the omentum of nonimmunosuppressed uninephrectomized Sprague-Dawley (host) rats. Metanephroi were either implanted directly or suspended in ice-cold University of Wisconsin (UW) preservation solution with or without added growth factors for 3 days before implantation. The size and extent of tissue differentiation preimplantation of E15 metanephroi implanted directly were not distinguishable from the size and differentiation of metanephroi preserved for 3 days. In contrast, E16 metanephroi were larger than E15 metanephroi preserved for 3 days. E16 metanephroi or E13 metanephroi grown in organ culture for 3 days contained more differentiated nephron structures than those in E15 metanephroi preserved for 3 days. By 4 wk posttransplantation, metanephroi that had been preserved for 3 days had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts with normal structure had developed. At 12 wk posttransplantation, inulin clearances of preserved metanephroi were comparable to those of metanephroi that had been implanted directly. Addition of growth factors to the UW solution enhanced inulin clearances. Here we show for the first time that functional kidneys develop from metanephroi transplanted from rat embryos to adult rats after as long as 3 days of preservation in vitro.

Diaphragm atrophy and weakness in cortisone-treated rats

1989 ◽  
Vol 67 (6) ◽  
pp. 2420-2426 ◽  
Author(s):  
B. J. Moore ◽  
M. J. Miller ◽  
H. A. Feldman ◽  
M. B. Reid
Keyword(s):  
Steroid Treatment ◽  
Force Frequency ◽  
Sprague Dawley ◽  
Direct Stimulation ◽  
Cross Sectional ◽  
Sprague Dawley Rats ◽  
Twitch Characteristics ◽  
Frequency Relationship ◽  
And Function

Despite frequent therapeutic use, the potential of corticosteroids to produce respiratory muscle myopathy is unknown. We studied effects of chronic steroid treatment on diaphragm mass and function. Eleven Sprague-Dawley rats were treated with cortisone acetate (100 mg.kg-1.day-1 im) for 10 days. Controls (injected with vehicle) included 11 freely eating rats and 11 animals pair fed to match food intake of cortisone rats. Steroid treatment depressed body weight 30% compared with controls. Mass of diaphragm, gastrocnemius, and extensor digitorum longus showed significant atrophy (30%); heart and soleus were unaffected. Isometric contractile properties of costal diaphragm strips were studied in vitro using direct stimulation. The force-frequency relationship was markedly depressed by steroid treatment, both at low and high frequencies. However, force developed per unit cross-sectional area was similar among all three groups, as were twitch characteristics. When stimulated every minute, forces developed by control strips fell progressively, whereas the forces of cortisone-treated strips remained unchanged. When stimulated every 5 s, the fall in force was not different between groups. We conclude that cortisone weakened the diaphragm by decreasing muscle mass but made the diaphragm more resistant to one form of fatigue in vitro.

scholarly journals Liver sinusoidal endothelial cell ICAM-1 mediated tumor/endothelial crosstalk drives the development of liver metastasis by initiating inflammatory and angiogenic responses

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Aitor Benedicto ◽  
Alba Herrero ◽  
Irene Romayor ◽  
Joana Marquez ◽  
Bård Smedsrød ◽  
...  
Keyword(s):  
Liver Metastasis ◽  
Tumor Cells ◽  
Cell Activation ◽  
Tumor Burden ◽  
Host Cells ◽  
Liver Sinusoidal Endothelial Cell ◽  
Metastatic Progression ◽  
Liver Sinusoidal Endothelial Cells

Abstract The prometastatic stroma generated through tumor cells/host cells interaction is critical for metastatic growth. To elucidate the role of ICAM-1 on the crosstalk between tumor and primary liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), implicated in tumor adhesion and angiogenesis, we performed in vitro cocultures and an in vivo model of liver metastasis of colorectal cancer (CRC). ICAM-1 blockade in the LSECs decreased the adhesion and transmigration of tumor cells through an LSEC in vitro and vivo. Cocultures of C26 cells and LSECs contained higher amounts of IL-1β, IL-6, PGE-2, TNF-α and ICAM-1 than monocultures. C26 cells incubated with sICAM-1 secreted higher amounts of PGE-2, IL-6, VEGF, and MMPs, while enhanced the migration of LSECs and HSCs. HSCs cultures activated by media from C26 cells pretreated with sICAM-1 contained the largest amounts of VEGF and MMPs. C26 cell activation with sICAM-1 enhanced their metastasizing potential in vivo, while tumor LFA-1 blockade reduced tumor burden and LSECs and HSC-derived myofibroblasts recruitment. In vivo ICAM-1 silencing produced similar results. These findings uncover LSEC ICAM-1 as a mediator of the CRC metastatic cascade in the liver and identifies it as target for the inhibition of liver colonization and metastatic progression.

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