5-ALA-mediated fluorescence of musculoskeletal tumors in a chick chorio-allantoic membrane model: preclinical in vivo qualification analysis as a fluorescence-guided surgery agent in Orthopedic Oncology

Background Fluorescence-guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) and other contrast agents has shown its efficacy in improving resection margins, local recurrence and survival rates in several medical disciplines. It is the objective of this study to analyze the engraftment rate of musculoskeletal tumor specimens on the chick chorio-allantoic membrane (CAM), the rate of tumor fluorescence (PDD), and the effects of photodynamic therapy (PDT) after exposure of tumors to 5-ALA in an in vivo environment. Methods A total of 486 CAMs were inoculated with macroscopic tumor grafts (n = 26; n = 478 eggs) and primary cell culture suspensions (n = 2; n = 8 eggs) from 26 patients on day 10 of egg development. On day 16, 2 mg/200 µl 5-ALA were topically applied per egg. After 4 h of incubation, Protoporphyrin IX was excited using blue light (420 ± 10 nm). Tumor fluorescence (PDD) was photo-documented. A subgroup of specimens was additionally exposed to red light (635 nm ± 10 nm; PDT). After the termination of the experiment, CAM-grown tumors were histopathologically analyzed. Results Benign and borderline tumors (chondroblastoma, giant cell tumor of bone and atypical chondrogenic tumor) presented with high rates of detectable fluorescence. Comparable results were found for chondrosarcoma, osteosarcoma and Ewing’s sarcoma among bone and dedifferentiated liposarcoma, myxofibrosarcoma and undifferentiated pleomorphic sarcoma among soft tissue sarcomas. Overall, tumor fluorescence was negative for 20.2%, single-positive (+) for 46.9% and double-positive (++) for 32.9% of macroscopic xenografts, and negative in 20% and (+) in 80% of primary cell culture tumors. Macroscopic tumor xenografts (n = 478) were identified as viable in 14.8%, partially viable in 2.9% and partially to completely regressive in 45.2%. All (n = 8) tumors grown from primary cell culture were viable. After PDT, tumor samples were found viable in 5.5%, partially viable in 5.5% and partially to completely regressive in 68%. Egg survival increased with decreasing PDT doses. Conclusions The CAM model proves to be a suitable in vivo model for the investigation of short-term observation questions in musculoskeletal tumors. The findings of this study warrant further investigation of PDT effects on musculoskeletal tumors and a possible incorporation of 5-ALA FGS in clinical Orthopedic Oncology care.

Amelioration of oxidative stress-mediated cytotoxicity and genotoxicity induced by copper and flubendiamide in-vivo and in- vitro by potent antioxidants

Present study was designed to assess the toxicity of copper @ 33 mg/kg and flubendiamide @ 200 mg/kg in vivo in male Wistar rats orally once daily for 90 days and protective effect of α-tocopherol, resveratrol, curcumin and catechin and in vitro cyto-genotoxicity in primary cell culture of thymocytes. In vivo study showed significant (p<0.05) increase in AST, total bilirubin and uric acid, creatinine and BUN levels while decrease in total proteins, GSH, SOD and GST levels and increased LPO and GPx with severe degenerative changes were observed in liver and kidney tissues in intoxicated groups. In vitro thymocytes were exposed to 40 µM concentration of flubendiamide and/or showed significant increase in TUNEL+ve cells, micronuclei, DNA shearing, and comet formation per 100 cells. Concurrent treatment with α-tocopherol in xenobiotics intoxicated groups showed almost normal values of the biochemical parameters and decreased LPO production and improved antioxidant enzymes activities and histoarchitecture of liver and kidney tissues suggest ameliorative potential of α-tocopherol whereas, resveratrol, curcumin, catechin or α-tocopherol in vitro decreased TUNEL+ve cells, micronuclei induction and comet formation and effect of antioxidants was concentration-dependent and their order of potency on equimolar concentration (10 µM) basis is: curcumin > resveratrol >catechin = α-tocopherol.

Isolation And Culture of Rat Intestinal Mucosal Microvascular Endothelial Cells Using Immunomagnetic Beads And Their Marker Expression

Microvascular endothelial cells (MVECs) have been an important tool in many research fields, while their purification remained challenging. This study aimed to establish a method for isolating rat intestinal mucosal MVECs using immunomagnetic beads, and characterize their proliferation ability and marker expression. Rat jejunum mucosas were scraped and digested by collagenase type II to obtain primary cell culture by the differential adhesion method. Magnetic beads were coated with anti-CD31 antibodies and incubated with the single-cell suspension of the primary cell culture. The CD31+ cells were separated using an automatic magnetic separation system, and their proliferation ability were assayed at different passages. The expression of factor VIII (FVIII), CD31, and CD34 was detected by immunofluorescence staining. Results indicated that rat intestinal mucosal MVECs grew into a contact-inhibited cobblestone-like monolayer after about 6 days, whose proliferation ability had no significant decline at passage 20. Three markers were generally expressed in the cells, and the fluorescence intensity of FVIII was higher after magnetic separation than before, and those of CD31 and CD34 remained similar. In conclusion, highly pure MVECs can be isolated from the primary cell culture of rat intestinal mucosas using magnetic beads coated with anti-CD31 antibodies, and magnetic separation may influence the expression of FVIII.

Multimodal characterization of the murine neurovascular niche using a novel microvascular isolation protocol

The blood-brain barrier (BBB) is central to separate blood from the extracellular fluids of the brain. To understand disease-related changes in the BBB is pivotal and such changes can increasingly be studied by single-cell RNA sequencing (scRNAseq), which provides high-resolution insight into gene expression changes related to the pathophysiological response of the vasculature. However, analysis of the vascular cells in the brain is challenging due to the low abundance of these cells relative to neuronal and glial cells, and improved techniques for enrichment of the vascular component is therefore warranted. The present study describes a method whereby panning with CD31-coated magnetic beads allows isolation of brain vasculature without the need for transgenic reporter lines or FACS sorting. The protocol was tested in three modalities: isolation of cells for scRNAseq, western blot (WB) analysis, and primary cell culture. For scRNAseq, a total of 22,515 single-cell transcriptomes were generated from 12-months old mice and separated into 23 clusters corresponding to all known vascular and perivascular cell types. The most abundant cell type was endothelial cells (EC) (Pecam1- and Cdh5-positive), which dispersed into clusters of arterial, capillary, and venous EC according to previously established BBB arterio-venous zonation markers. Furthermore, we identified clusters of microglia (Aif1-positive), one cluster of fenestrated endothelial cells (Plvap-positive; Cldn5-negative), a cluster of pericytes (Kcnj8- and Abcc9-positive) and a cluster of vascular smooth muscle cells (VSMC) (Acta2- and Tagln-positive). WB analysis using established markers for the different cell types (CD31 (EC), SM22 (VSMC), PDGFRB (pericytes), GFAP (astrocytes), and IBA1 (microglia) confirmed their presence in the isolated vascular component and suggests that the protocol is suitable for future proteomic analysis. Finally, we adapted the isolation protocol to accommodate primary culture of brain vascular cells. In conclusion, we have successfully established a simple and fast method for isolating microvasculature from the murine brain independent of cell sorting and alleviating the need to use reporter mouse lines. The protocol is suitable for a multitude of testing modalities, including single-cell analyses, WB and primary cell culture.

Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells

Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 μM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.