Optimization of primary hepatocyte isolation for the pharmacological characterization of metabotropic glutamate receptor (mGluR) s ubtype 5: A study on Reduction

To minimize the number of animals used during experiments, it is important to choose the suitable enzyme according to the final goal. In our work, we demonstrated the superiority of collagenase IV in the maintenance of functional transmembrane receptor, thus the pharmacological activity, in isolated rat hepatocytes.

Rapid Isolation of Functional ex vivo Human Skin Tissue-Resident Memory T Lymphocytes

Studies in animal models have shown that skin tissue-resident memory T (TRM) cells provide enhanced and immediate effector function at the site of infection. However, analyses of skin TRM cells in humans have been hindered by the lack of an optimized isolation protocol. Here, we present a combinatorial strategy-the 6-h collagenase IV digestion and gentle tissue dissociation – for rapid and efficient isolation of skin TRM cells with skin tissue-specific immune features. In comparison with paired blood circulating memory T cells, these ex vivo isolated skin T cells express typical TRM cell markers and display higher polyfunctional properties. Moreover, these isolated cells can also be assessed for longer periods of time in ex vivo cultures. Thus, the optimized isolation protocol provides a valuable tool for further understanding of human skin TRM cells, especially for direct comparison with peripheral blood T cells at the same sample collection time.

Isolation of rat hepatocytes for pharmacological studies on metabotropic glutamate receptor (mGluR) subtype 5: a comparison between collagenase I versus collagenase IV

Isolated hepatocytes can be obtained from the liver by collagenase infusion, a procedure that could affect cell isolation as well as the integrity of membrane receptors. In this respect we compared metabotropic glutamate subtype 5 receptor (mGluR5) protein expression and activity in rat hepatocytes isolated by two collagenases, type I and type IV. Hepatocytes were isolated from male Wistar rats (200-250 g) using collagenase I or collagenase IV and after isolation, viability and morphology of rat hepatocytes were assessed measuring mGluR5 protein expression by Western blot analyses. mGluR5 activation was evaluated by inositol-1-phosphate (IP-1) accumulation after treatment with the mGluR5 orthosteric agonist ACPD or the selective antagonist MPEP. No difference in cellular viability and morphology was observed using collagenase I when compared with collagenase IV. An increase in mGluR5 protein expression was observed in hepatocytes isolated using collagenase IV with respect to collagenase I. Moreover, hepatocytes treated with ACPD and with MPEP presented higher levels of IP-1 when isolated using collagenase IV compared to collagenase I. These results indicate that collagenase IV better preserves the activity of surface proteins such as mGluR5 in isolated rat hepatocytes, an in vitro model useful to reduce the use of experimental animals, in line with the 3R ethical framework.

GSK-3β Inhibition Induced Neuroprotection, Regeneration, and Functional Recovery after Intracerebral Hemorrhagic Stroke

Hemorrhagic stroke is a devastating disease that lacks effective therapies. In the present investigation, we tested 6-bromoindirubin-3′-oxime (BIO) as a selective glycogen synthase kinase-3β (GSK-3β) inhibitor in a mouse model of intracerebral hemorrhage (ICH). ICH was induced by injection of collagenase IV into the striatum of 8- to 10-week-old C57BL/6 mice. BIO (8 μg/kg, IP) was administered following either an acute delivery (0–2 h delay) or a prolonged regimen (every 48 h starting at 3 days post-ICH). At 2 days post-ICH, the acute BIO treatment significantly reduced the hematoma volume. In the perihematoma regions, BIO administration blocked GSK-3β phosphorylation/activation, increased Bcl-2 and β-catenin levels, and significantly increased viability of neurons and other cell types. The prolonged BIO regimen maintained a higher level of β-catenin, upregulated VEGF and BDNF, and promoted neurogenesis and angiogenesis in peri-injury zones at 14 days after ICH. The BIO treatment also promoted proliferation of neural stem cells (NSCs) and migration of nascent DCX+ neuroblasts from the subventricular zone (SVZ) to the lesioned cortex. BIO improved functional outcomes on both the neurological severity score and rotarod tests. The findings of this study corroborate the neuroprotective and regenerative effects of BIO and suggest that the Wnt/GSK-3β/β-catenin pathway may be explored for the treatment of acute or chronic ICH.

Effects of ovarian disaggregation on adult murine follicle yield and viability

Follicles are isolated from ovaries for numerous reasons, including IVM, but adult murine yields are <2 follicles mg−1. The aim of the present study was to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n = 7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV; 590 U mL−1) or animal origin-free collagenase IV (AOF) at 590 or 1180 U mL−1. Isolated follicles were stained with 4′,6′-diamidino-2-phenylindole (DAPI; nuclei), chloromethyl-X-rosamine (CMXRos; mitochondria) or fluorescein isothiocyanate-conjugated anti-α-tubulin antibody. Follicle diameters and staining were measured and analysed using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles (17 ± 10 follicles mg−1 ovarian tissue). All disaggregation methods released more secondary follicles (86 ± 20 per ovary; P < 0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles, whereas AOF disaggregation caused more damage (P < 0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (P < 0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. In conclusion, we improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function.