Actin cytoskeleton reorganization correlates with polarization of secretory vesicle and cell morphology in the degranulation of mast cell subtypes in human colon tissues.

Mast cells play a central role in the intestinal immune response. To investigate the relationship between degranulation, cell polarization and the reorganization of actin cytoskeleton of mast cells, we used fluorescence or gold labeling methods to identify different mast cell subtypes in human colon. The reorganization of filamentous actin was visualized and then the polarization of secretory vesicles, as well as cell surfaces, was analyzed by fluorescence microscopy and electron microscopy. Our results first showed a diversity of filamentous actin assembly or disassembly within the contacting cell membrane of different mast cell subtypes. The polarization and degranulation of secretory vesicles was not only accompanied with the assembly and disassembly of filamentous actin at the cell periphery, but also with changes of cell surface polarization. Our study provides an insight into the local membranous structures and suggested correlations of cytoskeleton arrangement with the polarization of secretory vesicles and cell surface configuration during mast cell degranulation.

[Mechanism of rat sciatic nerve regeneration induced by human hair keratin].

OBJECTIVE: To evaluate the effect of human hair keratin (HHK) in peripheral nerve repair and explore the mechanism of sciatic nerve regeneration. METHODS: Rat models of sciatic nerve damage was established by creating a 10-mm gap in the sciatic nerve, which was bridged with a HHK implant. Histological examinations of the nerve tissues were performed at different time points after the surgery. RESULTS: During the period from 2 days to 2 weeks following HHK implantation, Schwann cells were found to undergo dedifferentiation and proliferate along the HHK implant. Three weeks after HHK implantation, numerous macrophages and megakaryocytes occurred around the HHK, and a large quantity of regenerated Schwann cells aligned in orderly fashion was seen between the fine filaments of partially degraded HHK, where axons and capillaries were also observed. Six weeks later, massive nerve fibers and capillaries developed around the HHK, and at 9 weeks, the HHK implant was substantially degraded and numerous regenerated nerve fibers occurred characterized by obvious epineurium and perineurium. Till 12 weeks after HHK implantation, HHK was almost completely degraded and replaced by the newly regenerated nerve fibers that had grown across the nerve defect. CONCLUSIONS: HHK is an ideal material for nerve injury repair. Apocytosis plays a key role in the differentiation process of highly differentiated Schwann cells into immature Schwann cells following nerve injury. As a protective mechanism, the axons undergo enclosure and dissociation following injuries, and the intact axons give rise to growth cones that extend fibers of growing buds to competitively bind the one or more Schwann cells, but only one such but finally develops into a complete axon. The nerve fiber barrier membrane is derived from the capillary menchymal stem cells and the outmost vascular barrier membrane. The regeneration of the Schwann cells, axons and the nerve membrane is the result of self-organization through a well synchronized and coordinated mechanism.

Filamentous-actins in human hepatocarcinoma cells with CLSM.

AIM: To establish a method for optical sections of HepG2 human hepatoblastoma cells with confocal laser scanning microscope (CLSM) and to study the spatial structure of filamentous actin (F-actin) in HepG2 cells. METHODS: HepG2 cells were stained with FITC-phalloidin that specifically binds F-actin, with propidium iodide (PI) to the nucleus, and scanned with a CLSM to generate optically sectioned images. A series of optical sections taken successively at different focal levels in steps of 0.7 microm were reconstructed with the CLSM reconstruction program. RESULTS: CLSM images showed that the FITC-stained F-actin was abundant microfilament bundles parallel or netted through the whole cell and its processes. Most F-actin microfilaments extended through the cell from one part toward the other or run through the process. Some microfilaments were attached to the plasma membrane, or formed a structural bridge connecting to the neighboring cells. CONCLUSION: A method for double labeling HepG2 human hepatoblastoma cells and CLSM imaging F-actin microfilaments and nuclei by image thin optical sections and spatial structure was developed. It provides a very useful way to study the spatial structure of F-actin.

Autophagy of neuron axon during regeneration of rat sciatic nerves.

OBJECTIVE: To investigate the autophagic clearance of degenerated neuron axon during regeneration of rat sciatic nerves. METHODS: Wallerian degeneration model was established in rats by sciatic nerve transection. Samples from the distal stump were collected at different time points after the transaction and ultrathin sections prepared for electron microscopic examination and acid phosphatase (AcPase) activity detection. RESULTS: Neuron axon degeneration occurred after transection of rat sciatic nerve, presenting predominantly swelling of the axoplasm and separation of the axon from the myelin sheath seen 5 h to 2 d after the transection. On day 4, axoplasm condensation took place and the axons were completely separated from the myelin sheath to form dissociative axon body. Vacuoles of various sizes were identified in the axon in the early stage after operation and later when the axons were completely dissociated from the nerve sheath, larger dissociative axon bodies occurred. The axolemma surrounding the axon body was derived from the neuronal cytomembrane, and the condensed axoplasm contained numerous autophagic vacuoles at all levels along with large number of neurofilaments, microtubules and microfilaments arranged in a crisscross pattern. The autophagic vacuoles exhibited acid phosphatase (AcPase) activities. Since the day 7, the axon bodies were absorbed after degradation and macrophages could be spotted occasionally. CONCLUSION: The degenerated axons were cleared mainly through autophagy during regeneration of rat sciatic nerve and macrophages only assist in this process.

[Autophagic effect of Schwann cells in the regeneration of rat sciatic nerves].

OBJECTIVE: To investigate the autophagic effect of Schwann cells in the process of rat sciatic nerve regeneration. METHODS: Wallerian degeneration model was established by transecting the rat sciatic nerve. Samples from the distal stump were obtained 0, 0.5, 1, 1.5, 2, 3, 4, 5, 7, 10, and 15 days respectively after the transaction, and ultrathin sections were prepared for examination with electron microscope. RESULTS: Axons started to separate from the myelin sheath at day 0.5 after the transection, followed by rapid vacuolar degeneration. Since day 2, the myelin sheath folded and broke into fragments, and in the Schwann cells large cell membrane-bound myelin debris and many scattered small fragments could be seen to fuse with the lysosomes to form ACPase reaction-positive autophagic vacuoles. Immature cells were occasionally seen in the endoneural space, appearing in large amount a week later. After day 7, the number of autophagic vacuoles began to diminish. In the entire course of the observation, macrophages containing autophagic vacuoles, were spotted only occasionally. CONCLUSION: The degenerated myelin debris are cleared mainly through the mechanism of autophagy by the Schwann cells during regeneration of rat sciatic nerve. Schwann cells dedifferentiate into Schwann cell precursors, which then proliferate and differentiate to take part in the regeneration of the nerves.