Sabiha Hayath Basha

Microwave irradiated collagen tubes as a better matrix for peripheral nerve regeneration

Collagen is one of the best materials used for nerve guide preparation due to its biocompatibility and desirable tensile strength. In this work, we have compared regeneration and functional reinnervation after sciatic nerve resection with bioresorbable crosslinked collagen guides in 10 mm gap. The crosslinking was carried out either with glutaraldehyde (GTA) or microwave irradiation (MWI). The multilayered collagen membrane used for nerve guides are prepared by lamellar evaporation technique. Functional evaluations of the regenerated nerves were performed by measuring the sciatic functional index (SFI), nerve conduction velocity (NCV), and electromyography (EMG). Transmission electron microscopic studies showed growth of axonal cable with fewer myelinated axons, Schwann cells and more unmyelinated axons present in the case of group treated with uncrosslinked collagen tubes after 1 month of implantation. However, we have observed more myelinated axons in the case of autograft, GTA, and MWI crosslinked collagen tube implants across the gap of 1 cm after the same period of implantation. Smaller myelinated fiber diameter was observed in the case of GTA crosslinked collagen tube group when compared with the autograft and MWI collagen tube groups. There were more myelinated axons during the 3rd and 6th months postoperatively using these conduits as substantiated by light microscopic studies of the regenerated nerve. The conduction velocity and recovery index improved significantly after 5 months reaching the normal values in the autograft and MWI crosslinked collagen groups compared to GTA and uncrosslinked collagen tubes.

Initial upregulation of growth factors and inflammatory mediators during nerve regeneration in the presence of cell adhesive peptide-incorporated collagen tubes

Abstract  Neurotrophic factors play an important modulatory role in axonal sprouting during nerve regeneration involving the proliferation of hematogenous and Schwann cells in damaged tissue. We have exposed lesioned sciatic nerves to a collagen prosthesis with covalently bonded small cell adhesive peptides Arg‐Gly‐Asp‐Ser (RGDS), Lys‐Arg‐Asp‐Ser (KRDS), and Gly‐His‐Lys (GHK) to study local production of growth factors and cytokines in the regenerating tissues. Western/enzyme‐linked immunosorbent assay (ELISA) studies were performed after 10 days of regeneration, when the tubular prosthesis is filled with fibrous matrix infiltrated by hematogenous cells and proliferating Schwann cells with growth factors produced locally. Regeneration was also analyzed by morphometrical methods after 30 days. The quantification of growth factors and proteins by ELISA revealed that there was an enhanced expression of the neurotrophic factors nerve growth factor (NGF) and neurotrophins (NT‐3 and NT‐4) in the regenerating tissues. This was further established by Western blot to qualitatively analyze the presence of the antigens in the regenerating tissues. Schwann cells were localized in the regenerating tissues using antibodies against S‐100 protein. Other growth factors including growth‐associated protein 43 (GAP‐43), apolipoprotein E (Apo E), and pro‐inflammatory cytokine like interleukin‐1α (IL‐1α) expression in the peptide groups were evaluated by ELISA and confirmed by Western blotting. Cell adhesive integrins in the proliferating cells were localized using integrin‐αV. The combined results suggest that the early phase of regeneration of peripheral nerves in the presence of peptide‐incorporated collagen tubes results in the enhanced production of trophic factors by the recruited hematogenous cells and Schwann cells, which in turn help in the secretion of certain vital trophic and tropic factors essential for early regeneration. Furthermore, hematogenous cells recruited within the first 10 days of regeneration help in the production of inflammatory mediators like interleukins that in turn stimulate Schwann cells to produce NGF for axonal growth.

In-Vitro Culture Morphology of Spermatogonial Stem Cells (SSCs) in Mice

Spermatogonial Stem Cells (SSCs) from pre and post-pubertal age groups of mice were isolated by two step enzymatic digestion method and cultured in six-well plates. The in-vitro culture morphology of the SSCs was observed upto passage 2. In primary culture (P0), on day two, the cells were single and transparent. On day 4, the colony forming unit consisted of four to five cells. Three dimensional colonies were observed on day seven post incubation. From day eight onwards, the cells had spindle shape and showed varied morphology till twelve days post incubation. Double nucleolus is prominent in cells on day eight post incubation. In passage 1, though the cells exhibited different morphology viz., round, stellate, polyhedral, and triangular, the stellate cells were found to be more. In passage 2, the spindle-shaped cells with prominent nucleoli were often observed. Other cell morphology like round, stellate, polyhedral and triangular cells were also observed.

Microarchitecture of the Palatine Tonsil in Sheep (Ovis aries)

SUMMARY: The tissue pieces of palatine tonsil were collected from different postnatal age groups of sheep from the Corporation Slaughter House, Perambur, Chennai. The palatine tonsil consisted of a surface epithelium, capsule, tonsillar lobes, crypts, crypt epithelium and tonsillar follicles. The surface epithelium over the palatine tonsil was made up of non-keratinized stratified squamous epithelium in all the postnatal age groups studied. The palatine tonsil was clearly demarcated from the surrounding structures by a distinct connective tissue capsule and one septa dividing the tonsil into two lobes. The surface epithelium was invaginated into the substance of the tonsil to form primary and secondary crypts in each lobe. The crypt epithelium covered the regions of lymphoid follicles became lymphoepithelium. The macrophages were also observed in the epithelium. In the areas of lymphoepithelium the basement membrane was interrupted since lymphocytic infiltration was heavy into the epithelium. Numerous secondary tonsillar follicles with germinal centers separated by interfollicular areas were observed in the palatine tonsil. The tonsillar follicles consisted of a mantle zone, which was heavily populated with small darkly stained lymphocytes. These mantle zones were always oriented towards the crypts. The tonsillar follicles of young sheep showed many medium and small sized lymphocytes, lymphoblasts and also reticulocytes. The reticular cells usually appeared larger than lymphocytes and had a more abundant and organized cytoplasm with vacuoles. ˚