Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Summary

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Abstract

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors ^1,2 and providing growth promoting adhesion molecules ³ and extracellular matrix molecules ⁴. In addition they myelinate the demyelinated axons at the site of injury ⁵.

However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes ^6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules ^8-13.

In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.

These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.

The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer ^14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.

Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) ^16,17, FGF/Heparin ¹⁸, Eph/Ephrins¹⁹.

This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Protocol

1. Boundary Assay:

1. Basic preparation: Chamber slides are coated with Poly-D-Lysine overnight and sterile glass slides are prepared before starting the experiment. Medium is prepared using Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal calf serum (FCS), and 1% Penicillin-Streptomycin-Fungizon(PSF). Also a bottle of Calcium/Magnesium free Hanks balanced salt solution (HBSS) is prepared.
2. Primary rat Schwann cells cultured in flask are trypsinized for 3 minutes using 0.1% Trypsin. Primary rat astrocyte culture is trypsinized for 5 minutes using 0.1% trypsin.
   Trypsin is inactived by adding DMEM supplemented with 10% FCS a....

Discussion

The assays described above have been used in various studies demonstrating the role of multiple factors involved in boundary formation between the Schwann-astrocytes and limited Schwann cell migration in astrocytic environment.

Understanding the mechanisms underlying these events is essential as it would allow development of strategies to optimise and enhance the integration of Schwann cell grafts following transplantation and in doing so facilitate the exit of the regenerating axons from the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	41966-029
HBSS	Invitrogen	14170-088
FCS	Invitrogen	10091-148
PSF	Invitrogen	15240-062
BPE	Invitrogen	13028-014
Forskolin	Calbiochem	344273
Coverlips	VWR international	631-0149
Coverlips(rectangle)	Menzel-Glaser	BB022050A1
Chamber slides	Nalge Nunc international	177380
4 well plates, 6 well plates	Nalge Nunc international		4 well plates6 well plates
rat p75 antibody	EMD Millipore	MAB365
GFAP antibody	Dako	Z0334
Secondary antibodies (Alexa-conjugated)	Invitrogen	A-11004 A-11034	Goat anti mouse 568Goat anti Rabbit 488
Secondary biotinylated	Vector Laboratories		Goat anti mouse
DAB tablets	Sigma-Aldrich	D4418
ABC elite kit	Vector Laboratories	PK-6100
Fine Forceps	Fine Science Tools	11295-10
Paraformaldehyde	Sigma-Aldrich	P6168
Fluorosave	Calbiochem	345789