Análisis compositivo simbólico-formal y espacial

2.2 6.1. Preparation of fibronectin fibres

Ultrathin fibronectin fibres were made by using the method of Ejim et al, 1993 as described in Section 2.1.2. Individual fibres were pulled out and attached to sterilised round coverslips (BDH), which were coated with 1 mg/mL poly-L-lysine. The attached fibres were air dried for 24 hours before gamma irradiation.

2.2.6 2. Schwann cell culture on fibronectin strands

Rat Schwann cells were cultured fi’om a modified method of Brockes et al, 1979 as described in Chapter 2.1.8. Schwann cells at passage 4-5 were plated at a density of 5 X lO'* cells/coverslip in 4-well plates for culture onto fibronectin fibres and left for 24 hours.

2.2.6.3. Measurement of the speed of Schwann cell movement on fibronectin fibres

Cell cultures were placed on the stage of the inverted phase-contrast microscope and incubated at 37®C. The movement of Schwann cells along Fn-fibres was viewed under an inverted microscope (Nikon, Surrey, England) and recorded by time-lapse video using a CCD high performance camera (Sony CCDXC-77CE, Sony UK) and an Openlab Image analysis software package (Improvision, Coventry, England). The

position of each individual cell, defined as the position of the cell nucleus, was recorded every hour for 10 hours and then all the distances between the subsequent positions were summed to give the length of the cell path (S).

Parameters measured:

The methodology for measuring various parameters of the behaviour of cells have been described earlier by Wojciak et al., 1996. Briefly:

a. The mean speed of cell movement (Veffective) was calculated using the following equation:

Veffective S / t

Where S = the length of the total cell path t = the duration of the recording b. The real speed of cell movement ( V r e a i ) :

V ^ a l = S / t - N

Where N = the number of time intervals when the cell did not move (hours).

For calculation of V^ai we took into account only the time intervals when Schwann cells were seen to be moving.

c. The total cell translocation which is defined as the distance between the first and the last point of the cell path was calculated.

d. Persistence parameter (Pr):

Pr = total cell translocation/ total length of cell path (S).

Pr would equal 1 for the cell moving persistently along one straight line in one direction.

2.2.6.4. Light microscopy and orientation analysis

Binary images of Schwann cells on strands captured from the Image analysis system, as described above, were used to calculate the orientation of cells in relation to the strand. An orientation index devised by Herman (ref; Guido and Tranquillo, 1993) was used to reflect the orientation of cells. As Schwann cells display a polarised appearance, the angle of alignment can be measured directly in relation to the strand axis and is given by the equation: S = 2 (cos^a)-l, where a is equal to the angle between the central plane of individual cells to the axis of the strand. The term (cos^a) denotes the square cosine of (a) averaged over all measured cells. An S value of 1 would indicate perfect alignment and a value of 0 would indicate total random orientation of cells. Values of S were calculated for 100 cells along each fibre.

Cultured cells grown on fibres for 24 hours were fixed in 2.5% gluteraldehyde and attached to microscope slides using DPX and left to dry at room temperature. Samples were stained with haemotoxylin and eosin for routine histology. Images of cultures were scanned into a computer to give Schwann cells orientation after 24 hours as described above.

2.2.6.5. Measurement of cell area and cell extension

Binary image captured from the various time points were used to calculate the area of cells (i.e. cell spreading) and cell extension on the different substrata using the same technology as described earlier. Calculations of cell area and ceU extension was performed by the published methods of Dunn and Brown, 1986. Binary images captured from the various time points were used to calculate the area of cells (i.e. cell

spreading) and cell extension on the different substrata using the same technology as described earlier. Calculations of cell area and cell extension was performed by the published methods of Dunn and Brown, 1986 in which moments of a shape are used to calculate cell spreading parameters as follows:

L Calculation o f moments

Moments o f a shape. For each cell, the zero-order, the two first-order and the three second-order moments of its shape were calculated. The zero order moment is calculated as the number («) of pixels in the digitized image of the cell, and the remaining five moments (w) were calculated by obtaining the following sums fi'om the

X and y pixel co-ordinates of all « pixels:

moo = n mio = Dc moi = l y m2o = mji = 2)cy mo2 =

Central moments. Central moments, defined as moments referred to the centroid of the shape as the origin of the co-ordinate system. These moments are invariant to translation i.e. they do not change with changes in the position of the object. The second order central moments were calculated for each cell as:

fi20 = m2o - (m?lo/moo) pn = nin - (mwmoi/moo) po2 = mo2 - (m?oi/moo)

Normalised central moments. Normalised central moments are invariant to changes in size of the shape. For the second order, these were calculated as follows:

t]2o — p 2 o / m ^ 00 JJii — p i j / f n ^ o o J]02 = ^ 02/1^100

These three moments are the basis for the following measures.

2. Calculation o f the measures o f cell shape

The measures of cell shape was calculated fi'om two second order measures of cell shape defined by Hu, 1962, which are invariant to rotation:

<!>i = tjo2 + rj20 ( Vo2 - rj2o f 4 rf n

From these, the following two rotational invariants were calculated:

X i = 2n((l> i + (<l>2)) X2 = 2 jt((l)i - ((jn))

These were used to calculate, for each cell, the cellular extension: Extension = log2 (Xi)

This is a measure of how much the cell shape differs from a circle and takes the value of zero if the shape is circular and increases without limit as the shape becomes more compact.

Cellular area was pre-calculated and displayed by the image analysis system.

2.2.6 6. Scanning electron microscopy (SEM)

Coverslips containing Fn-fibres and cultured cells grown for 24 hours were fixed in 2.5% gluteraldehyde (Agar Scientific) for 10 minutes, then in 1% osmium tetroxide in PBS for 15 minutes. The cultures were then hydrated in a series of alcohols (10 minutes per wash) and two 5 minute washes in 100% hexamethyldisilazane (HMDS, Sigma), the second of which was allowed to evaporate at room temperature (Chissoe et al., 1994). The samples were then sputter coated and examined under a scanning electron microscope at lOkV.

2.2.6.7. Immunostaining of F-actin

Cultured cells on strands were rinsed in PBS and fixed in 1% gluteraldehyde for 15 minutes. Background staining was reduced by using 20mM sodium borohydride in PBS for 1 hour at 37°C. Coverslips were washed three times in PBS and permeabilized

in 0.5% Triton X-100 (Sigma) for 15 minutes. After washing three times in PBS cells were incubated in Ifig/mL TRITC-phalloidin solution in PBS for 45 minutes at 37°C and washed three times in PBS containing 1% GOC solution (0.18 mg/ml catalase; 0.5 mg/ml glucose oxidase and 0.1 mg/ml glucose in MHB) to enhance staining

{Methodology courtesy o f Dr. Wemer Baschong. Labelled samples were then mounted in Mowiol-4-88 (Hoechst) and examined under a Bio-Rad MRC-600 confocal laser scanning microscope, equipped with an Argon-ion laser working with the Bio-Rad Image System (COMOS 7.0, Bio-Rad UK). Cells stained for actin were examined under 529nm excitation wavelength with a 60x objective.