D en sity A n alysis

Camera lucida drawings of skin innervation in regions A-D (see Fig. 2.3) immunolabelled in the hindpaw (Chapter 4) were scanned with an AGFA Arcus II flatbed scanner. Digitised images were imported into the program Photoshop 3.0. A new layer (layer-1) was superimposed on the image and the outline of the epidermis traced in a different colour as guide lines. This epidermal outline was then rubbed out from the original drawing.

The outline of the epidermis was selected on the original drawing according to the guide lines in layer 1. The image was then inverted so that the nerve fibres drawn in black were now white on a black background. This step was taken, since some of the nerve fibres would be detected as shades of grey and not absolute black. The histogram function was utilised and the number of absolute black pixels recorded in addition to the total number of pixels for the selection. Values were substituted into a spreadsheet program (Microsoft Excel 4.0). The number of pixels enveloped by the nerve fibres were then calculated by subtracting the number of black (i.e. non-innervation) pixels from the total.

Since density analysis was undertaken at different developmental ages it was important to consider the area of the region under analysis, therefore a calibration value

was calculated from a scale bar on the camera lucida drawing. If the scale bar on the drawing was 100|im, then the number of pixels within a square equivalent to lOOOOgm^ was determined. Any region subsequently selected for area analysis was recorded in pixels and automatically converted to pm^ using the calibration value in the spreadsheet program.

The worksheet in the spreadsheet program was organised as follows to calculate the density of innervation in pixels per pm^of tissue:

i) The first column contained a constant derived from our calibration value, i.e. the total number of pixels in a standard area of 10,000pm^ (Stdpix)

ii) A column representing the total number of pixels in the epidermal area (Totpix) iii) A column with the total number of background pixels in the epidermal

area (BGpix)

To obtain the density of innervation, the computer calculated the number of pixels corresponding to innervation (Innpix), the surface where the measurements were performed (Surface) and finally the density (Dens).

iv) Innpix= Totpix-BGpix

v) Surface= (Totpix x 10,000)/ Stdpix

vi) Dens = Innpix / Surface (in pixels / pm^).

Stdpix Totpix BGpix D ens

P ixels / 10,000 pm^ P ixels in epidermis Background pixels

in epidermis

2.4 Methods for Chapter 6

2.4.1 In Situ H ybridisation

Both isotopic and non-isotopic methods of in situ hybridisation were applied to the detection of CGRP mRNA in embryonic spinal cord and DRG. The isotopic detection with S-35 labelled probe was carried out in collaboration with Dr. Greg Michael at UMDS, London. The protocol performed by Dr. Michael is detailed in appendix I and was conducted on embryonic tissue aged E14-E18. Non-isotopic detection with a DIG-labelled CGRP riboprobe was conducted on tissue aged E17 and E l 8. The probe was prepared by Richard Mannion, UCL and the protocol is detailed in Appendix II.

Hybridisation Protocol

Slides were removed from storage at -70®C, and placed in 4% paraformaldehyde

in O.IM PBS for 10 minutes. After fixation sections were washed in O.IM PBS three times, acetylated for 10 minutes and permeabilised in 1% triton X-100 (Sigma) for 30 minutes. They were washed again in O.IM PBS three times and 1ml hybridisation buffer added to each slide and left for 6 hours in a humidified chamber at room temperature.

Sections were then incubated in hybridisation buffer (probe concentration 250-500ng/ml) overnight in a humidified chamber at 45-72°C, and washed in decreasing concentrations of SSC (5x-0.1x) for 1 hour in total. Sections were placed in buffer B 1 for 5 minutes then buffer B2 for one hour, before being incubated overnight at 4°C in buffer B2 with anti-digoxygenin alkaline phosphatase-conjugated fab fragments (Boehringer Mannheim; 1:500). After hybridisation with the antibody, they were washed in buffer B1 three times for five minutes, buffer B3 for 5 minutes then the colour reaction product was visualised in buffer B4. The reaction was stopped with TE, sections were washed in de-ionised H^O and coverslipped using aquamount (BDH).

Solutions

IM phosphate buffer: 0.75M Na^HPO^JH^O (206.675g/litre) 0.25 M NaH^PO^ (31.975g/litre)

4% paraformaldehyde: see 2.1.3 3M sodium chloride: 175.71g/litre

Buffers

E l: O.IM tris pH 7.5, 0.15M NaCl

B2: 5g blocking reagent (Boehringer Mannheim) and 2.5g BSA in 500ml B1. Heat to 60°C then aliquot into 25 ml universals and store at -20°C.

B3: O.IM tris pH 9.5, O.IM NaCl, 0.05M MgCl^

B4: 75pg/ml NBT (Boehringer Mannheim), 50pg/ml BCIP, 0.24mg/ml levamisole (Sigma) in buffer B3.

H ybridisation buffer 50% formamide 5x SSC

5x Denhardts solution 250|Xg/ml baker’s yeast RNA 500pg/ml herring sperm DNA SSC (1 litre): 175.32g sodium chloride

88.23g sodium citrate

TE: lOmM tris, Ph 8.0

ImMEDTA

2.5 Methods for Chapter 7

2.5.1 A n ti-N G F T reatm ent

The protocol for embryonic injections of anti-NGF was modified from Ruit et al, 1992. Briefly, pregnant dams of fifteen days gestation were deeply anaesthetized under

anaesthesia) and a midline surgical incision made. The abdominal wall was opened and the uterine horns exposed. Two embryos from each horn were selected for injection. The 5pl injections of sheep anti-NGF (gift from Professor Clifford Woolf, UCL) were made with a Hamilton syringe with a 30 gauge sterile needle inserted into the mmp region of the embryo (Fig. 2.4). The embryo was first visualised through the uterine wall and membranes using a fibre optic light. After the injection was completed the needle was kept in place for some seconds to prevent leakage. The animal was sutured and allowed to recover for three days. At day 18 of gestation the mother was reanaesthetized and a second injection of lOpl was administered and the mother allowed to recover. Finally at E 2 1, the mother was terminally anaesthetized after the injected embryos were harvested and perfused.

2.5.2 S ize -fre q u e n c y H istogram see 2.2.3

2.5.3 C ell C ounts

Counts of DRG cells from control (n=2) and anti-NGF treated (n=3) embryos were conducted on three lumbar DRG from each animal. Counts were conducted using a variation of the physical dissector method (Coggeshall, 1992), referred to as the fractionator technique.

The lumbar vertebral column was transversely and serially sectioned at a thickness of 7pm. Sections throughout each of the three ganglia were identified and the number of sections in each calculated. The total number of sections in each DRG were summed and sequentially numbered, and the new total for the combined ganglia divided by 5 to produce the section separation (k) for the 5 required dissector pairs. For example, if the total number of sections (N) for the three ganglia was 100 (numbered 1-100), then the section separation =20. Section separation dictates that counts would be conducted on every 20th section. A random number (R) generated between 1 and k (e.g. 4) would decide which section the analysis would begin at. Counts are conducted on 5 pairs of sections equally distributed (every 20th) throughout the numbered sections, beginning

with the section number equivalent to the random number. In this example selected pairs would be numbered 4-5, 24-25, 44-45, 64-65, 84-85.

The first section of each pair is referred to as reference section and the second as the look-up section. Up until this stage, this method follows the physical dissector protocol. The fractionator technique departs from the established protocol as the area of the section and volume of the ganglion are not required. Instead, the ganglion section is divided into four approximately equal quadrants. One of the quadrants are randomly selected, and the number of cell “tops” counted. A cell top is defined as a cell that is in the reference section but not the look-up section. The number of tops are recorded (T,), then the process reversed and the number of tops calculated from the look-up to the reference section (Tj). These tops are averaged (T), multiplied by four (4T) to give the number of cells per whole ganglion section (S) and then multiplied by the total number of sections (N), to provide the number of cells in the three ganglia. The number of cells were finally expressed per lumbar ganglia (G).

Cells per ganglia(G): (T, + T2)/2 x 4=S (S X N)/3=G