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The lumbar region of the spinal cord was carefully

dissected out. This region is easily identified as it

appears as an obvious enlargement. Each vertebra was

carefully removed to reveal the underlying spinal cord. A

large proportion of the cord containing the lumbar region

was removed and placed in a small volume of the 2.5%

gluteraldehyde for postfixing for 2-4 hours at 4°C. The cord

was then transferred to a solution of 30% sucrose in

Millonig's phosphate buffer, pH 7.3, and kept overnight at

4°C in order to wash out all traces of the fixative and to

Using a dissecting microscope, the lumbar region was

excised from the rest of the cord. This can be done in an

exact manner because the L6 ventral and dorsal roots are

easy to identify as they are very much thinner than the

preceding L5 roots. The spinal cord was cut with a sharp

scalpel blade just below L2 and L6. In order to mark the

control side of the cord, a nick was made on the dorsal

surface of the left dorsal horn. The block of spinal cord

tissue was then cut into transverse sections. It was

mounted onto a freezing microtome (Pelcool) and surrounded

by dry ice to keep it fully frozen. Serial transverse

sections were cut at 50 nm and collected into individual

wells in a staining tray containing Millonig's phosphate

buffer and then processed for HRP histochemistry.

2.3.4. HRP-Histocheniistry

A widely used technique for HRP-histochemistry is the

Hanker-Yates method (Hanker et. al., 1977). Using this

method, the staining is less intense than that obtained by

other methods, for example, the Tetramethylbenzidine Method

reaction product tends to mask the nucleolus and nucleus.

Thus, using the Hanker-Yates method it was possible to

visualise the nucleolus and the Nissl substance by

counterstaining and this allowed accurate cell counting and

morphometry. A detailed schedule of the modified Hanker-

Yates method used in these experiments is described in

Appendix II. After processing for HRP the sections were

carefully mounted onto gelatinised slides (0.5%) and

allowed to dry overnight at 37°C.

2.3.5. Counterstaining

When the sections had fully dried, the RNA and Nissl

substance was counterstained with Gallocyanin (Culling,

1963) (see Appendix II for the protocol and preparation of

the stain). The sections were stained until the nucleolus

and the Nissl substance were clearly visible. The sections

were then dehydrated, cleared and coverslipped using DPX as

a mounting medium. The slides were left to dry overnight at

2.4. Microscopy

The sections were examined under a light microscope (Carl

Zeiss) for HRP-labelled motoneurone on both the operated

and control sides of the spinal cord. The control side of

the spinal cord was identified by the nick on the dorsal

horn made prior to sectioning. The microscope was fitted

with a camera lucida, which allowed cell profiles to be

drawn.

2.4.1. Cell Counts

Each section was first carefully scanned under a low power

objective (x2.5) to locate the motoneurone pool under study

and to ascertain whether there had been any obvious spread

of the HRP to motor pools other than that under study. The

labelled cells were then examined under a high power

objective (x40). Only those motoneurones containing a

nucleolus were counted in order to prevent the same cell in

consecutive sections being counted twice. The number of

labelled cells on each side of the cord were noted

separately. When all the motoneurones which were labelled

added, and the number of motoneurones on the operated side

was expressed as a percentage of the number on the control

side where possible.

2.4.2. Cell Morphometry

i. Drawing of Cell Profiles

After the number of labelled cells present in each side of

the spinal cord had been counted, the cells were then

scanned for a second time and the outline of each labelled

cell was drawn with the aid of a camera lucida. This was

done without any reference to the cell counts already made,

and once all the labelled cells have been drawn, the total

number of cells drawn were counted. This provided a backup

to the initial counts, and if a discrepancy appeared, the

section in question was reexamined to assess whether the

nucleolus was present, and thus whether the cell should be

included in the counts or not.

The outline of each positively stained cell was brought

into focus under a x40 objective. If the nucleolus was

present the cell outline was traced onto paper using the

cell was magnified by around 80Ox. A rather subjective

decision was made about the boundary of the soma as the

cellular processes were not included. Each cell was

numbered as it was drawn, to allow back reference at a

later stage if necessary.

ii. Computer Analysis

After the cell profiles have been traced onto paper, the

area of the cell body was measured by transferring the

camera lucida drawings onto a Summasketch III

(Summagraphics, Newbury, UK) graphics tablet interfaced

with a personal computer. Using a stereometry program

(Tablyt, designed by Dr. J.E. Cook, UCL) , the areas of all

the labelled cells from the operated and control side were

measured and the accompanying statistical analysis program

sorted the data into a frequency distribution histogram

for each side. The histogram of the operated and control

sides for individual animals was superimposed (FreeLance

Graphics version 4) and thus, any shift in the

distribution of the areas of the motoneurones could be

2.5. Electrophysiology