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(a) Group 1.

Eight hinds were fitted with 12 Alzet 2ML4 osmotic minipumps (Charles River U.K. Ltd. Margate, Kent, U.K.) in each experiment (see Section 3.2.4 for surgical details).

The 12 minipumps were filled with three treatments:

(i) Treatment A (N=4) - 6 mg/ml (High dose) ovine PRL solution.

(ii) Treatment B (N=4) - 0.6mg/ml (Low dose) ovine PRL solution. (iii) Treatment C (N=4) - Saline vehicle.

The pumps were distributed among 16 sites on the flanks of 8 hinds (Tables 3.1 and

3.2), on 18 November 1993 and 19 January 1994. Each animal received a pump containing either treatment A or B and half of the animals received a second pump containing treatment C in a contralateral site. In an attempt to assess the extent of the infusion, a single animal received a pump filled with methylene blue dye diluted with vehicle in an additional contralateral site in experiment 1.

(b) Group 2.

A second group of 6 hinds remained untreated and acted as controls.

3.2.3. Preparation of Minipumps:

3.2.3.I. Experiment 1:

The PRL solution was prepared by dissolving 120mg of ovine PRL (oPRL-18, AFP- 8277E; NIDDK, NHPP, University of Maryland School of Medicine, Rockville, MD, USA) in 5ml NIADDK buffer (0.03M sodium bicarbonate, 0.15M sodium chloride, 1%

BSA. pH 10.8), and was made up to 20ml by the addition of phosphate buffered saline (Gibco-BRL, Life Technologies Ltd, Paisley, U.K.). The final pH of the solution was adjusted to 7.4 by dropwise addition of 2M hydrochloric acid. The solution was divided into two 1 0 ml aliquots, one was retained for immediate use and the other was stored at

-70°C. A 1ml aliquot of the original 6 mg/ml (High dose) PRL solution was further

diluted in 9ml of phosphate buffered saline to give a 0.6mg/ml (Low dose) solution. Before filling, each of the twelve osmotic minipumps used in the experiment was assembled (Fig. 3.1). Surgical gloves were worn when handling the pumps and strict

Delivery portal Removable cap Flange Flow moderator Impermeable reservoir wall Osmotic agent Semi-permeable membrane Aqueous environment Reservoir

FIGURE 3.1: Cross section of an Alzet 2ML4 osmotic minipump showing design, components and mechanism of operation. (Redrawn from Alzet Technical Information Manual). Magnification 2 X

sterile technique was observed. The translucent end-cap of the flow moderator was removed and each empty minipump was weighed. The minipumps were filled using a 5ml syringe and the blunt-tipped 25-gauge filling tube, ensuring that the whole apparatus remained free of air bubbles throughout the procedure. After filling, the flow moderator was reinserted into the minipumps and any overflow displaced during the filling or by the insertion of the flow moderator was wiped off. Each filled pump was re-weighed to calculate the net weight of solution loaded. A 5cm length of vinyl catheter tubing (0.76mm i.d. x 1.22mm o.d.: Dow Coming Corporation, Michigan, USA) was then attached to the metal stub of the flow moderator. The free end of the catheter was blocked with medical grade silastic adhesive (Dow Coming Corporation, Michigan, USA) and two holes were pierced in the tubing, 1cm apart and 0.5cm from the sealed end. The seal was then left ovemight to set.

The pump apparatus was placed in sterile 0.9% saline at 37°C, 4h before surgery. This allowed the pump to begin operating before implantation and reduced the chance of an occlusion or clot forming in the catheter.

3.2.3.2. Experiment 2:

The same doses of PRL were retained for the second experiment. The remaining 10ml aliquot of the original (6 mg/ml) PRL solution was thawed ovemight at 4°C. A 1ml aliquot was removed and freshly diluted 1 : 10 in phosphate buffered saline for the

low (0.6mg/ml) PRL dose. The pumps were assembled, weighed and filled as before, but were not fitted with vinyl catheters before implantation.

3.2.4. Minipump Surgery:

3.24.1. Experiment 1:

Minipumps were implanted on 18 November 1993 in an area of the flank just behind the front leg of the animal (Fig. 2.1), according to the protocol in Table 3.1. Throughout the procedure the animals were restrained using the methods previously described in section 2 .2 .2 .

Table 3.1: Distribution of osmotic minipumps in Experiment 1 (18.11.93). Animal Left Flank Right Flank #1 High PRL Saline #2 Low PRL Saline #3 High PRL Saline

#4 High PRL Saline + Dye

#5 - High PRL

#6 - Low PRL

#7 Low PRL Saline

#8 - Low PRL

The surgical site was shaved with electric clippers and the skin thoroughly rubbed with sterile gauze soaked in pevidine surgical scrub (BK Vetinary Products Ltd., Bury St. Edmunds, Suffolk, U.K.). Approximately 2ml of lignocaine hypochlorite (Lignavet: C-Vet Ltd., Bury St. Edmunds, Suffolk, U.K.) local anaesthetic was injected in two or more pools subcutaneously in the centre of the sterilised patch. A scalpel was then used to make a 5cm vertical incision through the epidermal and dermal layers of the skin to the level of the subcutaneous fat layer. A cavity for the minipump was made between the dermal layer and fat layer underneath using blunt forceps. An additional opening was created with the blunt seeker leading away from the main cavity in an anterior direction. The minipump was placed horizontally into the cavity with the vinyl catheter attached to the pump run through the opening created with the blunt seeker. The wound was closed with at least two sutures using a 40mm reverse cutting needle with vicryl suture. Any bleeding was immediately staunched with sterile gauze. The surgical scar was then washed and sprayed with an aerosol antibiotic (Alamycin: Norbrook laboratories (G.B.) Ltd., London, U.K.), before an intramuscular dose of long acting antibiotic (Terramycin LA: Pfizer Ltd., Sandwich, Kent, U.K.) was administered at a dosage of 1ml per 1 0kg live weight.

During minipump implantation the position of each of the minipumps was monitored to ensure that the point of infusion remained constant. The minipumps were removed 27 days later, on 15 December 1993, using the same general surgical procedure, with the incision made posteriorly to the minipump, well removed from the point of infusion.

The cavity left after minipump removal was treated with powdered antibiotic (Aureomycin: Cyanamid Animal Health Division, Gosport, Hampshire, U.K.) before the incision was closed with a single suture. Any hair growth within the surgical site in the intervening 27 days was not shaved.

On removal the minipumps were labelled and retained for further analysis. The residue left in each minipump was removed using the original filling tube and measured, and the volume dispensed over 27 days was calculated. This volume expressed as a percentage of the original, provided an estimate of the relative efficiency of each minipump.

3.2.4.2. Experiment 2:

A separate set of 12 osmotic minipumps were implanted in the same animals in the second experiment that started two months later, on the 19 January 1994. Due to the reuse of animals for the second experiment, 11 of the original 13 surgical sites received minipumps again (Tables 3.1 and 3.2).

Table 3.2: Distribution of osmotic minipumps in Experiment 2 (19.1.94)

Animal Left Flank Right Flank #1 Saline High PRL #2 Saline Low PRL #3 Saline High PRL #4 - High PRL #5 - Low PRL #6 Saline High PRL #7 - Low PRL #8 - Low PRL

Attempts were made to limit animals receiving PRL-filled minipumps in the same site in the second experiment. Only 3 sites received PRL-filled minipumps in both experiments, and of those only one the same concentration of PRL both times. Precautions were taken to ensure that the effects of the two experiments did not overlap.

(i) The surgical incision made in the second experiment was at least 15cm above the scar of the previous experiment. This distance was chosen after examination of the area of effect of PRL treatment in the first experiment and represented over three times the maximum extent of induced hair growth. (ii) The area around and between the two sites was shaved repeatedly allowing

long-term monitoring of the local area of skin in response to PRL infusion. The extent of the infusion effects was revealed by the shaving which also clearly showed any interference between the two experimental sites.

Without the vinyl catheter, the minipumps were implanted with much less disruption of tissue within the wound site and discomfort to the animal. The surgery was modified to reflect this change. The positioning of the pocket formed for the minipump was almost entirely anterior to the incision, maximising the distance between the infusion point and wound (Fig. 3.2b). This is in contrast to the arrangement in the first experiment where the incision straddled the pocket formed beneath (Fig. 3.2a), and the point of infusion was removed clear of the wound with a vinyl catheter.

The minipumps were monitored as before to ensure the point of infusion remained constant. The short metal catheter protruding from the minipump gave a more consistent point of infusion than the flexible vinyl catheter. The minipumps were removed after 28 days on 16 February 1994.