Mulesing, the surgical removal of wool bearing skin and skin folds from the breech region of lambs and weaners, is an effective breech flystrike preventative measure that benefits the financial viability of the sheep industry and the long term welfare of extensive sheep populations in Australia. The procedure, performed without analgesia or anaesthesia, has been scrutinised by animal welfare groups, who successfully campaigned overseas retailers to boycott Australian wool.
The aim of this project was to develop an effective, safe and economical topical treatment to replace surgical mulesing that is acceptable for animal welfare. Therefore, this project aimed to identify potential skin necrotising agents, and combine them with lidocaine, a rapidly acting, widely used, cost effective, local anaesthetic drug.
As few in vitro transdermal studies have been undertaken on sheep, a series of studies were undertaken using Franz-type diffusion cells. Initially a study was performed to investigate methods for lidocaine extraction from transdermal study samples, for analysis by high performance liquid chromatography. A simple method using methanol to extract lidocaine from receptor solution and skin samples was devised.
Another methodological study examined the effect of freezing sheep skin on transdermal hydrocortisone (steroidal anti-inflammatory) movement. This study found that skin permeability to hydrocortisone after freezing for 1 month was not significantly different compared to fresh skin. However skin that had been frozen for 6 months had increased permeability to hydrocortisone compared to fresh skin and skin frozen for 1 month. As skin was opportunistically collected from sheep as they were mulesed or euthanased, this study determined appropriate storage guidelines for skin frozen before use.
Studies were then undertaken to investigate transdermal movement of lidocaine (log P 2.36) and hydrocortisone (log P 1.45) through the thoracic and breech region of lambs and weaners, to characterise sheep skin with respect to topically applied drugs and determine the ability to provide local analgesia transdermally to sheep. Like other species, regional and age differences exist in transdermal lidocaine movement. While both drugs penetrated sheep skin, the more lipophilic drug lidocaine had greater flux and reached greater skin concentrations compared to hydrocortisone, probably due to the greasy nature of sheep skin.
A series of in vitro studies were performed to screen chemical agents that fulfilled the initial criteria (non-carcinogenic, non-teratogenic, operator safety and economy), for their ability to induce skin necrosis sufficient to replicate surgical mulesing. Of the thirty-one agents and four vehicles tested, only sodium and potassium hydroxide consistently achieved the desired level of skin necrosis (grade 3/3), according to a devised histological scale, within one hour. This time frame was derived from the approximate duration of therapeutic activity of lidocaine.
A study then investigated the effect of eight vehicles to determine their influence on lidocaine skin penetration. Altering the vehicle used to deliver lidocaine had a profound influence with dimethyl sulphoxide (DMSO) achieving the greatest flux and skin concentration of lidocaine in the shortest time. Efficacy of DMSO as a vehicle was due to disruption of the skin barrier as represented by an increase in post-application transepidermal water loss. The results demonstrated that provision of analgesia by a topical mulesing alternative could be driven by altering the formulation and by disrupting skin integrity.
Further studies examined the effect of potential skin damaging agents and formulation on transdermal lidocaine penetration. Acids and alkalis with differing strength and chemical properties, with and without a vehicle, were tested over three hours. This study was designed to test the hypothesis that disruption of the integrity of the stratum corneum, the principle barrier to transdermal drug movement in other species, would increase transdermal lidocaine movement. Contrary to expectations, lidocaine flux decreased as the strength of the acids and alkalis increased. A possible reason for these findings was that skin damage created a barrier to drug movement by closing transdermal pathways.
An in vivo study was then performed over three hours to determine if weaker agents could achieve a similar effect while permitting lidocaine skin penetration. The results demonstrated that sodium hydroxide was the most timely and effective skin necrotising agent, with dodecylbenzenesulphonic acid and triethylamine inducing the most profound inflammatory reactions. This study validated the in vitro studies.
This research project contributed to knowledge of transdermal drug delivery in sheep. Sheep skin was further characterised to enable better prediction of transdermal drug movement, and it was shown that analgesia was able to be provided topically to sheep. However, the results showed the local anaesthetic lidocaine was not able to penetrate sheep skin when combined with a rapidly acting skin damaging agent, and therefore it was not possible to develop a treatment that would accomplish the aim of replicating surgical mulesing and yet be ethically acceptable and safe. Future studies could investigate other anaesthetics, the longer term effects of weaker agents, confocal microscopy in sheep or microneedles to deliver a topical mulesing alternative.