Assessing The Effects Of Commonly Used Surgical Antiseptics and Time Delays on RNA Concentration and Integrity from Peripheral Nerve Samples
Matthew B Wilcox, BSc (Hons)1; Tom J Quick, MBBS MA(Cantab) FRCS(Tr&Orth)2; James B Phillips, BSc (Hons), PhD1
1University College London, London, United Kingdom, 2Royal National Orthopaedic Hospital, London, United Kingdom
Real Time quantitative Polymerase Chain Reaction (RT-qPCR) has enabled researchers to identify the cellular and molecular mechanisms that underpin the regenerative capacity of the peripheral nervous system. However, this understanding remains largely limited to rodent models of nerve regeneration. Translation of these findings into human paradigms of regeneration is awaited. This is at least partially attributable to the practical challenges associated with extracting and processing human nerve samples liberated in the surgical environment. Based on intra-operative observations, this study characterised challenges associated with studying human nerve samples:
1) Time delays between surgical liberation of the nerve sample and cryopreservation
2) The interaction of samples with Chlorhexidine and Iodine based antiseptic surgical reagents
Human nerve samples liberated during reconstructive nerve procedures (where Iodine and Chlorhexidine based antiseptic reagents were utilised for pre-operative skin preparation) were stratified into 3 experimental groups:
Group 1: Samples whereby the time between sample liberation and cryopreservation was less than 3 minutes
Group 2: Samples where this time interval was greater than 3 minutes.
Group 3: Samples liberated and cryopreserved within 3 minutes but with a change of gloves and surgical equipment to minimise exposure to antiseptic reagents.
In order to isolate the effects of the antiseptic reagents, 9 terminally anaesthetised Sprague Dawley rats had their sciatic nerves excised and sectioned (in an environment known to be otherwise free of antiseptic reagents) into sizes representative of that liberated in the human model of nerve liberation (0.75cm length). 100uL of each of the aforementioned antiseptic reagents was applied to the sample prior to cryopreservation. The quantity and quality of RNA extracted was assessed using UV spectroscopy, electropherograms and gel electropheresis.
It was found that time delays of greater than 3 minutes between surgical liberation and cryopreservation of human nerve samples significantly (p<0.01) decreased RNA yields to levels sub-optimal for downstream qPCR applications (<5ng/uL). Similarly, the exposure of samples to antiseptic reagents reduced RNA yield from rodent nerve samples by an average of 88%.
This study will inform the development of qPCR protocols that aim to explore the cellular and molecular mechanisms that underpin the regenerative capacity of the human peripheral nervous system. Future studies should characterise the effects of these reagents on the regenerative capacity of peripheral nerves. Together, this will inform the development of surgical protocols to maximise functional outcomes for peripheral nerve injury patients.
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