Edited by: Kirsten Haastert-Talini, Hannover Medical School, Germany
Reviewed by: Joost Verhaagen, Netherlands Institute for Neuroscience, Netherlands; Erik Walbeehm, Radboud University Nijmegen Medical Centre, Netherlands
This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Peripheral Nerve Injury (PNI) is common following blunt or penetrating trauma with an estimated prevalence of 2% among the trauma population. The resulting economic and societal impacts are significant. Nerve regeneration is a key biological process in those recovering from neural trauma. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) and RNA sequencing (RNA seq) are investigative methods that are often deployed by researchers to characterize the cellular and molecular mechanisms that underpin this process. However, the ethical and practical challenges associated with studying human nerve injury have meant that studies of nerve injury have largely been limited to rodent models of renervation. In some circumstances it is possible to liberate human nerve tissue for study, for example during reconstructive nerve repair. This complex surgical environment affords numerous challenges for optimizing the yield of RNA in sufficient quantity and quality for downstream RT-qPCR and/or RNA seq applications. This study characterized the effect of: (1) Time delays between surgical liberation and cryopreservation and (2) contact with antiseptic surgical reagents, on the quantity and quality of RNA isolated from human and rodent nerve samples. It was found that time delays of greater than 3 min between surgical liberation and cryopreservation of human nerve samples significantly decreased RNA concentrations to be sub-optimal for downstream RT-qPCR/RNA seq applications (<5 ng/μl). Minimizing the exposure of human nerve samples to antiseptic surgical reagents significantly increased yield of RNA isolated from samples. The detrimental effect of antiseptic reagents on RNA yield was further confirmed in a rodent model where RNA yield was 8.3-fold lower compared to non-exposed samples. In summary, this study has shown that changes to the surgical tissue collection protocol can have significant effects on the yield of RNA isolated from nerve samples. This will enable the optimisation of protocols in future studies, facilitating characterisation of the cellular and molecular mechanisms that underpin the regenerative capacity of the human peripheral nervous system.
Peripheral nerve injury (PNI) is a common outcome following blunt or penetrating trauma with an estimated prevalence of 2% among the trauma population (
In some circumstances it is possible to liberate human nerve tissue for study, for example during reconstructive nerve repair. Samples that can be extracted are often finite and are exposed to the complex surgical environment, which includes chemical and physical environmental factors, time pressures and other priorities which are not present when sampling animal tissues in a laboratory setting. This affords numerous challenges when optimizing protocols for the extraction of RNA with sufficient quantity and quality for quantitative analysis, therefore this study is dedicated to exploring these challenges.
The extraction of RNA in sufficient quantity and quality is a critical step toward obtaining valid RT-qPCR/RNA seq results (
Based on experiences within our research unit and others, there appears to be a differential between the RNA extraction ratio [mean total RNA (μg) divided by initial tissue sample mass (mg)] of healthy and denervated nerve liberated from rats. Typical values range from 0.09 μg/mg for healthy sciatic nerve and 0.27 μg/mg for denervated sciatic nerve (
Accepting that the concentration of RNA that can be isolated from peripheral nerve tissue is likely to be lower than other tissues, it is pertinent to optimize surgical protocols in order to conserve whatever RNA is available. One variable that has been shown to be predictive of the quality and quantity of RNA extracted from samples is the time interval between sample liberation and cryopreservation (
While a number of past studies of other surgically liberated tissues for qPCR analysis have been optimized by manipulating variables such as time delays and RNA extraction protocols (
Protocols that detail how human nerve samples should be handled to optimize RNA yields for subsequent RT-qPCR and RNA seq analysis are not documented. This study aimed to explore the time course of RNA degradation in nerve tissue in order to establish an ideal time frame for the liberation of human nerve samples and cryopreservation (snap-freezing in liquid nitrogen). Additionally, this study aimed to investigate for the first time the effect of exposure of human nerve samples to surgical antiseptic reagents.
A total of 12 denervated human nerve samples were harvested from 12 different patients who underwent reconstructive surgery at the Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital after informed consent for the therapeutic procedure and for tissue donation (
Demographic of patients and samples included in this study.
Mechanism of injury | Intra-operative findings | Details of surgery | Nerve assessed | Denervated/ Innervated | Approximate time delay between surgical liberation and cryopreservation (min) |
---|---|---|---|---|---|
Motorbike accident | Axonotmesis of the tibial nerve | Below the knee amputation | Tibial | Denervated | 3 |
Motorbike accident | Right C5/6 Avulsion | Oberlin’s nerve transfer | Biceps branch of musculocutaneous | Denervated | 3 |
Fall on to sharp cast iron railing | Axonotmesis of superficial peroneal nerve | Excision of nerve | Superficial common peroneal nerve | Denervated | 3 |
Motorbike accident | Left C6-T1 root avulsion | Somsak’s nerve transfer | Medial head of triceps branch of the radial nerve | Denervated | 3 |
Iatrogenic nerve injury secondary to humeral fracture repair | Neurotmesis of the axillary nerve | Somsak’s nerve transfer | Axillary | Denervated | 5 |
Car v Tree | Right C4 - T1 avulsion | Intercostal nerve transfer to musculocutaneous nerve | Biceps branch of musculocutaneous | Denervated | 10 |
Motorbike accident | Right C5/6 Avulsion | Oberlin’s nerve transfer | Biceps branch of musculocutaneous | Denervated | 15 |
Car v Lorry | Axonotmesis of the accessory nerve | Fascicle of C7 transfer to accessory nerve | Fascicle of C7 to pectoralis muscles to accessory nerve | Denervated | 15 |
Motorbike accident | C5/6/7 Avulsion | Double Oberlin’s nerve transfer | Biceps branch of musculocutaneous | Denervated | 20 |
Iatrogenic nerve injury secondary to left neck lymph node biopsy | Neurotmesis of the spinal accessory nerve | Supraclavicular nerve transfer to spinal accessory | Supraclavicular and Spinal accessory | Supraclavicular (innervated) and Spinal accessory (denervated) | 3 |
Trampoline accident | Neurotmesis of the ulnar nerve | Sural nerve autograft to ulnar | Ulnar and Sural | Ulnar (denervated), Sural (innervated) | 3 |
Moped v Lampost | C5-8 Avulsion | Intercostal nerve transfer to triceps division of radial nerve | Radial and Intercostal | Intercostal (innervated), Radial (denervated) | 3 |
In all cases the site of operation was prepared with chlorhexidine or iodine based antiseptic reagents in concordance with standard surgical protocol (
Samples harvested were often heterogeneous in size, morphology and innervation, so they were dissected into sections measuring 0.5 ± 0.2 cm in the longitudinal orientation. The dimensions of the samples were chosen to allow comparisons with other RT-qPCR studies of rodent nerve samples which used similar dimensions (
Nerve samples were stratified into 3 experimental groups (shown in
Samples whereby the time between sample liberation and cryopreservation was less than 3 min.
Samples where the time interval between surgical liberation of the nerve sample and cryopreservation was greater than 3 min ranging up to 20 min.
Since RNA yields from nerve samples remained lower than that reported in rodent studies following optimisation of handling times, the exploration of other peri-operative variables was necessitated. This informed the development of a third experimental group to explore the effect of minimizing the exposure of nerve samples to antiseptic reagents.
Samples liberated and cryopreserved within 3 min but utilizing a “clean change” of surgical gloves and surgical equipment for harvest and handling of the sample to minimize exposure to antiseptic reagents. This group included healthy nerve samples in addition to denervated nerves.
Standard international operating protocols dictate that iodine and/or chlorhexidine based antiseptic reagents should be used to prepare the site of surgical incision as detailed by the World Health Organization (
All materials used in this process of RNA isolation were treated with RNase Zap (Invitrogen). Rodent and human nerves were placed into a 5 ml tube and snap frozen in liquid nitrogen. The time between tissue isolation and freezing was monitored as well as the interaction of samples with antiseptic surgical reagents. RNA was isolated from all nerve samples using the Qiagen RNeasy® Fibrous Tissue Mini Kit. The total volume of eluted RNA for each sample was 40 μl.
The quantity of RNA was determined using a TecanTM Infinite 200 PRO multimode reader. Quality of RNA was measured using a NanoDropTM spectrophotometer to ascertain 260/280 ratios for each sample. Samples were also analyzed using Bio-rad ExperionTM RNA analysis kits to assess Ribosomal Integrity Number (RIN), and obtain electropherogram data and automated agarose gel readings from samples using the ExperionTM Automated Electrophoresis System.
The effect of time between tissue extraction and freezing on the yield of RNA isolated from human nerve samples was investigated (Group 1 and Group 2).
The effect of time between tissue liberation and cryopreservation on RNA yield in human nerve tissue in a surgical environment utilizing standard antiseptic protocols. The duration between nerve tissue removal and freezing was monitored and samples were grouped according to whether the delay was more than (
The quality of RNA extracted from these nerve samples was concurrently determined quantitatively using 260/280 absorbance ratios (
The effect of time delays and surgical antiseptic reagents on the quality of RNA isolated from human nerve tissue.
Using the data from the electropherogram reports, a RIN ranging from 1 to 10 (with 10 being predictive of high quality RNA) was assigned to each sample. RIN is generated using an algorithm that selects features from the electropherograms and constructs regression models based on Bayesian learning techniques. This assessment has been validated in a number of studies and has been shown to be highly predictive of RNA quality (
Since RNA yields from human denervated tissue remained lower than that reported from rodent studies (
Even when the time delay is minimized/equivalent between samples there is still a large differential In RNA yield due to exposure to surgical antiseptics. Denervated samples were liberated under a standard (
Using nerve tissue freshly harvested from rats under carefully controlled environmental conditions enabled the effects of antiseptic reagents to be studied in isolation. A significant decrease in the yield of RNA (approximately 8.3 fold lower in exposed nerves compared to the untreated group) (
RNA yield from rodent nerves is reduced following exposure to surgical antiseptic reagents There was a statistically significant difference between the untreated (
In order to establish a protocol for the reliable extraction of RNA from human nerve samples, this study set out to characterize peri-operative variables predictive of RNA yield. The effect on RNA yield of time delays between liberation of the nerve sample and snap freezing was investigated and results suggested that nerve samples should be snap frozen within 3 min to preserve RNA quantity and quality. This time interval is considerably shorter than that cited in other studies that have extracted RNA from surgical specimens which have shown that time delays of several hours between surgical liberation of a sample and cryopreservation is detrimental to RNA quantity and quality (
Even when delay was minimized, RNA yields from human nerves in this study remained lower than those reported in rodent studies of denervated nerve tissue (
It was evident that healthy nerve samples yielded significantly lower quantities of RNA than that from denervated tissue, which concurs with rodent studies (
In order to isolate and further investigate the effects of exposure to antiseptic reagents, this study used a rodent model of peripheral nerve liberation which showed that these antiseptic reagents reduced RNA yields significantly. Chlorhexidine and iodine based reagents can be found in abundance in operating theaters around the world where they are often deployed for preoperative skin preparation. This finding, together with experimental evidence that has shown iodine and chlorhexidine based reagents to have cytotoxic effects on
In addition to influencing RNA extraction, iodine and chlorhexidine based reagents may have downstream effects on qPCR assays. These reagents have been shown to inactivate the Human Immunodeficiency Virus through a mechanism thought to be at least partially attributable to the ability of these reagents to manipulate the viral DNA reverse transcriptase (
In summary, this study reports new experimental evidence from human and animal studies that reveals the effects of time delays and surgical antiseptics on the RNA yield obtained from nerve tissue. This information can help to inform the development of improved methodology, specifically limiting time delay between sample liberation and cryopreservation to less than 3 min whilst utilizing a “clean change” surgical protocol to reduce antiseptic exposure. These findings provide new information about the response of fresh nerve tissue following isolation, including differences between healthy and denervated samples. This understanding will enable more effective use to be made of valuable human nerve tissue samples, addressing the knowledge gaps that currently exist in studying cellular and molecular mechanisms that underpin human nerve regeneration.
This study was carried out in accordance with the recommendations of ‘HTA guidelines, Biobank Ethical Review Committee’ with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the UCL Biobank Ethical Review Committee.
MW designed the concept and experimental methods used to assess quality and quantity of RNA yields, executed the experiments, performed analysis, and wrote the manuscript. TQ contributed to experimental design and clinical data detailed in the manuscript, made comments on the manuscript, and involved in writing up. JP contributed to experimental design and data analysis and informed the writing of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor is currently co-organizing a Research Topic with one of the authors JP, and confirms the absence of any other collaboration.