Serum neurofilament light chain measurements following nerve trauma

Abstract Background Optimal functional recovery following peripheral nerve injuries (PNIs) is dependent upon early recognition and prompt referral to specialist centres for appropriate surgical intervention. Technologies which facilitate the early detection of PNI would allow faster referral rates and encourage improvements in patient outcomes. Serum Neurofilament light chain (NfL) measurements are cheaper to perform, easier to access and interpret than many conventional methods used for nerve injury diagnosis, such as electromyography and/or magnetic resonance imaging assessments, but changes in serum NfL levels following traumatic PNI have not been investigated. This pre‐clinical study aimed to determine whether serum NfL levels can: (1) detect the presence of a nerve trauma and (2) delineate between different severities of nerve trauma. Methods A rat sciatic nerve crush and common peroneal nerve crush were implemented as controlled animal models of nerve injury. At 1‐, 3‐, 7‐ and 21‐days post‐injury, serum samples were retrieved for analysis using the SIMOA® NfL analyser kit. Nerve samples were also retrieved for histological analysis. Static sciatic index (SSI) was measured at regular time intervals following injury. Results Significant 45‐fold and 20‐fold increases in NfL serum levels were seen 1‐day post‐injury following sciatic and common peroneal nerve injury, respectively. This corresponded with an eightfold higher volume of axons injured in the sciatic compared to the common peroneal nerve (p < .001). SSI measurements post‐injury revealed greater reduction in function in the sciatic crush group compared with the common peroneal crush group. Conclusions NfL serum measurements represent a promising method for detecting traumatic PNI and stratifying their severity. Clinical translation of these findings could provide a powerful tool to improve the surgical management of nerve‐injured patients.


| INTRODUCTION
Traumatic peripheral nerve injuries (PNIs) can have devastating impacts on the quality of life of patients and represents a major global health challenge. 1,2 Patients describe deficits in the control, coordination and power of movement, reduction and alteration in appreciation of touch and sensation, and often experience chronic pain following injury. 3,4 Early detection of severe PNI and appropriate referral to specialist nerve injury clinicians is essential to ensure surgical intervention is offered where it may lead to clinical benefit. [5][6][7] This is important since functional recovery following PNI is heavily influenced by the time elapsed between injury and functional muscle reinnervation.
Incremental increases in the denervation time of the distal stump and end organ(s) lead to a microenvironment which becomes progressively antagonistic to nerve regeneration. 7,8 This observation is thought to be largely responsible for sub-favourable functional outcomes seen in cases where delayed surgical intervention is deployed. 9,10 A study which measured cellular and molecular markers of human nerve degeneration reported that the tissue microenvironment may deteriorate as soon as 3-month post-injury. 7 Investigations of referral times of nerve injuries requiring nerve reconstruction to specialist centres have shown that many patients are not referred until around 1-year post-injury. 11,12 This means the tissue microenvironment may no longer be optimal for nerve regeneration and functional reinnervation. 5,7 These data accentuate the need for improved diagnostic tools to identify PNI and facilitate prompt referral. Unfortunately, diagnostic tools used today afford a number of challenges to achieving this objective. Clinical diagnostic tools such as electromyography (EMG) and magnetic resonance imaging (MRI) analysis have emerged as the mainstay investigations to detect and monitor severe nerve injury. [13][14][15] However, they are expensive to perform and difficult to interpret 15,16 without input from highly trained specialised clinicians.
In many cases, it is not possible to delineate between different severities of nerve injury using these tests. 5 Due to this issue, delays to appropriate surgical treatment are often encountered leading to worse outcomes than had earlier intervention been made. 5,17 Together, this highlights an unmet clinical need for improved diagnostic tools that are accessible in different healthcare settings, costeffective and straightforward to interpret. Neurofilament light chain (NfL) is a protein that is released following axonal damage. 18 The advent of serum NfL assays has allowed highly sensitive and specific quantification of serum NfL levels which correlate with cellular markers of neuroaxonal damage in pathologies such as traumatic brain injury (TBI) and acquired axonal neuropathies. [18][19][20][21][22] Serum NfL assays are also significantly cheaper, less invasive and easier to interpret than EMG and MRI. 18 Studies which link NfL assays to a volume of axonal injury could form the basis for an improved quantitative classification system of nerve injury severity that has the power to inform clinical management at an earlier stage post-injury than is currently possible with conventional technologies. 15,23 Data which characterise: (1) the serum NfL signal seen following different severities of nerve trauma and (2) the time course of serum NfL changes after nerve injury will provide essential information that will inform the clinical utility of serum NfL in the context of nerve trauma.
Identification of appropriate human models of nerve injury through which these questions can be addressed is difficult. The broad spectrum of different trauma mechanisms means that human nerve injuries are often incomplete and mixed. [24][25][26] Retrieval of human nerve tissue for study in the laboratory is often fraught with ethical and practical challenges. 26 Low follow-up rates among trauma patients (reported to be as low as 2%) afford difficulties to the design and deliverability of longitudinal studies. 27 Controlled animal models of nerve injury allow researchers to retrieve tissues and monitor functional recovery at standardised endpoints for laboratory analysis. 28 The sciatic and common peroneal nerve crush are simple and well-controlled injuries widely used in rodents to model different volumes of axon injury. 28,29 The former represents a more proximal nerve with a larger cross-sectional area and a longer distal segment throughout which axons will degenerate post-injury when compared with the latter.
Using these controlled animal models of sciatic and common peroneal nerve injury, this pre-clinical study set out to determine whether serum NfL assays can: (1) capture the presence of a sciatic or common peroneal crush and (2) accurately delineate between different severities of PNI. Forty-six adult female Wistar rats (250-300 g) were included in the study. The rats were assigned randomly into groups housed in cages with soft bedding with free access to food and water. Each animal was deeply anaesthetised by inhalation of isoflurane, and the left sciatic nerve was exposed at mid-thigh level. This was done by making a 3-cm incision parallel to the femur between the knee and hip, followed by separation of the muscle layers to expose the nerve.
Under the microscope (Zeiss Stemi DV4), the sciatic nerve and its branches were released from the surrounding tissue. All animals received the same level of interaction throughout the study. They were handled before surgery and throughout the study for training and completion of functional testing. All animals were given 5 min resting time before functional measurements were recorded.
After sciatic or common peroneal nerve crush, the following protocols were deployed at 1 (n = 5 in each group), 3 (n = 5 in each group), 7 (n = 5 in each group) and 21 days (n = 3 in each group) following sciatic or common peroneal nerve crush. Ten rodents did not receive any nerve injury and were used as uninjured healthy controls.

| Nerve length measurements
Nerve samples were then removed from the 4% PFA. The following measurements of the length of nerve trunk distal to the injury site were taken using a ruler with mm divisions: (1) for the sciatic nerve injuries: the distance between the injury site and the most distal end of the harvested nerve; (2) for common peroneal nerve injuries: the distance between the injury site and the end of the common peroneal nerve. Nerve samples were then stored at 4 C in phosphate-buffered saline until analysis.

| Serum NfL analysis
NfL levels in serum were measured using a SIMOA ® SR-X analyser (Quanterix Corporation) with a SIMOA ® NF-light Advantage (SR-X) Kit (Quanterix, No. 103400) according to protocols well described elsewhere. 30 All samples were run in duplicate and operators were blinded to the experimental conditions. All samples measured were above the lower limit of detection (0.038 pg/mL) with a mean coefficient of variation of 9.15% between duplicates. Furthermore, two control samples were run on each 96-well plate with a mean coefficient of variation of 6.90%.

| Neurofilament staining and imaging
Cross-sections of the sciatic and common peroneal nerve immediately proximal to the injury site were taken. Samples were incubated in formalin for 5 min, followed by 70% ethanol for 10 min, 100% ethanol

| Static sciatic index
Functional recovery was measured using the static sciatic index (SSI).
The hind paw of each rat was imaged (a minimum of three images were obtained from below using a Samsung Galaxy A5 camera with the animal placed in an elevated polyethylene box) every 3-4 days following the crush injury until the endpoint of the experiment.
Images were analysed using ImageJ 31 to obtain the following parameters: Toe Spread Factor (TSF)-the distance between the first and the fifth toes.
Intermediary Toe Spread Factor (ITSF)-the distance between the second and the fourth toes.
These values were then used to calculate the SSI using the fol-

| Statistical analysis
Data are presented as mean ± standard deviation (SD) unless otherwise stated. A Shapiro-Wilk normality test was conducted where appropriate. A mixed-effects analysis was used to compare the different injury models in terms of serum NfL and SSI changes over time. In addition, ANOVA was used to compare serum NfL in the injury groups to baseline uninjured control animals. An unpaired t test was used to compare the distal axon volume in the two models. In all cases, p value of <0.05 was determined to be statistically significant. The study was powered to show twofold higher serum NfL concentration in the sciatic nerve injury group compared with the common peroneal nerve injury cohort with 90% power and an overall type I error rate of 0.05.

| RESULTS
The mean serum NfL concentration seen in healthy uninjured controls was detectable at a baseline level of 0.50 ± 0.2 pg/mL (Table 1).
Serum NfL measurements were significantly higher than baseline measurements at 1-, 3-and 7-days post-injury in the sciatic nerve crush group and at 1-day post-injury in the common peroneal nerve crush group (Figure 1 and Table 1). By 21-days post-injury, serum NfL concentration had returned to baseline levels in both injury groups.
The highest serum NfL levels in both the sciatic and common peroneal nerve injury models were recorded 1-day post-injury; mean serum NfL levels were 45-fold higher than baseline for sciatic nerve injuries and 20-fold higher than baseline in the common peroneal injury group ( Figure 1 and Table 1).
In the sciatic nerve injury group, serum NfL readings were significantly higher compared with the common peroneal nerve injury group

| DISCUSSION
For the first time, this study set out to obtain pre-clinical proof of concept data for serum NfL measurements as a biomarker of nerve injury.
In healthy controls, serum NfL levels were around 0.5 pg/mL, which concurs with a number of studies which have reported serum NfL readings in healthy controls. [18][19][20][21][22] This study demonstrates serum NfL measurements can distinguish between different severities of traumatic PNI, with severity in this case being defined by the volume of axonal material distal to the crush site that would degenerate following axotomy (sciatic nerve crush yielding a 15-fold greater volume of axonal damage and thus being classified as more severe than the smaller and shorter distal segment in the common peroneal nerve crush model). At 1-and 3-day post-injury, respectively, serum NfL readings taken from the sciatic T A B L E 1 Serum NfL measurements from uninjured controls and following nerve crush injury.  shown to remain persistently elevated (albeit to a lesser degree) over more chronic time points. 20,36 An additional factor that may contribute to the greater magnitude and speed of changes seen in serum NfL recordings following traumatic PNI ( Figure 1) compared with other neurological injury mechanisms is differentials in the interface between neuronal structures and systemic circulation. Nerve crush injuries lead to instant loss of the structural integrity of the nerve-blood barrier 28,37 meaning that proteins such as NfL are released directly into the systemic circulation following injury. By extension, NfL can be detected in the serum rapidly following injury and metabolised.
Conversely, in most central nervous system (CNS) injuries, NfL concentrations are significantly higher in the cerebrospinal fluid compared with serum. 18,38,39 This differential is thought to exist since a significant proportion of NfL released following CNS insult is prevented from entering the peripheral circulation by the bloodbrain barrier. 18 Similarly, in acquired axonal neuropathies, the nerve-blood barrier often retains structural integrity. 40,41 This may explain why in some studies, serum NfL readings lag behind key cellular and molecular events associated with CNS injury and peripheral neuropathy. [18][19][20][21][22]30  A limitation of this study is that the volume of axons injured in the sciatic and common peroneal crush models was only an estimate based on the dimensions of the nerve rather than a direct quantification of the degenerating axons in the distal nerve tissue. Future studies may wish to incorporate other conventional metrics of PNI such as nerve conduction studies to compare the sensitivity/specificity of these readings with serum NfL assays. Clinical translation of data presented in this paper will require serum NfL concentrations to be tested in human paradigms of nerve injury to establish the range and timing of changes associated with nerve injuries of differing severity.
One way to approach that would be to use scenarios where a controlled nerve injury is conducted following an earlier traumatic injury, for the purposes of autograft or nerve transfer. 5 Assuming sufficient time had elapsed for any serum NfL from the initial injury to have cleared, the increase in NfL associated with the controlled nerve injury procedure could be used to establish a correlation between nerve injury severity, that is, volume of degenerated axonal tissue, and serum NfL in humans. It is often possible to retrieve excess nerve F I G U R E 3 Static sciatic index (SSI) following rat sciatic and common peroneal nerve crush. SSI measurements were obtained at baseline then at various times following nerve crush. Mixed-effects analysis indicated significant ( p < .0001) effects of time, type of crush and interaction between the two factors. # compares each time point to Day 0 baseline in the same group, using Dunnett's post hoc test (# p < .05, ###p < .001, ####p < .0001). * compares sciatic to CP at each time point, using Sidak's multiple comparisons test (**p < .01, ****p < .0001).
tissue from the donor nerve during these surgeries which would otherwise be discarded, 7,26 providing an opportunity for histological analysis and approximations of axonal volume loss to be made in a similar manner to the present study.
In summary, this study in a rat model has shown for the first time