Review of Chinese General Medicine: Exploration of the application value of neurofilament light chain protein in peripheral neuropathy

Nerve axonal injury and loss are the pathological basis of many acute and chronic neurological diseases that lead to permanent disability, neurofilaments (Nfs) are highly specific for neuronal cell damage and death, and neurofilament light chain protein (NfL) has been identified as a biomarker of axon damage in many central nervous system (CNS) diseases
.

Cerebrospinal fluid NfL (cNfL) and/or serum NfL (sNfL) levels have shown great advantages in diagnosing neurodegenerative diseases, central nervous system demyelinating lesions, traumatic brain injury, infectious diseases such as Creutzfeldt-Jakob disease, genetic disorders such as Huntington's disease, and other neurological/psychiatric disorders, assessing disease activity, monitoring response to treatment, and determining prognosis, and, The evidence for cNfL and sNfL as very promising biomarkers for CNS disease is increasing
.

In recent years, the application value of NfL in peripheral neuropathy has gradually attracted attention, and is summarized as follows
.

Structure and function of 01Nfs

According to its diameter, cytoskeletal proteins can be classified into 6 nm actin filament, 10 nm intermediate filaments (including Nfs and glial filaments), and 15 nm myosin filaments
composed of motor proteins.
Nfs is a highly specific main cytoskeletal component in central and peripheral neurons, expressed by large-diameter myelinated axons, which structurally maintains axon stability, provides tensile strength for dendrites and axons, and enables axons to grow radially.
Functionally, NFs assemble each other and provide cross-bridging with actin filaments and metallothionein
.

Dynamic cytoskeleton is essential
in increasing the pulse conduction velocity of axons in intracellular axon transport, as well as in the process of post-translational modifications that regulate neurite growth.
NFS consists of light chains of neurofilament, middle chains of neurofilament, heavy chains of neurofilament, and α-interconnectins, all of which have a conserved α-helix rod domain with variable amino terminal and carboxyl terminal regions, and different lengths of variable regions give subunits different molecular weights of 61.
5, 102.
5, 112.
5, 55.
4 thousand daltons (kDa),
respectively 。 NfL is considered the most important part of Nfs, the only Nfs that can self-assemble into functional fibers, and the most abundant and soluble subunit in Nfs, which makes NfL the most reliable Nfs subunit
that can be measured.

02NfL detection method, reference range and physiological influencing factors

Many studies use enzyme-linked immunosorbent assay (ELISA) and/or commercial kits to detect NfL
.
The ELISA assay kit uses two highly specific non-competitive monoclonal antibodies (mAB47:3 and mAB2:1) to quantify soluble NfL, which is considered reliable in the detection of cNfL, but the sensitivity of ELISA detection is generally only pg/ml level, and the detection of ultra-low abundance proteins in blood often shows certain deficiencies, while electrochemiluminescence (ECL)-based assays have high sensitivity, wide dynamic range, and small sample size
。 But neither technique can detect small changes
in disease-related NfL.

Single molecule array (Simoa) technology is a newly developed ultra-high sensitivity in recent years, based on the double antibody sandwich ELISA method of paramagnetic beads, the sensitivity of which is more than 1 000 times that of ELISA technology, and the lower limit of detection reaches fg/ml
.
This method measures proteins
in a variety of different matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.
).
Several studies have demonstrated that Simoa technology can reliably detect NfL in blood samples (in the full concentration range), including in healthy individuals, thereby changing the way
brain injury and neurodegenerative diseases are diagnosed.

Normally, low levels of NfL are continuously released
from axons in an age-dependent manner.
The level of cNfL increased by 3.
30% per year in the normal control population, and cNfL was higher
in men.
STUDIES SUCH AS VAGBERG HAVE SHOWN THAT CNFL LEVELS IN NORMAL, HEALTHY ADULTS UNDER 30 YEARS OF AGE ARE ABOUT 187 ng/L; 30~<40 years old is 274 ng/L; 40~<60 years old is 466 ng/L; Higher levels of cNfL are released in old age, 693 ng/L
for those aged 60 years and older.
The results of a study of 335 healthy individuals aged 38.
5~85.
6 years showed that the median sNfL level was 32.
30 (23.
15, 43.
95) pg/ml, which is not very consistent with the results in other studies, which may be related to the lack of globally standardized certified reference materials and/or methods, and also highlights the need
for standardized testing and cut-off value determination before laboratory indicators can be reliably used in clinical practice.

03NfL with peripheral neuropathy

1.
NfL and Guillain Barré syndrome (GBS)

GBS is an immune-mediated, acute peripheral neuropathy characterized by flaccid paralysis that results in a high
risk of severe disability and death.
In the early stages of GBS disease, there is an urgent need to be able to identify serum or cerebrospinal fluid biomarkers with a poor prognosis
.
Previous studies have found significantly elevated
sNfL and cNfL levels in GBS patients.
ALTMANN ET AL.
STUDIED THE CORRELATION BETWEEN CLINICAL OUTCOMES AND SNFL IN 27 GBS PATIENTS, WITH A MEDIAN SNfL level of 85.
5 pg/ml AT ADMISSION AND 9.
1 pg/ml IN THE CONTROL GROUP, AND THE INCREASE IN BASELINE SNFL LEVEL WAS ASSOCIATED WITH
LONG HOSPITAL STAY, ADMISSION TO INTENSIVE CARE UNIT (INTENSIVE CARE UNIT), AND POOR SHORT-TERM PROGNOSIS AFTER DISCHARGE 。 KÖRTVELYESSY ET AL.
HAVE SIMILAR FINDINGS IN RECENT STUDIES, GBS PATIENTS WITH SNfL and cNfL levels are higher than control groups, sNfL in patients admitted to the icu is significantly increased, and sNfL is significantly correlated
with disability function scores in gbs patients.

MARTÍN-AGUILAR ET AL.
MEASURED NFL IN 98 SERUM SAMPLES AND 24 CEREBROSPINAL FLUID SAMPLES OF GBS PATIENTS BY SIMOA TECHNOLOGY, AND EVALUATED THE LONG-TERM PROGNOSIS OF GBS PATIENTS, AND THE RESULTS SHOWED THAT THE LEVELS OF SNfL and cNfL in gbs patients were significantly higher than those in the control group.
Furthermore, multivariate regression analysis showed that sNfL levels were associated with functional disability after 1 year in GBS patients, suggesting that sNfL levels could also be used as biomarkers to assess the long-term prognosis of GBS
.
AXELSSON et al.
used the ELISA method to detect cNfL in 18 GBS patients and found that high cNfL levels at onset predicted long-term disability and poorer quality of life
.
Based on the above studies, it can be found that the elevated baseline sNfL and cNfL levels in GBS patients are related to the severity of the disease.
The levels of sNfL and cNfL have independent predictive value
on the short-term and long-term prognosis of GBS patients.
Early detection of NfL in GBS patients can achieve later individualized disease risk stratification and prognosis judgment
.

IN THE OBSERVATION OF GBS OF DIFFERENT CLINICAL SUBTYPES, MARTÍN-AGUILAR ET AL.
FOUND THAT THE SNFL LEVEL OF PATIENTS WITH ACUTE MOTOR AXONAL NEUROPATHY (AMAN) WAS HIGHER THAN THAT OF OTHER PATIENTS (199.
53 pg/ml AND 46.
77 pg/ml, P=0.
006).

Patients with acute kinesthetic axonal neuropathy (AMSAN) and unclear electrophysiological classification had higher sNfL than those with acute inflammatory demyelinating polyradiculoneuropathy (AIDP) (107.
15, 104.
7, 33.
88 pg/ml, respectively).

The sNfL levels of patients with pure motor variant and Miller Fisher syndrome were higher than those in sensorimotor GBS (162.
18, 95.
50, and 38.
02 pg/ml, respectively).

IN THE KÖRTVELYESSY ET AL.
COHORT, ELECTROPHYSIOLOGICAL RESULTS SHOWED THAT THE CNfL/sNfL ratio of patients with mixed neuropathy type or pure axonal injury type was lower than that of normal control, while the cNfL/sNfL ratio of three patients with demyelinating type was similar
to that of normal control.
The above studies all suggested that NfL was different in patients with different subtypes of GBS, and the elevated sNfL level and decreased cNfL/sNfL ratio were more suggestive of the predominantly axonal injury subtype, showing the potential
of NfL as a biomarker of axonal injury.

The correlation between sNfL levels and axonal injury has also recently been validated in biopsy studies of the gastrocnemic nerve, where sNfL levels are associated with axonal nerve axonal injury in xonal neuropathy-dominated studies, and there is an inverse relationship
between sNfL and fiber density.
HOWEVER, THE RESULTS OF ALTMANN ET AL.
, INCLUDING 27 PATIENTS WITH GBS (INCLUDING 17 AIDP, 5 AMAN, AND 5 PATIENTS WITH UNKNOWN CLASSIFICATION), SHOWED THAT SNfL levels did not differ
significantly between the aidp subtype and the aman subtype.
The researchers believe that this may be due to the continuous release of Nfs from damaged nerve fibers in the subtype dominated by demyelinating injury, but axon damage below the threshold
detectable by neuroelectrophysiology.
Therefore, the neuroelectrophysiological pattern of GBS patients is not representative of the pathological process
that really occurs.
sNfL can be used as a sensitive serum biomarker in GBS patients to make early assessment
of axon damage before definitive electrophysiological damage occurs.

2.
NfL and chronic inflammatory demyelinating radiculoneuropathy (CIDP)

CIDP is a rare acquired immune-mediated demyelinating neuropathy with strong heterogeneity in clinical presentation, prognosis, and response to treatment
.
To date, there are no reliable blood biomarkers for people with CIDP that can help diagnose and observe disease activity progression and determine prognosis
.
In addition, objective disease activity parameters are urgently needed to guide treatment in patients receiving maintenance therapy
.

Recent evidence suggests that significantly elevated sNfL levels can be observed in patients with highly disease-active CIDP; However, sNfL levels were not correlated with disease duration, and there was no significant difference in sNfL levels between newly diagnosed patients and relapsed patients; In addition, sNfL is related
to age and gender.
VAN LIEVERLOO ET AL.
EVALUATED THE RESULTS OF NERVE CONDUCTION DETECTION (NCS) BEFORE TREATMENT IN PATIENTS WITH CIDP AND FOUND THAT HIGHER SNfL level was associated with lower negative peak area of median nerve composite muscle action potential (cmap) and lower negative peak area of total cmp pool, suggesting a correlation
between sNfL level and axonal injury.
FUKAMI et al.
further confirmed this conjecture through NCS and pathological studies, and the level of sNfL was negatively correlated with the amplitude of CMAP changes in the tibial nerve, and positively correlated
with the degree of active axonal degeneration in pathology.
Therefore, sNfL can be used in conjunction with NCS to assess neuroaxonal injury in CIDP; sNfL has an advantage over NCS because sNfL is non-invasive and dynamically reacts to persistent axonal damage
.

HAYASHI ET AL.
OBSERVED 11 PATIENTS WITH CIDP BEFORE AND AFTER GAMMA GLOBULIN TREATMENT, AND FOUND THAT THE BASELINE SNfL level before treatment was significantly increased, and the sNfl level decreased significantly after treatment and during remission
.
VAN LIEVERLOO ET AL.
OBSERVED A SIGNIFICANT DECREASE AND NORMALIZATION OF SNFL LEVELS DURING THE FOLLOW-UP PERIOD IN 5 PATIENTS WITH TREATMENT RESPONSE, WHILE SNFL LEVELS WERE HIGHER IN PATIENTS WITH ACTIVE CIDP, SUGGESTING THAT SNFL CAN BE USED AS A BIOMARKER TO ASSESS CIDP DISEASE ACTIVITY
.
GODALAINE ET AL.
USED ENHANCED CHEMILUMINESCENCE (ECL) TO MEASURE SNFL IN 76 PATIENTS WITH CIDP AND FOUND THAT SNFL WAS ASSOCIATED WITH ONE-YEAR DISEASE PROGRESSION (REDUCED MRC TOTAL SCORE) OF CIDP DURING FOLLOW-UP, WHILE PATIENTS WITH HIGH SNFL LEVELS HAD AN INCREASED PROBABILITY OF POOR TREATMENT RESPONSE DURING FOLLOW-UP
.
These evidence further suggest that sNfL can be used as a biomarker to assess disease activity and prognosis in peripheral neuropathy
.
In addition, sNfL may also be of great value in the clinical decision-making of CIDP, identifying patients
who ultimately need to switch therapy or who should start receiving more intensive therapies (e.
g.
, combination therapy) immediately.

Recent studies have clarified that patients with IgG4 autoantibodies targeting Langfei's paranodal ligator components such as neuromyelin 155 (NF155) have a different
mechanism of neuropathy than patients with macrophage-induced demyelination-induced CIDP.
The CIDP study cohort of FUKAMI et al.
included 13 patients who were positive for anti-NF155 antibodies, and sNfL levels were significantly higher in patients who were
positive for anti-NF155 antibodies compared with those who were negative.
The sNfL level and anti-NF155 antibody titer were significantly reduced in the eight patients after treatment compared with baseline, and the change of sNfL level was positively correlated
with NF155 antibody titers.
The study suggests that autoantibodies may directly damage axon-Schwann cells, leading to extensive neuronal damage that leads to elevated
sNfL levels.
FUKAMI ET AL.
ALSO FOUND NO SIGNIFICANT DIFFERENCE
IN SNfL levels between classic and variant forms of cidp.
There are currently no studies that have looked at cNfL levels
in patients with CIDP alone.

3.
NfL and other immune-mediated/inflammatory peripheral neuropathy

In the cohort study of MARIOTTO et al.
, 12 patients with CIDP, 5 cases of GBS, 3 cases of multifocal motor neuropathy (MMN), 3 cases of antimyelin-binding glycoprotein (MAG) antibody positive peripheral neuropathy, and 1 case of non-systemic vasculitic neuropathy were included, and the results showed that the overall NfL level was significantly higher and the cNfL level was higher than that of sNfL.
In addition, sNfL is associated with disease activity and predicts subsequent disability progression
.
Unfortunately, the investigators did not conduct disease grouping studies
.
THE STUDY OF SSTASCHEIT ET AL.
DID NOT DIRECTLY COMPARE THE RELATIONSHIP BETWEEN SNFL LEVELS IN 6 MMN PATIENTS AND THE CONTROL GROUP, BUT THERE WAS NO DIFFERENCE BETWEEN MMN PATIENTS AND CIDP CLASSIC AND VARIANT; In this study, 63 patients with CIDP had significantly elevated sNfL levels, which indirectly suggested the possible
assessment role of sNfL levels in MMN disease activity.
In addition, the latest prospective observational study in 2022 included 24 patients who had not received anti-MAG peripheral neuropathy and found no association of sNfL with
disease activity.
Therefore, the relationship between sNfL and other immune-mediated peripheral neuropathy disease needs to be further studied
.

The gold standard for the diagnosis of vasculitic neuropathy (VN) is peripheral nerve biopsy, but invasive and other reasons limit its clinical
use.
BISCHOF ET AL.
RETROSPECTIVELY ANALYZED THE CLINICAL DATA OF 11 PATIENTS WITH VN AND FOUND THAT SNfL levels could be used as markers of vasculitic injury and/or disease activity, and sNfL levels were significantly higher than during
remission at the time of VN diagnosis.
Therefore, sNfL is very promising
in the application of VN as a biomarker that can be rapidly screened and suggest acute axonal injury.

4.
NfL and hereditary peripheral neuropathy

SANDELIUS ET AL.
AND MILLEE ET AL.
CONDUCTED TWO PERONEAL MUSCULAR ATROPHY (CMT) COHORTS (INCLUDING CMT1A, CMTX1 AND SPTLC1), RESPECTIVELY, AND FOUND THAT SNfL levels were significantly elevated and correlated with disease severity.
Patients with CMTX1 have higher sNfL levels than CMT1A and other CMT disease types
.
Furthermore, sNfL levels are similar
in patients with CMT with a predominantly axonal or demyelinating type.
The authors note that this result is reasonable given the importance of Schwann cell-axon interactions in the maintenance of neuronal function and the previously observed degree of disability in CMT1A (demyelinating-based) is
determined by the degree of axonal damage rather than the degree of slowing conduction velocity.

The variability in sNfL levels in 9 patients with CMT was 16.
4% after 1 year of follow-up, which was in line with the expectation of slow and constant progression of hereditary diseases, indicating that it could be used as a standard to
measure disease progression.
MAIA et al.
have found that sNfL can distinguish between asymptomatic mutation carriers and early symptomatic patients, as well as sensory neuropathy patients and motor neuropathy patients, and can be used as a disease-oriented treatment efficacy indicator
in clinical and preclinical trials.
Subsequent studies also validated the conclusions of MAIA et al.
, and found that sNfL levels were elevated in patients with amyloid light chain degeneration peripheral neuropathy and ATTR peripheral neuropathy, and were also associated with
the severity of the latter.
Recent studies such as LUIGETTI have further validated the previous conclusions, and sNfL can be used to assess the severity of ATTR peripheral neuropathy and monitor its progression
.

Based on the available evidence, sNfL can be used as a biomarker of hereditary peripheral neuropathy activity and a possible indicator
of therapeutic efficacy.

5.
NfL and chemotherapy drug-associated peripheral neuropathy (CIPN)

Recent studies on CICN and NfL have been reported
.
In animal models of CIPN induced by vincristine chemotherapy, sNfL levels continued to rise significantly during vincristine administration, and increased nearly 4-fold
when axonal signs and intraepidermal nerve loss appeared.
While elevated sNfL levels in animal models of CIPN induced by cisplatin and paclitaxel chemotherapy, the timing of the elevation was also related to
the severity of morphological and functional changes in axon structures.
Patients with oxaliplatin-induced CICN had increased
sNfL levels during oxaliplatin administration.
HUEHNCHEN ET AL.
OBSERVED NFL CHANGES THROUGH CELL ASSAYS (EXPOSURE OF INDUCED PLURIPOTENT STEM CELL-DERIVED SENSORY NEURONS TO PACLITAXEL) AND IN BREAST OR OVARIAN CANCER PATIENTS RECEIVING PACLITAXEL CHEMOTHERAPY, RESPECTIVELY, AND FOUND THAT SNfL was associated with the development and severity of cipn, and monitoring sNfL during chemotherapy can indicate the ongoing neural axonal injury and the severity
of cipn.

6.
NfL and diabetic peripheral neuropathy (DPN)

DPN versus NfL has been studied less
.
CELIKBILEK ET AL.
FIRST PROPOSED THAT SNfL-mRNA can be used as a surrogate marker for early prediction of pre-diabetic peripheral neuropathy
.
RECENTLY, MORGENSTERN ET AL.
FOUND THAT DNN PATIENTS HAVE ELEVATED SNfL levels, and the increase in sNfL may be associated with
the development of hyperalgesic phenotypes.

04cNfL and sNfL differ in the origin and diagnostic value of the central nervous system and peripheral nervous system

MILLE ET AL.
POINTED OUT THAT SEVERAL ISSUES STILL NEED TO BE ADDRESSED IN THE FUTURE OF NFL AS A BIOMARKER: THE AREA OF OVERLAPPING NFL LEVELS BETWEEN DISEASE PATIENTS AND CONTROLS, THE STANDARDIZATION OF DETECTION METHODS AND CUT-OFF VALUES, THE NONSPECIFICITY OF NFL, AND THE GENERAL MODERATE CORRELATION WITH
DISEASE SEVERITY.

In CNS disease, cell destruction leads to the release of axonal cytoskeletal protein family Nfs into the cerebrospinal fluid and into the blood circulation through arachnoid granules, so cNfL is considered to be of CNS origin, in which case there is a certain correlation
between cNfL and sNfL 。 The latest systematic review of cNfL shows that high levels of cNfL have been observed in cognitively impaired AIDS (HIV) patients, amyotrophic lateral sclerosis, frontotemporal dementia and Huntington's disease in existing studies; 33.
3% of studies observed that cNfL levels were higher in men than in women, and cNfL levels increased with age; Most CNS diseases with comparable levels of cNfL are measured, but cNfL has the potential to
help distinguish frontotemporal dementia from Alzheimer's disease and Parkinson's disease from atypical parkinsonism.

In peripheral nervous system diseases such as GBS/CIDP, lesions affect the intrathecal and extrathecal parts of the peripheral nervous system, and a significant correlation
between cNfL and sNfL in GBS patients has also been observed in related studies.
Elevated sNfL levels reflect nerve root and/or peripheral nerve damage, and sNfL is presumed to originate from peripheral nervous system injury
in most acutely active GBS patients.
The cNfL/sNfL ratio can roughly assess the peripheral or central origin of sNfL
.
KÖRTVELYESSY ET AL.
SHOWED THAT THE AVERAGE CNfL/sNfL ratio in the control group was 26.
7, INDICATING THAT THE CNS ORIGIN OF NFL WAS DOMINANT UNDER NORMAL PHYSIOLOGICAL CONDITIONS, WHILE THE CNfL/sNfL ratio in gbs patients with axonal or mixed axonal demyelinating lesions was significantly reduced, suggesting that sNfL may originate more often in the peripheral nervous system
in pathological conditions 。 When the blood-nerve barrier (BNB) and the blood-cerebrospinal fluid barrier (BCB) are disrupted, axonal damage to nerve roots releases NfL into cerebrospinal fluid; When BNB/BCB exacerbates extensively, NfL in peripheral blood can also enter the cerebrospinal fluid system
.
In other peripheral neuropathy, any change in sNfL level reflects only a change in
neuropathy.
Therefore, sNfL levels may be more sensitive
than cNfL in monitoring peripheral nervous system disease activity and tissue damage.

In summary, the evidence suggests that sNfL quantification can be used as a very promising biomarker for dynamic measurement of axon damage, dynamic monitoring of peripheral nervous system disease activity, and future clinical trials and monitoring of therapeutic effects
.