Increased Cerebrospinal Fluid Adenosine 5′-Triphosphate Levels in Patients with Guillain–Barré Syndrome and Chronic Inflammatory Demyelinating Polyneuropathy

Background Extracellular adenosine 5′-triphosphate (ATP) acts as a signaling molecule in the peripheral nerves, regulating myelination after nerve injury. The present study examined whether the cerebrospinal fluid (CSF) ATP levels in patients with Guillain–Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are related to disease severity. Methods CSF ATP levels in 13 patients with GBS and 18 patients with CIDP were compared with those in a control group of 16 patients with other neurological diseases (ONDs). In patients with CIDP, CSF ATP levels were compared before and after treatment. The correlations between CSF ATP levels and other factors, including clinical data and CSF protein levels, were also evaluated. Results Median CSF ATP levels were significantly higher in patients with GBS and CIDP than in those with ONDs. When patients with CIDP were classified into two groups depending on their responsiveness to immunotherapy, median CSF ATP levels were significantly higher in good responders than in ONDs. CSF ATP levels tended to decrease after treatment in patients with CIDP. In patients with CIDP, there is a negative correlation between CSF ATP and CSF protein levels. Conclusions CSF ATP levels were increased in patients with GBS and CIDP. In particular, CSF ATP levels tended to decrease following treatment in patients with CIDP. CSF ATP levels may be useful biomarkers for the diagnosis or monitoring of therapeutic effects in patients with GBS and CIDP.


Introduction
Guillain-Barré syndrome (GBS) and chronic infammatory demyelinating polyneuropathy (CIDP) are both immunemediated demyelination neuropathies.In GBS, leukocytes infltrate peripheral nerves via an autoimmune mechanism associated with antecedent infection, leading to the rapid progression of limb paralysis and sensory disturbance [1].CIDP is characterized by slowly progressive limb weakness and sensory disturbances lasting for >2 months.Although the immunological pathogenesis of this disease remains poorly understood, evidence suggests that aberrant cellular and humoral immunity cause peripheral nerve demyelination and axonal injury [2].Although patients with GBS and CIDP sometimes present with severe symptoms and poor prognosis, these are treatable diseases, for which early diagnosis and treatment are very important.Te diagnosis of these diseases is predominantly based on clinical symptoms and electrophysiological examinations; however, blood and cerebrospinal fuid (CSF) biomarkers can also be useful.Several blood and CSF proteins associated with demyelination have been investigated as biomarkers of GBS and CIDP.For example, CSF sphingomyelin levels, which are useful diagnostic biomarkers, are elevated in patients with acquired demyelinating neuropathies [3].In patients with GBS, serum neuroflament light chain (NfL) levels are correlated with clinical outcomes [4].Similarly, serum NfL levels were found to be increased in patients with CIDP and were decreased during the remission period [5].However, there are still no established biomarkers to refect disease severity or monitor the therapeutic efects in patients with GBS and CIDP.
Extracellular adenosine 5′-triphosphate (ATP) acts as a signaling molecule in the peripheral nervous system by binding to its specifc receptor.Under physiological conditions, neurons release ATP, which regulates myelination by activating Schwann cell proliferation via action on purinergic receptors.Under pathological conditions, ATP is released from dying cells and regulates myelination following nerve injury [6].Te relationship between CSF ATP levels and the pathophysiology of several neurological diseases has been investigated.In patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), CSF ATP levels have been found to refect disease severity and treatment efcacy [7].Other studies have further reported that CSF ATP levels are elevated in patients with neuromyelitis optica spectrum disorder (NMOSD), which induces neuropathic pain [8].Furthermore, elevated CSF ATP levels have been associated with disease severity in amyotrophic lateral sclerosis [9].However, the relationship among CSF ATP levels, disease severity, and response to treatment has not been investigated in GBS and CIDP.Te present study examined whether CSF ATP levels in patients with GBS and CIDP are related to disease severity or other clinical factors.

Patients and Ethics.
Tirteen patients with GBS and eighteen patients with CIDP admitted to the Toyama University Hospital between 2008 and 2022 were enrolled.GBS was diagnosed according to the Brighton criteria [10].All patients fulflled the diagnostic certainty level of 1 or 2 and tested positive for antiganglioside antibodies.Patients with CIDP were newly diagnosed according to the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) 2010 criteria [11] and had no history of prior immunotherapy.To compare CSF ATP levels before and after treatment in patients with CIDP, initial CSF specimens were sampled at the time of diagnosis, prior to the induction of immunotherapy at the Toyama University Hospital.Posttreatment CSF was collected when the patients were readmitted for the second course of immunotherapy.Te median interval between the CSF collections was 5 months.CSF samples from patients with other neurological diseases (ONDs) (n � 16), including idiopathic normal-pressure hydrocephalus (iNPH, n � 11), familial amyloid polyneuropathy (n � 4), and neuropathy with a liability to pressure palsy (n � 1), were used as controls.As CSF ATP levels increase due to pleocytosis and blood contamination, CSF samples collected by traumatic lumbar punctures were excluded.Tis study was conducted in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and was approved by the Ethics Committee of the University of Toyama (approval no.29-32).Written informed consent was obtained from all the patients.

Measurement of the
Extracellular ATP Levels.CSF samples were obtained from lumbar puncture and stored at −80 °C until measurement.A highly sensitive and automated ATP measurement device was used to measure extracellular ATP levels [12].Luciferin-luciferase reagent HS from the Lucifell HS Set (Kikkoman Biochemifa Co., Ltd., Tokyo, Japan) was selected as the luminescent reagent and diluted 10-fold with distilled water.Te CSF specimens were diluted 20-fold with distilled water before measurement to prevent the inhibitory efect of high concentrations of chloride ions on the luminescent reagent.Luminescence was measured using a luminometer for 10 s after the addition of 50 µL of luminescent reagent to 10 µL of the diluted specimen.Extracellular ATP levels were quantifed as the average relative light intensity (counts/s).Measurements were performed in triplicate for each sample.A calibration curve for the luminescence and ATP concentrations were obtained using a 10-fold dilution series of a standard ATP solution adjusted with a saline solution diluted 10-fold with distilled water.ATP concentrations were further calculated from the luminescence values obtained from the calibration curve [7].To confrm the precision and detection limit of the assay, we measured ATP standard solutions prepared using commercial ATP and CSF collected from patients using iNPH as the diluent.Tese results indicated that the measurement error reached up to 2.4%, while the ATP quantifcation limit in the CSF was at least 2.0 × 10 −11 mol/L.

Statistical Analysis.
Statistical tests were performed using the JMP Ⓡ Pro software 2019 ver 15.0 (SAS Institute Inc., Cary, NC, USA).Te Wilcoxon test with Bonferroni correction was used for comparison between groups.Fisher's exact test was used to compare the diferences in frequency distribution according to sex.Spearman's test was used to examine the correlations between variables.Te Wilcoxon signed-rank test was used to compare the paired data.Results were considered signifcant when the p values were <0.05.

Clinical Characteristics and CSF Profles of Patients with GBS and CIDP.
Te median ages of patients with GBS and CIDP were 49 and 62.5 years, respectively, both of which were lower than that of patients with ONDs.Patients with GBS were classifed according to Ho's electrodiagnostic criteria [13]; four cases (30.8%) were demyelinating, four  4).CSF ATP levels before and after treatment were compared in 11 patients with CIDP for whom CSF samples were available.Te CSF ATP levels tended to decrease in each patient with CIDP after treatment, although the diference did not reach statistical signifcance (Figure 5).

Discussion
Tis study demonstrated that CSF ATP levels were significantly higher in patients with GBS and CIDP than in controls, while CSF ATP levels were higher in patients with CIDP who responded well to immunotherapy and tended to decrease in each patient after treatment.To the best of our knowledge, this is the frst study to evaluate the CSF ATP levels in patients with GBS and CIDP.Previous studies reported that extracellular ATP is involved in the regulation of myelination after nerve injury.For example, extracellular ATP levels are increased in in vitro Wallerian degeneration models, where they inhibit myelin sheath degradation [14].Extracellular ATP secreted from degenerating neurons elicited an increase in cytosolic Ca 2+ levels in Schwann cells and activated Schwann cell regeneration in an in vitro model of the Miller Fisher syndrome, a subtype of GBS [15].In contrast, in vitro experiments have shown that a high concentration of extracellular ATP activates microglia and astrocytes by binding to specifc receptors and inducing neurotoxic infammation [16].Terefore, extracellular ATP receptor antagonists have shown to have potential as novel therapies for reducing microglial activity and neuroinfammation in several neurological diseases including GBS [17], epilepsy [18], Parkinson's disease [19], and Alzheimer's disease [20].Tese fndings suggest that increased extracellular ATP acts as a damage-associated molecule and promotes peripheral nerve regeneration or causes peripheral nerve damage in some demyelinating neuropathies such as GBS and CIDP.Although no previous studies have measured the CSF ATP levels in patients with infammatory neuropathy, Ishikura et al. measured CSF ATP levels in patients with NMOSD, an infammatory spinal cord disease [8].In that report, the mean CSF ATP level in patients with NMOSD was approximately 8,000 pmol/L, which was higher than that in our study (mean 1,853 pmol/L in patients with GBS and 3,209 pmol/L in patients with CIDP).Tis suggests that CSF ATP levels may vary depending on the degree and location of infammation.
In the present study, we identifed a negative correlation between CSF ATP and CSF protein levels in patients with CIDP although there was no correlation between CSF ATP levels and the MRC sum score or clinical scores, such as Hughes grade and INCAT, in patients with GBS and CIDP.Albuminocytological dissociation provided evidence supporting the diagnosis of CIDP [21].As albuminocytological dissociation indicates a disruption of the blood-nerve barrier due to nerve infammation [22], high CSF protein levels may suggest a high degree of nerve infammation and nerve injury.Furthermore, because high CSF ATP levels likely occur as a result of ATP secretion from injured neurons, a positive correlation is expected between CSF ATP levels and CSF protein levels.Although the reason for this negative correlation is unclear, the small number of patients may be the main reason.

Neurology Research International
In addition, CSF ATP levels tended to decrease after immunotherapy in patients with CIDP.Tis indicates that CSF ATP levels may be a therapeutic biomarker in patients with CIDP.To date, no therapeutic biomarkers have yet been established for immunotherapy in patients with CIDP.NfL is an abundant cytoskeletal component axons and a useful biomarker for peripheral nervous system diseases [23].Kapoor et al. further reported that the plasma NfL concentration was higher in patients with CIDP than in healthy controls and was signifcantly reduced after IVIg therapy [24].As they also showed that plasma NfL concentration was signifcantly higher in unstable patients with CIDP than in stable patients during IVIg treatment, plasma NfL was considered to be a useful biomarker refecting axonal injury in patients with CIDP.Sphingomyelin (SM), a myelin-rich lipid, is a component of myelin sheaths.In the CSF of patients with acquired demyelinating neuropathies, several pathological products are increased owing to their release from nerve roots or fow from peripheral blood, which is attributed to the disruption of the blood-nerve barrier that refects damage to peripheral nerves [25].In patients with GBS and CIDP, CSF sphingomyelin levels were found to be increased compared to those in patients with nondemyelinating neurological diseases and were further higher in patients with active CIDP than in those with stable disease [26].Tese data suggest that CSF sphingomyelin acts as a diagnostic and severity biomarker of GBS and CIDP.Compared with NfL and sphingomyelin, both of which indicate peripheral nerve injury, CSF ATP levels are elevated upon remyelination and may be a marker of remyelination.CSF ATP levels may be more useful than NfL and sphingomyelin levels in predicting therapeutic efcacy in patients with CIDP.
Tis study had some limitations.First, the sample size was relatively small, and the relationships between CSF ATP levels and clinical scores, disease severity, and electrophysiological examinations in patients with GBS and CIDP could not be elucidated.Furthermore, type 2 errors were likely to occur because of small sample sizes.Second, there were no data on the correlation between CSF ATP levels and other biomarkers such as CSF NfL.If elevated CSF ATP levels are associated with peripheral nerve regeneration, then a negative correlation may be observed between CSF ATP  and NfL levels in patients with GBS and CIDP.Tird, multivariate correlation analysis was not performed because of the small number of cases and due to the potential for unknown confounding factors that afect CSF ATP concentration, such as age and underlying disease.Fourth, the change in CSF ATP levels in patients with CIDP was only measured between two time points, before and after treatment, and there were no data on the long-term follow-up.As this was a pilot exploratory study, continuous sampling over longer treatment periods in larger cohorts is required to demonstrate the utility of CSF ATP levels as a therapeutic biomarker in patients with CIDP, similar to other biomarkers such as NfL and sphingomyelin.

Conclusion
Overall, the present study showed that CSF ATP levels were increased in patients with GBS and CIDP.CSF ATP levels may be useful biomarkers for the diagnosis or monitoring of therapeutic efects in patients with GBS and CIDP.

Figure 2 :Figure 3 :Figure 4 :Figure 5 :
Figure 2: Correlations between the CSF ATP levels and MRC sum score (a), Hughes grade (b), and CSF TP (c) in patients with GBS.Spearman's rank correlation was used for analysis.

Table 1 :
Demographics of the ONDs and patients with GBS CIDP.

Table 2 :
CSF data of the ONDs and the patients with GBS and CIDP.
*Te GBS group included patients whose CSF samples were collected within four weeks.Te CIDP group included patients whose CSF samples were collected prior to immunotherapy.Data are presented as the median (interquartile range).* p < 0.05 (Wilcoxon test with Bonferroni correction).