Pathological Study Of Skin Small Nerve Fibers In Parkinson’s Disease

Objective To observe the pathological characteristics of small nerve fibers in the skin of patients with Parkinson’s disease(PD),to explore the relationship between the pathological changes of small skin fibers and clinical features in patients with PD,to find accurate and reliable early biomarkers of PD,and to further explore the possible pathogenesis of PD by taking the pathology of small nerve fibers in the skin as the starting point.Methods Sixteen patients and sixteen healthy controls with gender and age matching were admitted to the Department of Neurology,Suzhou Hospital of Anhui Medical University and Shanghai Sixth People’s Hospital,Shanghai Jiao Tong University School of Medicine from November 2020 to February 2023 who met the inclusion and exclusion criteria were included.Clinical data assessments including gender,age,course of disease,medication,Unified Parkinson’s Disease Rating Scale Part III(UPDRS-III),Hoehn-Yahr(H-Y)scale,nonmotor symptoms scale(NMSS),Pin Sensation of the Utah Early Neuropathy Scale(Utah),activities of daily living(ADL),Autonomic Scale for Outcomes in Parkinson’s Disease(SCOPA-AUT),Supine position test and bladder residual urine volume were collected from all subjects.Neurophysiological assessments were performed on all subjects in 2 groups,including distal motor latency(DML),compound muscle action potentials(CMAP),motor nerve conduction velocities(MCV);distal sensory latency(DSL),sensory nerve active potential(SNAP)and sensory nerve conduction velocities(SCV)of bilateral ulnar,median,tibial and common peroneal nerve.All subjects underwent skin biopsy on distal right lower extremity(10 cm above lateral malleolus).Small nerve fibers of the skin were stained by standardized pan-axonal marker protein gene product 9.5(PGP9.5),growth associated protein-43(GAP-43)and anti-serine 129 phosphorylation-α-syn(anti-p-Ser129 α-synuclein)antibodies stain by a variety of immunohistochemistry.The morphology of small nerve fibers was observed under optical microscope and Intraepidermal nerve fiber density(IENFD)was quantified.SPSS26.0 and Graph Pad Prism v.9 were used for statistical analysis.Inform all subjects of the skin biopsy procedure,sign the informed consent form,and pass the ethical review of Suzhou Hospital Affiliated to Anhui Medical University.All subjects were informed of the skin biopsy process,signed the informed consent form,and passed the ethical review of Suzhou Hospital of Anhui Medical University.Results1.A total of 16 patients with PD who were clinically confirmed and met the inclusion and exclusion criteria were collected in this study,including 7 males and 9 females,with an average age of(66.000 ± 9.675)years.There were 16 healthy controls,7 males and 9 females,with an average age of(60.625 ± 13.336)years.There were no significant differences in age or sex between the two groups.In PD group,the average H-Y grade was(1.906 ± 0.612),mean course was(1.850 ± 1.463)years,mean UPDRSIII score was(29.875 ± 9.208),and all patients were treated with levodopa.2.The DSL of ulnar,median and sural nerve in PD patients was longer than that of healthy control group(p<0.05),and there were no statistical differences in DML,CMAP amplitude,MCV,SNAP amplitude and SCV of ulnar,median and tibial nerve between the two groups(P > 0.05).3.The IENFD of skin specimen for PGP9.5 and GAP-43 staining in the PD group were lower than those in healthy control(8.618 ± 3.984 fibers/mm vs 18.198 ± 5.387 fibers /mm,4.543 ± 3.363 fibers/mm vs 14.174 ± 5.485 fibers /mm;p<0.05)。 4.The IENFD for PGP9.5 staining in the PD group and the healthy control group were positively correlated with those for GAP-43 staining(r = 0.937,p< 0.05).5.In PD group,IENFD for PGP9.5,GAP-43 and GAP-43/PGP9.5 ratios were not correlated with gender,age and disease course,but were correlated with the severity of the disease.6.In PD group,IENFD for PGP9.5 staining was only positively correlated with SNAP of median nerve(r = 0.782,p > 0.05),but no correlation was found with DML,CMAP amplitude,MCV,SNAP amplitude and SCV of ulnar,median,tibia and common peroneal nerve.The IENFD for GAP-43 staining was only positively correlated with tibial MCV(r = 0.952,p>0.05),and no correlation was found in other indicators.7.There was no significant difference in IENFD for GAP-43/PGP9.5 ratio between PD and healthy control(0.575 ± 0.380 vs 0.787 ± 0.218),and the correlation was only significant with UPDRS-III.8.A small amount of phosphorylated α-syn deposition was observed around nerve bundle and blood vessels in 2 patients with PD.No deposits were found in the muscle arrector pilorum or the sweat glands,and no phosphorylated α-syn deposition was found in the normal control group.There was no significant difference in the positive rate of phosphorylated α-syn deposition between the two groups(p > 0.05).9.In PD patients with positive phosphorylated α-syn deposition,the IENFD was(5.270 ± 0.877)fibers/mm for PGP9.5 staining and(2.960 ± 2.192)fibers/mm for GAP-43 staining.In PD patients with negative phosphorylated α-syn deposition,IENFD was(9.631 ± 3.756)fibers/mm for PGP9.5,and(6.106 ± 3.668)fibers/mm for GAP-43.IENFD for PGP9.5 and GAP-43 of PD patients with positive phosphorylated α-syn deposition was lower than those of negative,and the difference was not statistically significant(p>0.05).Conclusion 1.PD patients have peripheral nerve damage,mainly involving small nerve fibers,while large nerve fibers are not obviously involved.Degeneration and impaired regeneration of small nerve fibers in the skin of PD patients may precede the large nerve fibers.Skin biopsy is a reliable method for diagnosing small fiber neuropathy in patients with PD.2.IENFD for PGP9.5 and GAP-43 staining was correlated with the clinical and severity symptoms of PD.The determination of IENFD by skin nerve biopsy technology can detect PD as early as possible,give timely diagnosis and treatment,evaluate the course of the patient’s disease,and alleviate the pain of patient.3.After excluding various influencing factors,the ratio of IENFD under GAP-43/PGP9.5 staining may not be used as a potential marker for the occurrence and development of PD.Phosphorylated α-syn deposition in small nerve fibers of skin may lead to injury of small nerve fibers in PD,which may be the best biomarker for diagnosing PD in vivo,and provides a certain theoretical basis for the further exploration of pathogenesis of PD.