Background Lewy bodies and neurites (LN), the two pathological hallmarks of Parkinson’s disease (PD), are found in the enteric nervous system (ENS). Previously, we have shown that whole mounts of submucosa obtained after microdissection of colonic biopsies can be used for the detection of LN in the submucosal plexus (SMP) of PD patients. Recent reports suggest that Lewy pathology may extend beyond the submucosa to involve the digestive mucosa. The aim of the present research was to determine whether the analysis of the mucosa obtained after microdissection may help improve the sensitivity of colonic biopsies to detect Lewy pathology in the colon of PD patients.
Methods Nine PD patients and 10 controls were included. Four biopsies were taken from the sigmoid/descending colon junction during the course of a rectosigmoidoscopy (short colonoscopy) in PD patients and during a total colonoscopy for colorectal screening in controls. Biopsies were microdissected, the mucosa was separated from the submucosa and both structures were analyzed by immunohistochemistry. Immunohistochemical analysis was performed using antibodies against phosphorylated alpha-synuclein to detect LN and neurofilaments NF200 kDa to label the neuronal structures.
Key Results Lewy neurites were present in the SMP of four patients and in the mucosa of three patients. Remarkably, among the patients who displayed LN within their mucosa, one was devoid of Lewy pathology in his SMP. No LN were observed in the mucosa and the SMP of controls.
Conclusions & Inferences The parallel analysis of colonic mucosa, along with the SMP, can help detect Lewy pathology in PD.
The degenerative process in Parkinson’s disease (PD) is characterized by the development of specific intraneuronal inclusions termed Lewy neurites (LN) in dendrites and axons and Lewy bodies in the somata.1 The main component of Lewy bodies and LN is aggregated and phosphorylated alpha-synuclein.2 Clearly, it has now been established that Lewy pathology affects the enteric nervous system (ENS).3–5 Lewy pathology has indeed been described in both the myenteric and submucosal plexus (SMP) in a large proportion of PD patients.3 Combining routine colonic biopsies and microdissection techniques,6 we have shown that whole mounts of submucosa can be used for a comprehensive assessment of the SMP thereby allowing identification of LN in 65% of PD patients when biopsies of ascending colon were analyzed and in 42% when biopsies of descending colon were performed.7 This led us to propose that the ENS is an original source of histopathological marker and a unique window to assess the neuropathology in living PD patients.
Comprehensive autopsy surveys performed by Braak and collaborators have shown that terminal ramifications of LN can, in some cases, cross the muscularis mucosa to reach the lamina propria and to ramify in the gastric glands.4,8 This suggests that the analysis of the mucosa in parallel with the submucosa could increase the sensitivity of colonic biopsies for detecting Lewy pathology. To test this hypothesis, mucosa and submucosa of PD patients, microdissected from colonic biopsies, were analyzed and compared for the presence of Lewy pathology.
Patients and Methods
Nine PD patients aged 40–75 years were enrolled. Diagnosis was made according to the United Kingdom PD Survey Brain Bank.9 Ten healthy patients requiring a total colonoscopy for colorectal cancer screening were included as controls. None of the control subjects had a history of neurological or psychiatric diseases. All controls underwent a neurological examination to rule out PD symptoms and cognitive deficiency. The study protocol was performed according to the Declaration of Helsinki, approved by the local Committee on Ethics and Human Research and registered on ClinicalTrials.gov (identifier NCT00491062). Written informed consent was obtained from each patient.
Rectosigmoidoscopy and colonoscopy biopsies
For PD patients, a rectosigmoidoscopy (short colonoscopy) was performed using a standard 130-cm flexible colonoscope that was introduced as far as tolerated by the patient. This allowed us to perform four biopsies at the junction between sigmoid and descending colon or in the distal part of the descending colon.10 Control subjects underwent total colonoscopy for colorectal screening during, which four biopsies were taken in the distal part of the descending colon. Biopsies were performed using standard biopsy forceps without needles (FB210K; Olympus co., Japan). Samples were immediately immersed in 4 °C saline solution and microdissected.
Microdissection and immunohistochemistry
Microdissection was performed as previously described.6 Briefly, biopsies were transferred in a Sylgard-coated Petri dish filled with 4 °C Hank’s buffered salt solution then stretched and pinned flat under a stereomicroscope with the mucosa oriented on the bottom of the dish. The submucosa was then mechanically separated from the mucosa with watchmaker’s forceps. Both submucosa and mucosa were fixed in phosphate buffered saline (PBS) with 4% paraformaldehyde for 3 h at room temperature or overnight at 4 °C. After fixation, the samples were rinsed three times for 10 min with PBS and kept at 4 °C in PBS with 1% sodium azide (PBS/NaN3) until further use.
Each whole-mount preparation of mucosa and submucosa obtained from a single biopsy were permeabilised for 3 h in PBS/NaN3 containing 1% Triton X-100 and 10% horse serum, and then incubated with the antibodies against phosphorylated alpha-synuclein (1 : 5000; WAKO, Osaka, Japan) and neurofilament 200 kDa (NF, 1 : 500; Chemicon, USA). Suitable secondary antibodies conjugated to Alexa Fluor 488, 594 and 647 were used (Invitrogen, Cergy-Pontoise, France). Specimens of submucosa and mucosa were viewed under a Zeiss Axiovert 200 m microscope fluorescence microscope.
Table 1 shows the main clinical features and pathological scores of all nine patients. Age and sex did not differ significantly between patients and the 10 control subjects (PD group: mean age 61.7 ± 7.6 years, four male; control group: mean age 59 ± 7.1 years, five male). For each subject, the four biopsies performed were stained with antibodies against phosphorylated alpha-synuclein and NF. A sample, either submucosa or mucosa, was considered positive when containing at least one LN immunoreactive for phosphorylated alpha-synuclein. Four out of nine PD patients had at least one positive biopsy when the SMP was analyzed (Table 1 and Fig. 1A, B, C), whereas three patients had at least one positive biopsy when the mucosa was screened for Lewy pathology (Table 1 and Fig. 1D, E, F). Among the three patients with a positive biopsy in the mucosa, one did not display alpha-synuclein immunoreactive lesions in the SMP (Table 1). No positive samples in the submucosa or mucosa were observed in controls. Morphological features of phospho-alpha-synuclein immunoreactive neurites were similar between mucosa and submucosa, with a dot-like aspect (Fig. 1B, D).
Table 1. Main clinical characteristics and immunohistochemical findings in patients. DD: disease duration; SMP: number of positive (containing at least one LN) samples out of 4 in the submucosal plexus; mucosa: number of positive (containing at least one LN) samples out of 4 in the mucosa
Using routine colonic biopsies and microdissection techniques, our study demonstrates that the mucosa should be analyzed along with the submucosa to detect Lewy pathology in living PD patients. Four out nine PD patients displayed LN within their SMP when biopsies were performed at the sigmoid/descending colon junction, a proportion comparable to that of our previous studies.7,11 The parallel analysis of the corresponding mucosa enabled to detect LN in three patients. Remarkably, one patient displayed LN in 3 out of 4 mucosa samples, whereas his submucosa was devoid of pathology.
Firstly, our results have implications regarding the pathophysiology of PD. The dendrites and axons found in the mucosa, which regulates secretion and microvasculature, originate primarily from the SMP and to a much lower extent from the myenteric plexus.12–14 The presence of widespread Lewy pathology in the mucosa along with absence or rarity of LN in the SMP in two patients (patients 1 and 6) suggests that in some cases, alpha-synuclein aggregates occur primarily in the most distal part of the neurites. This is consistent with a recent thorough autopsy survey in which accumulation of alpha-synuclein in the distal axons of the cardiac sympathetic nervous system was more severe and antedated that of neuronal somata or neurites in the paravertebral sympathetic ganglia.15 As a whole, this suggests that neurodegeneration of the autonomic nervous system and of the ENS could, in some cases, follow a centripetal pattern.
Secondly, our results have direct consequences on the use of colonic biopsies as a source of biomarker for PD.16,17 We have set up a microdissection technique that allows a comprehensive morphological analysis of the SMP and an accurate appraisal of Lewy pathology.6 Using this approach, LN were found in 65% of patients when submucosa of ascending colonic biopsies were analyzed, dwindling to 42% and 23% for biopsies of the descending colon and rectum, respectively.7 Rectosigmoidoscopy or short colonoscopy is a safer and easier procedure than total colonoscopy. When performed with a 130-cm flexible colonoscope, it allows to reach the junction between the sigmoid and the descending colon in almost all patients.10 Therefore, we took advantage of this procedure to determine whether LN can also be retrieved from the sigmoid/descending colon mucosa to improve the sensitivity of descending colon biopsies as a histopathological marker for PD. In one patient out of 9 (patient 1), the appraisal of the mucosa allowed to rectify the pathological diagnosis. This suggests that the analysis of the mucosa may be warranted when the assessment of the sole SMP does not show obviously detectable Lewy pathology.
Work in Michel Neunlist’s lab is supported by France Parkinson, FFPG (Fédération française des groupemets de parkinsoniens) and Parkinsoniens de Vendée. We are indebted to Philippe Hulin and the Cellular and Tissular Imaging Core Facility of Nantes University (MicroPICell) for the microscopy study.
This work was supported by a biomarker grant from the Michael J Fox Foundation for Parkinson’s research (to MN and PD) and by a grant from Nantes University Hospital (Direction de la Recherche Clinique, to PD).
The authors have no competing interests.
HP, TL, MN and PD designed the research study, performed the research and analyzed the data; EC and SBV performed the rectosigmoidoscopy and helped in analyzing the data; HP, TL and PD wrote the manuscript.