Modelling motor and non-motor signs of early-stage Parkinson's disease

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Abstract

Introduction. As Parkinson's disease (PD) develops, a number of non-motor signs precede motor symptoms, including gastrointestinal tract dysfunction. Modelling early-stage PD to comprehensively assess the pattern of morphofunctional changes in the gastrointestinal tract is important in order to develop methods of early disease diagnosis and more effective treatment of autonomic disturbances that are typical in PD, and to increase the patients' quality of life.

Study aim — to offer a model of early-stage PD through long-term oral administration of small doses of the neurotoxin rotenone to rats, and to study the functional and immunohistochemical changes in the gastrointestinal tract of the experimental animals, as well as changes in the substantia nigra.

Materials and methods. The experiment was conducted in male Wistar rats aged 3.0–3.5 months. The study group rats (n = 10) were given rotenone orally at a dose of 5 mg/kg, as a suspension in a 4% carboxymethyl cellulose solution, every second day for 7 months. The control group rats (n = 10) received only the 4% carboxymethyl cellulose solution.

The animals' mobility was assessed at the start and end of the experiment using the open field and narrowing beam-walking test. Gastrointestinal motility was assessed by measuring the passage of dye from the pylorus in a caudal direction along the small intestine. The rats were decapitated and immunohistochemistry was used to assess the density of dopamine neurons in the substantia nigra, nerve fibres, and glia in the Auerbach's plexus of the small intestine, and the location of the total and phosphorylated alpha-synuclein in the enteric nervous system.

Results. Rats in the study group had a statistically significant reduction in the number of dopamine neurons in the substantia nigra. Auerbach's plexus of the small intestine contained significantly less nerve fibres and glia, while fluorescence intensity for alpha-synuclein was increased. Phosphorylated alpha-synuclein was identified in the cholinergic and adrenergic fibres of Auerbach's plexus. Experimental animals had a statistically significant reduction in the gastric emptying rate and small intestine motility compared to the control group.

Conclusion. The presented model of early-stage PD enables the physiological and immunohistochemical symptoms of gastrointestinal dysfunction, similar to that of patients with PD, to be replicated. They are based on intestinal denervation changes and accumulation of abnormal forms of alpha-synuclein in the enteric nervous system.

About the authors

Mikhail V. Ivanov

Research Center of Neurology

Author for correspondence.
Email: ivanov@neurology.ru
ORCID iD: 0000-0001-5947-9093

junior researcher, Laboratory of neuromorphology

Russian Federation, 125367, Moscow, Volokolamskoye shosse, 80

Kristina A. Kutukova

Research Center of Neurology

Email: Chrisbiomag@mail.ru
ORCID iD: 0000-0002-5483-9157

junior researcher, Laboratory of neuromorphology

Russian Federation, 125367, Moscow, Volokolamskoye shosse, 80

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2. Fig. 1. Motor and neurodegenerative changes in rats after long-term administration of rotenone. A — number of paw slips with right and left limbs (% of total number of steps) in the control group and the rotenone group in the narrowing beam-walking test; B — number of errors in the narrowing beam-walking test, points; C — spread of Evans Blue dye from the pylorus caudally along the small intestine, 20 minutes after oral dye administration, cm; D — number of TH-positive neurons in pars compacta of the substantia nigra per field of view; E — TH-positive neurons in pars compacta of the substantia nigra of control animals, × 10; F — TH-positive neurons in pars compacta of the substantia nigra of experimental animals, × 10; G — number of β-III-tubulin positive nerve fibres in the Auerbach's plexus of the small intestine per field of view; H — number of TH-positive nerve fibres in the Auerbach's plexus of the small intestine per 100 μm2.

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3. Fig. 2. Total alpha-synuclein (α-syn) in the enteric nervous system of rats after long-term administration of rotenone. A–D — the location of total α-synuclein in the β-III-tubulin positive structures of the enteric nervous system in an experimental animal, × 20; А — immunofluorescence staining of cell nuclei (DAPI, blue); В — immunofluorescence staining of β-III-tubulin (red); C — immunofluorescence staining of total α-synuclein (red); D — the result of overlaying images А–С. Areas of β-III-tubulin and total α-synuclein colocalization are yellow and orange. E — intensity of immunofluorescence staining of total α-synuclein in the Auerbach's plexus ganglion cells, C.U. *р < 0.05.

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4. Fig. 3. Location of phosphorylated α-synuclein (α-synP) in the Auerbach's plexus of the small intestine. А–С — colocalization of phosphorylated α-synuclein in cholinergic neurons of the Auerbach's plexus (yellow arrows indicate cell bodies with colocali- zation), × 20; D, E — immunofluorescence staining for VIP (green colour, green arrows) and phosphorylated α-synuclein (red colour, red arrows) in the Auerbach's plexus: no colocalization, × 40; F — immunofluorescence staining for TH (green) and phosphorylated α-synuclein (red) in the Auerbach's plexus: colocalization in individual fibres (yellow arrows), × 40; G — immunofluorescence staining for GFAP (red, red arrow) and total α-synuclein (green, green arrow) in the Auerbach's plexus ganglion cells: no colocalization, × 40; H — intensity of immunofluorescence staining for GFAP in the Auerbach's plexus ganglion cells, C.U. * — р < 0.05.

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Copyright (c) 2022 Ivanov M.V., Kutukova K.A.

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