Vagus nerve stimulation improves locomotion and neuronal populations in a model of Parkinson's disease

Abstract

Background: Parkinson's disease (PD) is a progressive, neurodegenerative disorder with no disease-modifying therapies, and symptomatic treatments are often limited by debilitating side effects. In PD, locus coeruleus noradrenergic (LC-NE) neurons degenerate prior to substantia nigra dopaminergic (SN-DA) neurons. Vagus nerve stimulation (VNS) activates LC neurons, and decreases pro-inflammatory markers, allowing improvement of LC targets, making it a potential PD therapeutic.

Objective: To assess therapeutic potential of VNS in a PD model.

Methods: To mimic the progression of PD degeneration, rats received a systemic injection of noradrenergic neurotoxin DSP-4, followed one week later by bilateral intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine. At this time, a subset of rats also had vagus cuffs implanted. After eleven days, rats received a precise VNS regimen twice a day for ten days, and locomotion was measured during each afternoon session. Immediately following final stimulation, rats were euthanized, and left dorsal striatum, bilateral SN and LC were sectioned for immunohistochemical detection of monoaminergic neurons (tyrosine hydroxylase, TH), α-synuclein, astrocytes (GFAP) and microglia (Iba-1).

Results: VNS significantly increased locomotion of lesioned rats. VNS also resulted in increased expression of TH in striatum, SN, and LC; decreased SN α-synuclein expression; and decreased expression of glial markers in the SN and LC of lesioned rats. Additionally, saline-treated rats after VNS, had higher LC TH and lower SN Iba-1.

Conclusions: Our findings of increased locomotion, beneficial effects on LC-NE and SN-DA neurons, decreased α-synuclein density in SN TH-positive neurons, and neuroinflammation suggest VNS has potential as a novel PD therapeutic.

Keywords: Dopamine; Locus coeruleus; Norepinephrine; Parkinson's disease; Substantia nigra; Vagus nerve.

Figure 1. Experimental design timeline

Rats were randomly given either DSP-4 (50 mg/kg, i.p.) or saline. Seven days post-injection, the rats underwent vagus nerve cuff implantation, followed by intracranial 6-OHDA administration (5μg/μl, 2μl/site) or saline. Starting 11 days after lesioning, the rats with a vagus cuff received stimulation 2x a day for 30min each session. All animals, regardless of treatment group, underwent locomotor assessment (LA) during the pm session. After 10 days of stimulation, the rats were euthanized and brains processed for immunohistochemistry (IHC).

Figure 2. Locomotion was increased in lesioned rats with VNS

Total distance was measured in cm, and locomotion across all ten days shows decreased locomotor activity in lesion non-VNS animals compared to saline non-VNS animals (A). Average total distance traveled across the ten days was analyzed and displayed as a scatter plot with the group mean +SEM denoted by lines and error bars (B). Lesion non-VNS rats displayed less locomotion than saline non-VNS (***p<0.001), whereas VNS resulted in increased locomotion after lesion (*p<0.05).

Figure 3. VNS effects on TH in the LC, striatum, and SN of lesioned rats

Photomicrographs of LC (A–D, scale=100μm), striatum (E–H, scale=1mm), and SN (I–L, scale=0.5mm). Saline VNS rats had greater TH-positive cell counts than saline non-VNS rats in the LC (A–B, **p<0.01), but TH-ir was not increased in the striatum (E–F), nor increased TH-positive neurons in the SN of saline VNS rats (I–J). TH was significantly lower in lesion non-VNS rats in all three brain regions (LC counts: C, ****p<0.0001; striatal TH-ir: G, ****p<0.0001; SN counts: K, ***p<0.001). Lesion VNS rats had greater TH in all three regions (LC counts: D, **p<0.01; striatal TH-ir: H, **p<0.01; SN counts: L, **p<0.01). TH results are quantified in M (LC), N (Striatum), and O (SN).

Figure 4. α-synuclein immunoreactivity is reduced by VNS in SN TH-positive cells of lesioned rats

Lesion non-VNS rats had a phenotypic loss of TH compared to saline non-VNS rats (A, C), although lesion VNS rats had comparable DAPI staining to saline non-VNS rats (A, D). Lesion non-VNS rats also display greater overall α-synuclein-ir in remaining TH-positive neurons compared to saline non-VNS rats (E, G, *p<0.05). VNS treatment did not significantly alter α-synuclein-ir in saline rats (F), and lowered α-synuclein-ir in lesioned rats compared to lesion non-VNS rats (H, *p<0.05). The inset images in E–H show the localization of α-synuclein in the TH-positive cell denoted with the box. As can be seen in G, α-synuclein is localized to the TH-positive cell body in lesion non-VNS rats, with no visible localization of α-synuclein to TH-positive cells in the other treatment groups. Scale for A–D (shown in A) = 10μm. Scale for E–H (shown in E) = 25μm. Quantification of results in I.

Figure 5. GFAP-positive astrocytes were reduced by VNS in the LC and SN of lesioned rats

The measurement area for stereology is outlined for the LC (A) and the SN (E). Photomicrographs of LC (A–D, scale=50μm) and SN (E–H, scale=100μm). VNS did not alter GFAP-positive astrocytes in saline animals (LC: A, B; SN: E, F). Lesioned rats had significantly more GFAP-positive astrocytes than saline rats (LC: C, *p<0.05; SN: G, ****p<0.0001). Total astrocyte counts in lesioned rats were lower after VNS (LC: D, *p<0.05; SN: H, ***p<0.001). Quantification of total GFAP-positive cells in I (LC) and J (SN). Insets demonstrate primarily resting state astrocytes in saline and lesion VNS rats (A, B, D, E, F, H); however, in lesion non-VNS rats more astrocytes are activated than in the other groups (C, G).

Figure 6. Iba-1-ir was reduced by VNS in the LC and SN of lesioned rats

The measurement area for microglia density analysis is outlined for the LC (A) and the SN (E). Photomicrographs of LC (A–D, scale=50μm) and SN (E–H, scale=100μm). VNS had no effect on Iba-1-ir in the LC of saline rats (A, B), but resulted in reduced Iba-1-ir in the SN (E, F, *p<0.05). Lesioned rats had significantly higher Iba-1-ir (LC: C, **p<0.01; SN: G, ****p<0.0001). VNS reduced Iba-1-ir in lesioned rats (LC: D, **p<0.01; SN: H, ***p<0.001). Quantification of results in I (LC) and J (SN).