Deciduous DPSCs Ameliorate MPTP-Mediated Neurotoxicity, Sensorimotor Coordination and Olfactory Function in Parkinsonian Mice

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder defined by progressive deterioration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Dental pulp stem cells (DPSCs) have been proposed to replace the degenerated dopaminergic neurons due to its inherent neurogenic and regenerative potential. However, the effective delivery and homing of DPSCs within the lesioned brain has been one of the many obstacles faced in cell-based therapy of neurodegenerative disorders. We hypothesized that DPSCs, delivered intranasally, could circumvent these challenges. In the present study, we investigated the therapeutic efficacy of intranasally administered DPSCs in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Human deciduous DPSCs were cultured, pre-labelled with PKH 26, and intranasally delivered into PD mice following MPTP treatment. Behavioural analyses were performed to measure olfactory function and sensorimotor coordination, while tyrosine hydroxylase (TH) immunofluorescence was used to evaluate MPTP neurotoxicity in SNpc neurons. Upon intranasal delivery, degenerated TH-positive neurons were ameliorated, while deterioration in behavioural performances was significantly enhanced. Thus, the intranasal approach enriched cell delivery to the brain, optimizing its therapeutic potential through its efficacious delivery and protection against dopaminergic neuron degeneration.

Keywords: MPTP; Parkinson’s disease; behavioural analysis; dental pulp stem cells; intranasal delivery; tyrosine hydroxylase.

Figure 1

(i) Primary culture images obtained from 6 assays displaying the morphology of DPSCs (Magnification 4×; phase contrast images) (ii) Images of DPSCs expanded in FBS at subculture 3 (A: Magnification at 10×; B: Magnification at 20×; phase contrast images) (iii) Immunophenotype analysis of DPSCs expanded in FBS using flow cytometry. Cells were tested against human antigens CD34, CD45, CD73, CD90, CD166, and HLA-DR.

Figure 2

Detection of pluripotent indicators as well as neuronal markers. The Ct value of genes was analysed in the study using SYBR green-based qRT–PCR for DPSCs. Generally, the higher a fold change value, the more copies are present in the specific sample. Total RNA from brain was used as a positive control. Values are presented after normalization to 18s mRNA levels (p < 0.05).

Figure 3

Control, MPTP-induced and DPSC-administered MPTP mice were tested for sensorimotor coordination on the challenging beam traversal, spontaneous activity in cylinder and adhesive removal tests. (A) The time taken to traverse the beam, (B) the number of errors made per step, (C) the number of spontaneous rears made on the hindlimbs and (D) the time taken to make contact with the sensory stimuli were measured. MPTP mice took longer to traverse the beam and made more errors during steps compared with the control mice in the beam test. In addition, MPTP mice were less active in the cylinder test and were significantly slower to respond to sensory stimuli compared with the control mice in the adhesive removal test. However, performance was significantly improved in MPTP mice across all measures following DPSC delivery (red arrow) at Day 7 (p < 0.001). Values are expressed as mean ± SD.

Figure 4

Control, MPTP-induced and DPSC-administered MPTP mice were tested for olfactory function on the buried pellet test and the block test. The time taken to (A) discover the hidden pellet and (B–D) to discriminate between their own scent and to that of a conspecific were measured. The block test was divided into three levels with increased complexity. MPTP mice took longer to find the hidden pellet and recognize foreign odour when compared with control mice in the buried pellet and block tests respectively. However, olfactory function was significantly improved in MPTP mice across both tests following DPSC delivery (red arrow) at Day 7 (p < 0.001). Values are expressed as mean ± SD.

Figure 5

(A) Pictures displaying the effects of MPTP and intranasally administered DPSCs on tyrosine hydroxylase (TH) expression in the substantia nigra pars compacta (SNpc) of MPTP induced mice. (i–iii) Photomicrographs showing TH expression within the SNpc at low (5×), medium (10×) and high magnification (20×). (a): Control group at day 0 (b): Vehicle group: MPTP + saline at Day 7 (c) Treatment group: MPTP + DPSCs (two weeks after DPSC delivery) (d)Treatment group: MPTP + DPSCs (four weeks after DPSC delivery). The DPSCs labelled with PKH26 were delivered to MPTP mice at Day 7. Mice were lesioned with MPTP at Day 0. (B) Survival and migration of the PKH-labelled DPSCs within the SNpc following intranasal administration. PKH26 fluorescence visualization proved the existence of cell deposits in the SN (red arrow) of all grafted animals, indicating cell survival for at least four weeks after intranasal delivery (photomicrographs at high (20×) magnification).