Updates in the pathophysiological mechanisms of Parkinson’s disease: Emerging role of bone marrow mesenchymal stem cells

Abstract

AIM: To explore the approaches exerted by mesenchymal stem cells (MSCs) to improve Parkinson’s disease (PD) pathophysiology.

METHODS: MSCs were harvested from bone marrow of femoral bones of male rats, grown and propagated in culture. Twenty four ovariectomized animals were classified into 3 groups: Group (1) was control, Groups (2) and (3) were subcutaneously administered with rotenone for 14 d after one month of ovariectomy for induction of PD. Then, Group (2) was left untreated, while Group (3) was treated with single intravenous dose of bone marrow derived MSCs (BM-MSCs). SRY gene was assessed by PCR in brain tissue of the female rats. Serum transforming growth factor beta-1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1) and brain derived neurotrophic factor (BDNF) levels were assayed by ELISA. Brain dopamine DA level was assayed fluorometrically, while brain tyrosine hydroxylase (TH) and nestin gene expression were detected by semi-quantitative real time PCR. Brain survivin expression was determined by immunohistochemical procedure. Histopathological investigation of brain tissues was also done.

RESULTS: BM-MSCs were able to home at the injured brains and elicited significant decrease in serum TGF-β1 (489.7 ± 13.0 vs 691.2 ± 8.0, P < 0.05) and MCP-1 (89.6 ± 2.0 vs 112.1 ± 1.9, P < 0.05) levels associated with significant increase in serum BDNF (3663 ± 17.8 vs 2905 ± 72.9, P < 0.05) and brain DA (874 ± 15.0 vs 599 ± 9.8, P < 0.05) levels as well as brain TH (1.18 ± 0.004 vs 0.54 ± 0.009, P < 0.05) and nestin (1.29 ± 0.005 vs 0.67 ± 0.006, P < 0.05) genes expression levels. In addition to, producing insignificant increase in the number of positive cells for survivin (293.2 ± 15.9 vs 271.5 ± 15.9, P > 0.05) expression. Finally, the brain sections showed intact histological structure of the striatum as a result of treatment with BM-MSCs.

CONCLUSION: The current study sheds light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.

Key Words: Parkinson’s disease; Pathophysiology; Bone marrow derived mesenchymal stem cells; Rotenone; Anti-inflammatory action; Ovariectomy; Anti-apoptotic effect; Neurogenic potential

Core tip: The current study was planned to clarify the mode of action of mesenchymal stem cells (MSCs) in targeting multiple systems implicated in the pathophysiology of Parkinson’s disease (PD) in the rat model. For this purpose, the MSCs were isolated from bone marrow (BM) of rat femur bone and PD was induced in ovariectomized rats by rotenone administration for 14 d. Our results provided clear evidences for the therapeutic role of BM-derived MSCs against PD pathophysiology through their immunomodulatory properties, anti-inflammatory and anti-apoptotic effects as well as neurotrophic and neurogenic potentials.

INTRODUCTION

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, associated with extrapyramidal motor dysfunction[1] due to the progressive and specific loss of dopaminergic neurons in the substantia nigra pars compacta and declining levels of dopamine (DA) in the striatum[2]. It affects approximately seven million people globally[3]. The commonness of PD raises with age, as 1% of people over 60 years of age, 3.4% of those over 70, and 4% of those over 80 were affected by the disease[1]. Epidemiological studies and pathological investigations exhibit a mean period of onset of 70 in sporadic PD, which represents about 95% of patients[4,5]; but familial forms of the disease linked to transformation in a limited number of genes account for 4% and these patients suffer from early-onset disease before the age of 50[6].

Growing body of evidences have demonstrated that environmental factors play a critical role in the etiology of PD[7]. For example, the environmental toxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was identified as one the causative agents of Parkinsonism[8]. Also, herbicides or pesticides usage increase the risk of PD[9,10]. As, the pesticide rotenone and the herbicide paraquat reproduce the PD phenotype in animals[11]. Additionally, it has been suggested that exposure to organic solvents, carbon monoxide and carbon disulfide[12] play roles in the etiology of PD. Epidemiological studies have proposed a potential link between pesticide exposure and increased risk of PD. For example, agrarian laborers, particularly individuals who work with pesticides, are at increased risk for suffering from PD[13].

At present, there is no therapy clinically accessible to postpone neurodegeneration, thusly modulation of the disease course is an imperative unmet clinical need. Along these lines, understanding of the pathophysiology and etiology of the disease at cellular and molecular levels to find new targets against which neuroprotective/disease-modifying therapy may be developed is the pivotal issue in the field of PD research[7].

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that have the capacity of self-renewal and differentiation into mesodermal lineage cells and other embryonic lineages, including adipocytes, osteocytes, chondrocytes, hepatocytes, neurons, muscle cells, epithelial cells, etc.[14]. Additionally, these cells have several advantages, such as easy availability as well as few ethical concerns and low immunogenicity. An expanding number of data has demonstrated that MSCs not only depend on their differentiation capacity to repair damaged tissue, but also rely on their ability to modify local environment, activate endogenous progenitor cells, and secrete several factors[15]. The aforementioned properties make MSCs perfect candidate cell type for tissue engineering, regenerative medicine and autoimmune disease treatment[14].

The focus of our interest was to clarify the mode of action of bone marrow derived MSCs (BM-MSCs) in targeting multiple systems implicated in the pathophysiology of PD in the rat model.

MATERIALS AND METHODS

Preparation of BM-MSCs

BM was harvested by flushing the tibiae and femurs of 6-wk-old male Sprague Dawley rats with Dulbecco’s modified Eagle’s medium (DMEM; GIBCO/BRL, Grand Island, New York, United States, Cat. #42430-082) supplemented with 10% fetal bovine serum (FBS; GIBCO/BRL, Cat. #16000-044). Nucleated cells were isolated with a density gradient [Ficoll/Paque (Pharmacia)] and resuspended in complete culture medium supplemented with 1% penicillin–streptomycin (GIBCO/BRL, Cat. #10378-016). Cells were incubated at 37 °C in 5% humidified CO₂ for 12-14 d as primary culture or upon formation of large colonies. When large colonies developed (80%-90% confluence), cultures were washed twice with phosphate buffer saline (PBS; Gibco/BRL, Cat. #10010056) and the cells were trypsinized with 0.025% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA) (Gibco/BRL, Cat. #R-001-100) for 5 min at 37 °C. After centrifugation, cells were resuspended with serum-supplemented medium and incubated in 50 mL falcon tube. The resulting cultures were referred to as first-passage cultures. MSCs in cultures were characterized by their adhesiveness and fusiform shape[16].

Experimental set up

Twenty four adult female Sprague-Dawley rats weighing 130-150 g were obtained from the Animal House Colony of the National Research Centre, Giza, Egypt and acclimated in a specific area where temperature (25 °C ± 1 °C) and humidity (55%). Rats were controlled constantly with a 12 h light/dark cycles at National Research Centre Animal Facility Breeding Colony. Rats were individually housed with ad libitum access to standard laboratory diet consisted of casein 10%, salt mixture 4%, vitamin mixture 1%, corn oil 10%, cellulose 5% and completed to 100 g with corn starch and tap water. Rats were cared for according to the guidelines for animal experiments which were approved by the Ethical Committee of Medical Research at National Research Centre, Giza, Egypt.

After the acclimatization period (2 wk), the female rats were ovariectomized surgically in Hormones Department, Medical Research Division at the National Research Centre. Then, after one month from ovariectomy the animals were classified into 3 different groups (8 rats/ group). The first group (Ovariectomized control group) was untreated ovariectomized control group. While, the second and third groups were subcutaneously injected with rotenone (Sigma, United States, Cat. #R8875) in a dose of 12 mg/kg b. wt.[17] daily for 14 d for induction of PD. Thereafter, the second group (PD untreated group) was left untreated for 4 mo while, the third group (PD + BM-MSCs group) was infused intravenously with a single dose (3 × 10⁶ cells/rat) of BM-MSCs[18]. For MSCs infusion, the PD induced rats were deeply anaesthetized via diethyl ether and MSCs were suspended in 100 μL PBS before transplantation and then slowly injected into the tail vein in 5 min with a 27G needle. The needle was kept in the tail vein for another 5 min to avoid regurgitation and then withdrawn.

At the end of the experimental period (4 mo), all animals were fasted for 12 h and the blood samples were collected from retro-orbital venous plexus under diethyl ether anaesthesia. The blood samples were left to clot and the sera were separated by cooling centrifugation (4 °C) at 1800 ×g for 10 min and then stored immediately at -20 °C in clean plastic Eppendorf until analyzed. Moreover, the whole brain of each rat was rapidly and carefully dissected. Then, each brain was sagittally divided into two portions. The first portion was immediately frozen in liquid nitrogen and stored at -80 °C prior to extraction for molecular study and DA level determination. While, the second portion was fixed in formalin buffer (10%) for histological investigation and immunohistochemical study.

Detection of male-derived MSCs in the brain of females

The genomic DNA was isolated from the brain tissues of female rats which were treated with BM-MSCs using phenol/chloroform extraction and ethanol precipitation method according to Sambrook et al[19] with minor modifications. The presence or absence of the sex determination region on the Y chromosome male (SRY) gene in recipient female rats was assessed by PCR. Primer sequences for SRY gene (forward 5′-CATCGAAGGGTTAAA-GTGCCA-3′, reverse 5′-ATAGTGTGTAGGTTGTTGTCC-3′, Invitrogen) were obtained from published sequences[20] and amplified to a product of 104 bp. The PCR conditions were as follows: Incubation at 94 °C for 4 min; 35 cycles of incubation at 94 °C for 50 s, 60 °C for 30 s, and 72 °C for 1 min; with a final incubation at 72 °C for 10 min. PCR products were separated using 2% agarose gel electrophoresis and stained with ethidium bromide.

Biochemical analyses

Serum transforming growth factor beta-1 (TGF-β1) level was assayed by enzyme linked immunosorbent assay (ELISA) using kit purchased from DRG Diagnostics Co., Germany (Cat. #EIA-1864), according to the method described by Kropf et al[21]. While, serum monocyte chemoattractant protein-1 (MCP-1) level was determined by ELISA method using kit purchased from Bender MedSystems GmbH, Europe (Cat. #BMS631INST), according to the method described by Baggiolini et al[22]. Moreover, serum brain derived neurotrophic factor (BDNF) level was evaluated by ELISA method using kit purchased from Millipore Corporation, United States (Cat. #CYT306), according to the method described by Laske et al[23]. Finally, the quantitative determination of brain DA level was carried out according to the method described by Ciarlone[24] using a fluorometric method.

Detection of tyrosine hydroxylase and nestin genes expression level

Total RNA was isolated from brain tissues of female rats by the standard TRIzol^® reagent extraction method (Invitrogen, Cat. #15596-026). Then, the complete Poly(A)⁺ RNA was reverse transcribed into cDNA in a total volume of 20 μL using RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas, Germany, Cat. #K1631). An amount of total RNA (5 μg) was used with a reaction mixture, termed as master mix. The MM was consisted of 50 mmol/L MgCl₂, 5 × reverse transcription (RT) buffer (50 mmol/L KCl; 10 mmol/L Tris-HCl; pH 8.3; 10 mmol/L of each dNTP, 50 μmol/L oligo-deoxyribonucleotide primer, 20 U ribonuclease inhibitor (50 kDa recombinant enzyme to inhibit RNase activity) and 50 U M-MuLV reverse transcriptase. The RT reaction was carried out at 25 °C for 10 min, followed by 1 h at 42 °C, and the reaction was stopped by heating for 5 min at 99 °C. Afterwards the reaction tubes containing RT preparations were flash-cooled in an ice chamber until being used for DNA amplification through semi-quantitative real time PCR (sqRT-PCR). An iQ5-BIO-RAD Cycler (Cepheid, United States) was used to determine the rat cDNA copy number. PCR reactions were set up in 25 μL reaction mixtures containing 12.5 μL 1 × SYBR^® Premix Ex TaqTM (TaKaRa, Biotech. Co. Ltd., Germany, Cat. #RR820A), 0.5 μL 0.2 μmol/L forward primer, 0.5 μL 0.2 μmol/L reverse primer (Invitrogen), 6.5 μL distilled water, and 5 µL of cDNA template. Primer sequences were F: 5’-ACTGTGGAATTCGGGCTATG-3’, R: 5’-GACCTCAGGCTCCTCTGACA-3’ for tyrosine hydroxylase (TH)[25]; F: 5’-TGGAGCGGGAGTTAG-AGGCT-3’, R: 5’-ACCTCTAAGCGACACTCCCGA-3’ for nestin[26] and F: 5’-CTGTCTGGCGGCACCACCAT-3’, R: 5’-GCAACTAAGTCATAGTCCGC-3’ for β-actin[27]. The reaction program was allocated to 3 steps. First step was at 95.0 °C for 3 min. Second step consisted of 40 cycles in which each cycle divided to 3 steps: (1) denaturation at 95.0 °C for 15 s; (2) annealing at 58.0 °C for 30 s, 55.0 °C for 5 s and 60 °C for 30 s for TH, nestin and β-actin genes respectively; and (3) extension at 72.0 °C for 30 s. The third step consisted of 71 cycles started at 60.0 °C and then increased about 0.5 °C every 10 s up to 95.0 °C for melting curve analysis which was performed at the end of each sqRT-PCR to check the quality of the used primers. Each experiment included a distilled water control.

Immunohistochemical examination of brain survivin expression

Samples were taken from brain of rats of the different groups and fixed in 10% formalin buffer for 24 h. Washing was done in tap water then ascending grade of ethyl alcohol (30%, 50%, 70%, 90% and absolute) was used for dehydration. Specimens were cleared in xylene and embedded in paraffin (melting point 58 °C-60 °C) for 24 h. Sections were cut into 4 μ thick by sledge microtome then fixed on positive slides in a 65 °C oven for 1 h. Slides were placed in a coplin jar filled with 200 mL of triology working solution (Cell Marque, CA-United States, Cat. #920P-04) which combines the three pretreatment steps: Deparaffinization, rehydration and antigen unmasking. Then, the jar is securely positioned in the autoclave which was adjusted so that temperature reached 120 °C and maintained stable for 15 min after which pressure is released. Thereafter, the coplin jar is removed to allow slides to cool for 30 min. Sections were then washed and immersed in Tris-buffer saline to adjust the pH and these were repeated between each step of the immunohistochemical procedure. Quenching endogenous peroxidase activity was performed by immersing slides in 3% hydrogen peroxide for 10 min. Broad spectrum LAB-SA detection system (Invitrogen, Cat. #85-8943) was used to visualize any antigen-antibody reaction in the tissue. Background staining was blocked by putting 3 drops of 10% goat non immune serum blocker on each slide and incubating them in a humidity chamber for 10 min. Without washing, excess serum was drained and the working solution (1:100) of survivin mouse monoclonal (Thermo Scientific, United States, Cat. #RB-9245-P1) was prepared. Three drops of the working solution were applied and slides were incubated in the humidity chamber overnight at 4 °C. Henceforward, biotinylated secondary antibody from ultravision detection system anti-polyvalent HRP/3,3’-diaminobenzidine (DAB) (Thermo Scientific, Cat. #TP-015-HD) was applied on each slide for 20 min followed by 20 min incubation with the streptavidin HRP enzyme conjugate (Thermo Scientific, Cat. #TP-015-HD). Then, DAB chromogen (Thermo Scientific, Cat. #TP-015-HD) was prepared and 3 drops were applied on each slide for 2 min. DAB was rinsed, after which counterstaining with Mayer hematoxylin and cover slipping were performed as the final steps before slides were examined under the light microscope (Olympus Cx21 with attached digital camera)[28]. Image analysis was performed using the image J, 1.41a NIH, United States analyzer.

Histopathological investigation of brain tissue of rats

Samples were taken from brain of rats in different groups and fixed in 10% formalin buffer for 24 h. Washing was done in tap water then ascending grade of ethyl alcohol (30%, 50%, 70%, 90% and absolute) was used for dehydration. Specimens were cleared in xylene and embedded in paraffin (melting point 58 °C-60 °C) for 24 h. Paraffin wax tissue blocks were prepared for sectioning at 4 μ by sledge microtome. The obtained tissue sections were collected on glass slides, deparffinized and stained by hematoxylin and eosin (H and E) stain[29] for histopathological examination through the electric light microscope.

Statistical analysis

In the present study, all results were expressed as mean ± SE of the mean. Data were analyzed by one way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) program, version 14 followed by least significant difference (LSD) to compare significance between groups. Difference was considered significant when P value was < 0.05.

Animal care and use statement

The animal protocol was designed to minimize pain or discomfort to the animals. The animals were acclimatized to laboratory conditions (25 °C, 12 h/12 h light/dark, 55% humidity, ad libitum access to food and water) for 2 wk prior to experimentation. The animals were deeply anaesthetized via diethyl ether for intravenous infusion of MSCs. Also, blood samples were collected from retro-orbital venous plexus under diethyl ether anaesthesia.

RESULTS

BM-MSCs homing

To confirm that the intravenously transplanted MSCs derived from male bone marrow migrate and home to the female injured brain, DNA was isolated from the brain tissues of female rats and the presence or absence of the responsible region for sex determination on Y chromosome (SRY gene) was assessed by PCR. The agarose gel demonstrated that SRY gene was present in the brain tissues obtained from the group of rats treated with BM-MSCs. While, SRY gene was absent in the brain tissues obtained from the ovariectomized control rats (Figure 1).

Figure 1 An agarose gel electrophoresis of DNA fragments showed SRY gene in recipient female rats for bone marrow derived mesenchymal stem cells in Parkinson’s disease model. Lane (M) represents DNA ladder; Lane (1) represents ovariectomized control sample; Lane (2) represents sample from PD group treated with BM-MSCs. PD: Parkinson’s disease; BM-MSCs: Bone marrow derived mesenchymal stem cells.

Effect of treatment with BM-MSCs on inflammatory markers

Since, TGF-β1 has a pivotal role in the control of the transition between pro-inflammatory and anti-inflammatory response[30] and MCP-1 has a vital role in the migration of inflammatory cells across the blood-brain barrier as well as forms chemotactic gradients within the CNS to control the local inflammatory response[31]. Serum TGF-β1 and MCP-1 levels were determined by ELISA to evaluate the anti-inflammatory and immunomodulatory effects of the injected BM-MSCs in PD model.

Our data revealed that rotenone administration causes significant (P < 0.05) elevation in serum TGF-β1 (43.6%) and MCP-1 (27.2%) levels vs the ovariectomized control group (Table 1). While, treatment with BM-MSCs elicits a significant (P < 0.05) reduction in both serum TGF-β1 and MCP-1 levels by 29.2% and 20.1% respectively relative to the group of rats left untreated.

Table 1 Effect of treatment with bone marrow derived mesenchymal stem cells on serum transforming growth factor beta-1 and monocyte chemoattractant protein-1 levels in Parkinson’s disease model.

	TGF-β1 (pg/mL)	MCP-1 (pg/mL)
Ovariectomized control	481.5 ± 7.5	88.1 ± 0.9
PD untreated	691.2 ± 8.0a	112.1 ± 1.9a
PD + BM-MSCs	489.7 ± 13.0c	89.6 ± 2.0c

Data are represented as mean ± SE of 8 rats/group.

^aSignificant change at P < 0.05 in comparison with the ovariectomized control group;

^cSignificant change at P < 0.05 in comparison with the untreated PD group. PD: Parkinson’s disease; BM-MSCs: Bone marrow derived mesenchymal stem cells; TGF-β1: Transforming growth factor beta-1; MCP-1: Monocyte chemoattractant protein-1.

Effect of treatment with BM-MSCs on neurotrophic and neurogenic markers

Brain derived neurotrophic factor plays an important role in supporting the survival of existing neurons and encouraging the growth as well as differentiation of new neurons and synapses[32]. Thusly, serum BDNF level was estimated by ELISA to evaluate the neurotrophic capacity of the injected BM-MSCs in PD model. In view of the data of the current work, rotenone administration experiences significant (P < 0.05) decline in serum BDNF level by 21.5% (Table 2) as compared to the ovariectomized control group. In contrast, treatment with BM-MSCs elevates serum BDNF level significantly (P < 0.05) by 26.1% (Table 2) relative to the group of rats left untreated.

Table 2 Effect of treatment with bone marrow derived mesenchymal stem cells on serum brain derived neurotrophic factor and brain dopamine levels as well as brain tyrosine hydroxylase and nestin genes expression level in Parkinson’s disease model.

	BDNF (pg/mL)	DA (μg/g tissue)	Relative expression of TH gene (TH/β-actin)	Relative expression of nestin gene (nestin/β-actin)
Ovariectomized control	3700 ± 26.4	882 ± 20.3	1.19 ± 0.004	1.30 ± 0.004
PD untreated	2905 ± 72.9a	599 ± 9.8a	0.54 ± 0.009a	0.67 ± 0.006a
PD + BM-MSCs	3663 ± 17.8c	874 ± 15.0c	1.18 ± 0.004c	1.29 ± 0.005c

Data are represented as mean ± SE of 8 rats/group.

^aSignificant change at P < 0.05 in comparison with the ovariectomized control group;

^cSignificant change at P < 0.05 in comparison with the untreated PD group. PD: Parkinson’s disease; BM-MSCs: Bone marrow derived mesenchymal stem cells; BDNF: Brain derived neurotrophic factor; DA: Dopamine; TH: Tyrosine hydroxylase.

Brain DA level was determined by a fluorometric method, while brain TH and nestin genes expression level was detected by sqRT-PCR to evaluate the neurogenic potential of the injected BM-MSCs in PD model. It is well known that DA is a neurotransmitter released by nerve cells to play crucial role in motor control, motivation, arousal, cognition and reward[33]. Furthermore, TH enzyme catalyzes the conversion of L-tyrosine to L-3,4-dihydroxy-phenylalanine[34]. While, nestin is one of the markers of neural precursors[35]. The data of our work revealed that rotenone administration leads to significant (P < 0.05) depletion of brain DA level (32.1%) and significant (P < 0.05) down-regulation in the expression level of brain TH and nestin genes by 54.6% and 48.5% respectively (Table 2) as compared to the ovariectomized control group. However, treatment with BM-MSCs produces significant (P < 0.05) elevation in brain DA level by 45.9% and significant (P < 0.05) up-regulation in brain TH and nestin genes expression level by 122.2% and 92.5% respectively (Table 2) vs the group of rats left untreated.

Effect of treatment with BM-MSCs on anti-apoptotic marker

The anti-apoptotic action of the single intravenous dose of BM-MSCs in PD model was evaluated through the detection of brain survivin expression using immunohistochemical technique. As, survivin belongs to a family of endogenous cellular inhibitors of caspases that directly repress apoptotic cell death through interactions with pro-apoptotic caspases[36]. In view of the current data, rotenone administration causes insignificant (P > 0.05) decrease in the number of positive cells for survivin expression by 5.7% (Table 3 and Figure 2B) relative to the ovariectomized control group. While, treatment with BM-MSCs produces insignificant (P > 0.05) increase in the number of positive cells for survivin expression by 8.0% (Table 3 and Figure 2C) in comparison with the group of rats left untreated.

Figure 2 Immunohistochemical examination of survivin expression in Parkinson’s disease model groups. A: Ovariectomized control; B: PD untreated; C: PD + BM-MSCs. PD: Parkinson’s disease; BM-MSCs: Bone marrow derived mesenchymal stem cells.

Table 3 Effect of treatment with bone marrow derived mesenchymal stem cells on brain survivin expression in Parkinson’s disease model.

	Survivin (cell number)
Ovariectomized control	288 ± 16.5
PD untreated	271.5 ± 13.9
PD + BM-MSCs	293.2 ± 15.9

Data are represented as mean ± SE of 8 rats/group. PD: Parkinson’s disease; BM-MSCs: Bone marrow derived mesenchymal stem cells.

Effect of treatment with BM-MSCs on brain structure

The brain section photomicrograph of ovariectomized control rat shows congestion in the blood vessels in striatum area (Figure 3A). While, brain section photomicrographs of untreated rotenone administered rat show congestion in the blood vessels and capillaries (Figure 3B) in the striatum as well as hyalinization and plaques formation in the matrix of the striatum indicating the occurrence of neurodegeneration (Figure 3C). Finally, the brain section photomicrograph of rotenone administered rat treated with BM-MSCs shows intact histological structure of the striatum (Figure 3D).

Figure 3 Photomicrograph of brain section of: A: Ovariectomized control group shows congestion in blood vessels of striatum (v) (H and E × 80); B: untreated Parkinson’s disease (PD) group shows congestion in blood vessels and capillaries of striatum (v) (H and E × 80); C: Untreated PD: Parkinson’s disease group shows hyalinization with plaques formation in the matrix of striatum (H and E × 160); and D: PD group treated with bone marrow derived mesenchymal stem cells shows intact histological structure of the striatum (H and E × 80).

DISCUSSION

MSCs have been considered as an effective tool for regenerative cell therapy. These cells could be isolated from both healthy and patient tissues and expanded in vitro on a therapeutic scale without posing significant ethical or procedural problems[37]. Furthermore, it has been proposed that stem cells may replace lost cells by differentiating into functional neural tissue; provide source of trophic support for the diseased nervous system or alter the immune system to prevent further neurodegeneration[38]. Therefore, the current study was planned to elucidate the mechanisms by which BM-MSCs could attenuate PD pathophysiology in the experimental model.

In consistent with Yoon et al[39] who found that intravenously transplanted BM-MSCs could migrate and home into the brain, the data presented in this work demonstrated that the intravenously transplanted MSCs were able to migrate to the site of injury (brain). The homing property afforded by MSCs was likely attributable to their broader expression of homing molecules[40]. Furthermore, it has been reported that, chemokines released from tissue or endothelial cells may contribute to the activation of adhesion ligands, transendothelial migration, chemotaxis, and/or subsequent retention in surrounding tissue[41].

In view of the data of the current work, rotenone administration for 14 d in ovariectomized rats elevated the level of serum TGF-β1 and MCP-1 significantly. This finding is greatly supported by those of Rota et al[42] and Reale et al[43] who stated that both TGF-β1 and MCP-1 levels are increased in several chronic neurodegenerative pathologies such as PD. It has been reported that the inflammatory response due to Parkinsonism is characterized by activation of microglia in the brain. The proposed explanation in regards to the reason of degeneration in dopaminergic neurons is that PD is caused by activation of microglial cells as a result of increased levels of cytokines[44]. Activated microglia release a wide array of pro-inflammatory and cytotoxic factors as well as eicosanoids and nitric oxide[45], which work in concert to develop neurodegeneration[46]. Moreover, Gao et al[47] reported that the dopaminergic neurodegeneration enhanced by rotenone might be attributed primarily to the activation of microglia and consequently their release of superoxide free radicals that play an important role in the inflammation mediated oxidative damage to neurons. This effect might be ascribed to the known susceptibility of dopaminergic neurons to oxidative stress as a result of reduced antioxidant capacity, high content of iron and DA, and possible defect in mitochondrial function[48]. The release of cytokines from the brain into the peripheral blood supply through the blood brain barrier[49] could explain the observed increase in serum TGF-β1 and MCP-1 levels.

The results of the current study manifested that treatment with BM-MSCs lessen the level of serum TGF-β1 and MCP-1 significantly. This finding is in great accordance with our previous work on adipose tissue derived MSC[50] that proved its anti-inflammatory and immunomodulatory activities which are implicated in mitigating neuroinflammation characterizing PD. Accordingly, the observed role of BM-MSCs in depleting serum TGF-β1 and MCP-1 levels could be allied to the ability of BM-MSCs to modulate microglia/macrophage activation including inflammatory responses as documented by Németh et al[51] and Choi et al[52].

Growing body of evidence indicates that there is a link between pro-inflammatory cytokines and neurotrophic factors in the CNS[53]. It has been postulated that there is a balance between cytokine and neurotrophin in the brain and disruption of this balance cause injurious changes in the CNS[54]. Moreover, Borchelt[55] observed that astrocytes stimulated by mediators released from microglia down-regulate neurotrophic factors expression and release additional inflammatory mediators that in turn activate microglia. Parallel to these evidences, our results indicated that rotenone administration elicited significant decrease in serum BDNF level. This finding could be allied to the diminished level of brain BDNF due to inflammation. As, Klein et al[56] reported that BDNF level in the blood correlates with alteration in the level of BDNF in the brain.

In view of the current data, treatment with BM-MSCs experienced significant increase in serum BDNF level. This preferable effect could be related to the ability of MSCs to secrete BDNF as observed by Lattanzi et al[57] and Han et al[58]. Blandini et al[59] documented that MSCs have the ability to differentiate into glial cells that release diverse neurotrophic factors to provide protection against neurotoxin after their grafting into Parkinsonian rat brains. Additionally, there is an evidence that MSCs may modulate the expression of neurotrophic factors according to the environment in which they exist[60,61].

The data presented in this work revealed that rotenone administration led to significant down-regulation in brain TH gene expression level in concomitant with significant decline in brain DA level. This observation could be ascribed to the dopaminergic degeneration[62] due to elevated sensitivity of dopaminergic neurons to oxidative damage[47] as well as inhibition of complex I activity and decrement of the mitochondrial membrane potential as a result of rotenone administration[47,63].

Our previous findings indicated the neurotrophic and neuroprotective potentials of adipose tissue derived MSC against neurodegenerative insult of PD[50]. Similarly, the data of the present work demonstrated that treatment with BM-MSCs elicited significant increase in brain DA level as well as brain TH gene expression level. This finding comes in line with the study of Shetty et al[64] who demonstrated that BM-MSCs can be transdifferentiated efficiently into functional dopaminergic neurons capable of secreting DA and alleviating behavioral deficiencies. Moreover, the results of Bouchez et al[25] study showed that grafting of BM-MSCs caused an increase in the immunostaining of TH in striatum associated with elevation in the number of TH⁺ neurons in the substantia nigra pars compacta. Also, Blondheim et al[65] and Offen et al[66] stated that the transplantation of BM-MSCs into the animal model induced with 6-hydroxydopamine resulting in an increase in the level of TH in the striatal region thus improving motor behavior in a mouse model of PD. Since, TH is the rate-limiting enzyme in DA synthesis, the increase in the level of TH would increase the production of DA. Additionally, the observed increase in brain DA content and TH expression level as a result of treatment with BM-MSCs could be explained by the ability of MSCs to secrete a wide array of cytokines and growth factors, including BDNF[57] which exert neurotrophic and neuroprotective effects on DA neurons[67]. Furthermore, Trzaska et al[68] reported that BDNF has a crucial role in the functional maturation of MSC-derived DA progenitors.

In line with previous studies reported by Höglinger et al[69] and Abdipranoto et al[70], the current study manifested that rotenone administration caused significant down-regulation in brain nestin gene expression level. This finding could be imputed to the depletion in DA level due to degeneration of dopaminergic neurons as documented by Crews et al[71]. In contrast, treatment with BM-MSCs induced significant up-regulation in nestin gene expression level. Bouchez et al[25] found that rat MSCs express neuronal proteins such as nestin at the RNA and protein levels. Moreover, the study of Ye et al[72] indicated the presence of nestin positive cells in brain tissue of PD rat after transplantation of undifferentiated BM-MSCs. The suggested mechanism by which BM-MSCs treat PD rat model could be related to that transplanted BM-MSCs might become nestin-positive stem cells that differentiate into astrocytes or other non-dopaminergic neurons and participate in the reconstruction of dopaminergic neurons circuits[72].

The data of this work revealed that rotenone administration produced slight decrease in the number of positive cells for survivin expression. This finding harmonizes with that of Zhang et al[73] who reported that degenerating neurons lacked survivin expression. Jiang et al[74] results showed that survivin is critically required for the survival of developing CNS neurons. Moreover, Zhang et al[75] suggested that there is a connection between the expression of survivin and adult neurogenesis. Thus, the observed decrement in survivin expression might be attributed to the decreased neurogenesis due to DA depletion[71]. Another possible mechanism by which rotenone could decrease survivin expression might be related to its effect on p53 which was shown to be over expressed by rotenone[76]. Under normal conditions, p53 protein levels are low and regulated by IκB kinase (IKK) and prominently by mouse double minute 2 (Mdm2), an ubiquitin ligase responsible for p53 degradation. Cellular stress reduces the interaction between p53 and Mdm2 leading to accumulation of the former[77]. Wu et al[76] reported that the degeneration of dopaminergic neurons by rotenone was accompanied by an increase in p53 protein level which in turn induces p21 expression. Then, the increased level of p21 suppresses the expression of cycline dependent kinases leading to accumulation of hypophosphorylated retinoblastoma that interact with E2F (a transcriptional activator) to repress survivin expression[78].

In the light of our results, treatment with BM-MSCs caused insignificant increase in the number of positive cells for survivin expression. This increment is in agreement with Okazaki et al[79] and it could be imputed to the ability of MSCs to enhance neurogenesis and inhibit apoptosis through their secreted BDNF as documented by Ye et al[72]. Moreover, Kim et al[80] reported that grafted MSCs attenuate dopaminergic neuronal loss through their anti-apoptotic effects. Also, the increase in survivin expression by MSCs treatment might be related to their inhibitory action on P53 through the inactivation of ERK1/2[81].

In view of the histopathological investigations of brain tissues section of the current work, rotenone administration resulted in congestion in the blood vessels and capillaries of striatum. Also, there were hyalinization and plaques formation in the matrix of striatum indicating the occurrence of neurodegeneration. Sai et al[82] demonstrated that rotenone causes dopaminergic neurons degeneration in vivo and substantia nigra pars compacta and striatum are the main targets of rotenone in the rat brain. These findings could be allied to the inhibition of neuronal mitochondrial complex I activity[47] and consequently oxidative damage[83] as a result of rotenone administration.

Brain tissue sections examination indicated that single infusion with BM-MSCs resulted in intact histological structure of the striatum. This finding coincides with Dezawa et al[84] who reported that nerve system recovery after BM-MSCs transplantation could be related to their secretion of neurotrophic factors that restore the function of nervous system, promotion of local angiogenesis and vascular reconstruction and neuronal regeneration through promotion of autologous neuronal regeneration and differentiation of transplanted cells into neural cells.

In conclusion, the current study provided experimental evidences for the ability of BM-MSCs to mitigate PD pathophysiology through multi-mechanistic approaches (immunomodulatory, anti-inflammatory and anti-apoptotic effects as well as neurotrophic and neurogenic potentials). These promising results pave the way for the clinical trial application of MSCs in the treatment of neurodegenerative diseases particularly PD.

COMMENTS

Background

Parkinson’s disease (PD) is one of the neurodegenerative diseases, accompanied by extrapyramidal motor dysfunction due to the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta and declining levels of dopamine in the striatum. So, it is very important to stop or halt neurodegeneration. However, to date, there is no therapy clinically available that delays the neurodegenerative process itself, therefore modification of the disease course is an important unmet clinical need. Transplantation of mesenchymal stem cells (MSCs) for treating neurodegenerative disorders has received growing attention recently because these cells are readily available, easily expanded in culture, and when transplanted survive for relatively long periods of time.

Research frontiers

MSCs are a heterogeneous subset of stromal stem cells that have the ability of self-renewal and multipotency. In the area of neurodegenerative disorders treatment, the current research hotspot is how to modify the disease course by specifically target the pathophysiologic cascade, hoping to delay the onset of the disease and slow its progression.

Innovations and breakthroughs

Modern research has focused on discovering effective disease-modifying therapies, which specifically target the pathophysiologic cascade, hoping to delay the onset of the disease and slow its progression. The study provided a non invasive approach for mitigating PD pathophysiology via bone marrow derived MSCs (BM-MSCs) transplantation which has immunomodulatory, anti-inflammatory and anti-apoptotic effects as well as neurotrophic and neurogenic potentials.

Applications

The study results shed light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.

Terminology

PD is the second most common neurodegenerative disease, accompanied by extrapyramidal motor dysfunction which resulting from the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta and declining levels of dopamine in the striatum. MSCs are a heterogeneous subset of stromal stem cells that have the ability of self-renewal and multipotency, which could differentiate into cells of the mesodermal lineages and other embryonic lineages, including adipocytes, osteocytes, chondrocytes, hepatocytes, neurons, muscle cells, epithelial cells, etc.

Peer-review

This article is well written, clearly demonstrating the therapeutic effect of BM-MSCs for the treatment of PD. Authors also presented the molecular basis for the amelioration of PD pathology by showing decrements and increments in inflammatory mediators and neurotrophic factors in the serum, respectively. The overall data presented in this manuscript are sound.