First, the double lesion-induced MSA-P was established with co-injections of MPTP (10mg/day, total dose 90 mg/kg for 9 days) and 3- NP (total dose 450 mg/kg for 9 days, 12hr interval). At one day after last injection, hMSCs were injected into the tail vain (1X106cells/ml). Three groups of mice were compared (control group, only MPTP+3-NP group, hMSC treatment in MPTP+3-NP group) through histopathological and behavioral analysis. Compared to only MPTP+3-NP-treated mice, hMSCs treatment in double lesion
mice significantly increased survival of TH- and NeuN-immunoreactive cells in the substantia nigra and the striatum, respectively. Additionally, hMSC treatment significantly decreased Iba-1, GFAP and increased Calbindine immunoreactive cells in the substantia nigra and the striatum. Behavioral analysis showed that the decent times on the top of a vertical wooden pole was significantly decreased in hMSCs-treated double lesion mice, comparable to controls. This study demonstrates that hMSCs treatment had a protective effect on loss of neurons in the substantia nigra and the striatum induced by MPTP+3-NP through a variety of mechanisms, such as anti-inflammatory actions, anti-apoptotic effect.
Second, the neuroinflammation plays collectively suggest that excessive neutrophil infiltration and environmental factors, such as lower astrocyte density and higher BBB permeability, contribute to severe inflammation and neuronal death in the SNpc. At 4hour (4hr) after LPS injection, hMSCs were injected into the tail vain (1X106cells/ml) and three groups of rat were compared (control group, only LPS group, hMSC treatment in LPS group) through histopathological analysis after 12hr. Compared to only LPS-treated rats, hMSCs treatment in LPS-treated rats significantly showed increase of EBA-immunoreactive cells and reduction of Evans blue- immunoreactive cells in the substantia nigra. Interestingly, Compared to only LPS-treated rats, hMSCs treatment in LPS-treated rats significantly showed increase of the densities of astrocytes, assumed to significantly influence neurovascular structure and integrity and reduction of p-gp-immunoreactive cells, the one BBB transporter. Consequently, hMSC treatment significantly showed reduction of MPO-immunoreactive cells and increase of TH-MPO-immunoreactive cells in the substantia nigra. This
study demonstrates that hMSCs treatment had a protective effect on neuroinflammation plays in the substantia nigra induced by LPS through inhibition of BBB permeability.
Key words : Mesenchymal stem cells, multiple system atrophy, blood brain barrier.
TABLE OF CONTENTS
A. Human mesenchymal stem cells exerts neuroprotection in an animal model of double lesion-induced multiple system atrophy-parkinsonism (MSA-P) ···2
B. Inhibition of blood brain barrier (BBB) permeability by hMSCs ···3
. MATERIALS AND METHODS
. RESULTS
Ⅲ ··· 12
A. Human mesenchymal stem cells exerts neuroprotection in an animal model of double lesion-induced multiple system atrophy-parkinsonism (MSA-P)··· 12
1. Characterization of hMSCs ··· 12
2. Recovery of motor behavior by hMSCs ··· 12
3. Detection of hMSCs in the double toxin-treated SN and striatum··· 13
4. Histological analysis of transplanted hMSCs in double lesion-induced MSA-P model ··· 13
5. Effect of cell therapy with hMSCs on modulation of inflammation and gliosis in animals treated with double toxins··· 14
6. Effect of cell therapy with hMSCs on modulation of cell death signaling pathway ··· 15
B. Inhibition of blood brain barrier (BBB) permeability by hMSCs ··· 15
1. Effect of hMSCs on loss of dopaminergic neuron and modulation of inflammation in LPS-induced animal models··· 15
2. Effect of hMSCs on BBB permeability in LPS-induced animal models··· 16
3. Effect of hMSCs on modulation of the densities of astrocytes at the SN of animals treated with LPS··· 16
4. Effect of hMSCs on modulation of P-glycoprotein at the BBB of LPS-induced animal models ··· 17
5. Effect of hMSCs on MPO-1 neutrophil infiltration in the SNpc ··· 17
. DISCUSSION
Ⅳ ··· 33
A. Human mesenchymal stem cells exerts neuroprotection in an animal model of double lesion-induced multiple system atrophy-parkinsonism (MSA-P)··· 33
B. Inhibition of blood brain barrier (BBB) permeability by hMSCs ··· 36
. CONCLUSION Ⅴ ··· 40
A. Human mesenchymal stem cells exerts neuroprotection in an animal model of double lesion-induced multiple system atrophy-parkinsonism (MSA-P)··· 40
B. Inhibition of blood brain barrier (BBB) permeability by hMSCs ··· 40
REFERENCE ··· 41
국문요약 ···50
LIST OF FIGURES
Fig. 1. Schedule of MSA-P animal model··· 19
Fig. 2. Characterization of MSC··· 20
Fig. 3. Motor behavioral test ··· 21
Fig. 4. Detection of hMSCs in the double-toxin treated mice ··· 22
Fig. 5. Effect of cell therapy with hMSCs on animals treated with 3-NP and MPTP ···23
Fig. 6. Effect of cell therapy with hMSCs on modulation of inflammation and gliosis in animals treated with double toxins ··· 24
Fig. 7. Effect of cell therapy with hMSCs on modulation of cell death signaling pathway·· ··· 25
Fig. 8. Schedule of LPS-induced animal model ··· 26
Fig. 9. Effect of cell therapy with hMSCs on animals treated with LPS ··· 27
Fig. 10. Effect of cell therapy with hMSCs on modulation of inflammation
in animals treated with LPS ··· 28
Fig. 11. Effect of cell therapy with hMSCs on BBB permeability in animals treated with LPS ··· 29
Fig. 12. Effect of hMSCs on modulation of the densities of astrocytes
at the SN of animals treated with LPS··· 30
Fig. 13. Effect of hMSCs on modulation of P-glycoparotein at the BBB of animals treated with LPS··· 31
Fig. 14. Effect of hMSCs on MPO-1 neutrophil infiltration in the SNpc ···32
I. INTRODUCTION
Mesenchymal stem cells (MSCs) are present in adult bone marrow and represent
<0.01% of all nucleated bone marrow cells. MSCs are themselves capable of multi-potency, with differentiation under appropriate conditions into chondrocytes, skeletal myocytes, and neurons. MSCs have been also known to pose neuroprotective effects through secreting various cytotrophic factors. Previous our study in animal model of Parkinson’s disease (PD) demonstrated that hMSCs had a protective effect on progressive dopaminergic neuronal loss through a variety of mechanisms, such as anti-apoptotic effect, deceasing the polyubiquitinated proteins, and anti-inflammatory actions in addition to possible transdifferentiating effect of MSCs into dopaminergic neurons (Park et al, 2008).
Additionally, we also reported in LPS-induced animal model of PD that hMSCs had a neuroprotective property on dopaminergic neurons through a potent anti-inflammatory action (Kim & Park et al, 2009). Furthermore, we recently reported an open-label clinical trial of MSCs in patients with MSA, demonstrating that MSCs injection delayed progression of neurological deficits and improved cerebral glucose metabolism in cerebellum compared to the control patients (Lee et al, 2008).
Based on our previous studies demonstrating neuroprotective effect of hMSCs in animal model of Parkinson’s disease, we extended our investigations into other parkinsonian disease of multiple system atrophy (MSA). In addition, we also evaluate whether hMSCs may modulate blood-brain barrier, which is known to be an important key player in progression of neurodegenerative changes.
A. Human mesenchymal stem cells exerts neuroprotection in an animal model of double lesion-induced multiple system atrophy-parkinsonism (MSA-P).
Multiple system atrophy (MSA) is a sporadic neurodegenerative disease of the central and autonomic nervous system. Pathologically, MSA includes striatonigral degeneration, olivopontocerebellar degeneration, astrogliosis and microgliosis. TClinically, the cardinal features include autonomic failure, parkinsonism (MSA-P), cerebellar ataxia, and pyramidal signs in any combination, of which autonomic failure is an integral component in the diagnosis of MSA. Along with progressive supranuclear palsy and corticobasal degeneration, MSA is one of Parkinsonian disease and its prevalence is the second most common following PD. With an identification of α-synuclein-positive glial cytoplasmic inclusions (GCI) as a pathological hallmark, MSA has been regarded as a unique entity within the spectrum of oligodendrogliopathy. Since the prognosis of MSA is fetal, many in vivo and clinical trials have been conducted to archive neuroprotective strategies in MSA. Of those, Stefanova et al. (Stefanova et al, 2008) demonstrated that MAO-B inhibitor had a disease modifying activity in transgenic animal models of MSA, although other clinical trials have been failed to delay disease progression.
The animal model of MSA-P was based on the idea of applying selective degeneration in the nigral and striatal neurons by using 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) and 3-nitropropionic acid (3-NP), which had been previously used to mimic Parkinson’s disease and Huntington’s disease in rodents (Stefanova et al, 2003), respectively.
In this double lesion model, the MPTP is known to potentiate striatal damage and its related
with behavioral impairments induced by 3-NP intoxication in mice and thus constitute a useful model of MSA-P. In present study, we investigated whether MSCs has a protective effect on neuronal loss in the substantia nigra (SN) and striatum using double toxins-induced MSA-P animal model.
B. Inhibition of blood brain barrier (BBB) permeability by hMSCs.
The blood-brain barrier (BBB) provides a barrier from potentially toxic molecules, in addition to regulating ion balance and nutrient transport. Recently, BBB impairment has been detected in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, although the integrity of the BBB may depend on the disease severity or duration (Desai et al, 2007). The implications of BBB dysfunction have not been fully elucidated, however, this dysfunction may act as a modifier of disease progression.
Along with the dysregulation of tight junction proteins due to the accumulation of misfolded proteins in the BBB, neuroinflammation is known to be the main contributor to BBB dysfunction in neurodegenerative disease (Desai et al., 2007). Recent studies have suggested that a microglial reaction and inflammatory processes participate in the cascade of neuronal degeneration in neurodegenerative disease (Gao and Hong, 2008). There is in vivo evidence of BBB alterations in both AD and PD. Skoog et al, 1998 identified BBB alterations before the onset of clinical dementia in AD patients and Bowman et al, 2007 suggested that BBB alterations were a modifier of AD progression. In patients with PD, BBB
alterations seemed to depend upon clinical stage; the integrity of the BBB was relatively preserved in early-stage of PD, whereas the BBB was impaired in late-stage PD (Bartels et al, 2008; Kortekaas et al, 2005). Furthermore, we recently demonstrated in vivo evidence of BBB impairment and significant correlations between the BBB indices and clinical severity in patients with MSA, suggesting that BBB dysfunction in MSA may be closely coupled to the extension of MSA pathology (Song et al, 2010).
Based on previous studies, we hypothesize that (i) infiltrated neutrophils by BBB dysfunction are increased inflammation in the brain, (ii) MSCs treatment may exert modulation of BBB integrity through its immunosuppressive properties , and (iii) MSCs may have a neuroprotective effects on dopaminergic neurons though BBB stabilization.
In present study, we employed the LPS-treated animal model and investigated whether hMSCs treatment has a protective effect on inflammation and neuronal loss through BBB dysfunction.
. MATERIALS AND METHODS
Ⅱ
A. MATERIALS