Effect of hMSCs on MPO-1 neutrophil infiltration in the SNpc

To determine the effects of hMSCs on modulation of inflammation by neutrophil infiltration, brain tissue in the SN was immunostained with MPO, a marker for activated neutrophil. There was a markedly increase in MPO-ir cells in LPS-induced animal models (Fig. 14); however, hMSCs treatment in LPS-induced animal models markedly decreased

MPO-ir cells (Fig. 14-A). Stereological analysis revealed that the number of neutrophil infiltration was significantly decreased in hMSCs treatment group than in the group only treated with LPS in the SN (Fig. 14-B, p<0.01 ).

Fig. 1. Schedule of MSA-P animal model. C57BL/6 adult male mice aged 16 weeks were injected MPTP (10mg/day, total dose 90 mg/kg, i.p.) and 3- NP (10 mg/kg ´ 4, 20 mg/kg ´ 4, 30 mg/kg ´ 4, 40 mg/kg ´ 4 and 50 mg/kg ´ 1, total dose 450 mg/kg, 12 hrs interval, i.p.) for 9-day period (each group, n=5). At one day after last injection, hMSCs were injected into the tail vain (1 ´ 10⁶ cells/ml). The pole test was performed at baseline, then at day 1, week 1 and 2 after hMSCs transplantation.

Fig. 2. Characterization of MSC. Flow cytometric analysis (A) and immunofluorescent labeling of human mesenchymal stem cells (B) Scale bar: 100μm.

Fig. 3. Motor behavioral test. The pole test was performed at baseline, then at day 1, 10 and 20 after hMSCs transplantation. The total time until the mouse reached the floor with its four paws was significantly increased in mice of double-toxin treated mice than controls (p<005, n=5). Compared to double toxin-treated mice, hMSCs administration in double toxins-treated mice had a significant decrease in the total time and this significant difference was maintained for 10 days after hMSCs administration (n=5, *p<0.05).

Fig. 4. Detection of hMSCs in the double-toxin treated mice. ^The

existence of hMSCs in the substantia nigra (SN) and striatum (ST) of the double toxin-treated mice was identified by human specific NuMA staining (A). The number of NuMA-ir cells in the SN and striatum was 20120 ± 825 and 37859 ± 25, respectively, which corresponded to about 2.0% and 3.8% of the total injected hMSCs (n=5, B). Scale bar:100mm

Fig. 5. Effect of cell therapy with hMSCs on animals treated with 3-NP and MPTP. Immunohistochemical analysis showed that hMSCs treatment significantly decreased the decline in the number of TH-ir and NeuN-ir cells in the substantia nigra (SN) and striatum (ST) of double-toxin treated animals (A). Stereological analysis revealed that the number of TH- ir and NeuN-ir cells was significantly higher in hMSCs treatment group than in the group treated with double toxin alone (n=5; *p<0.05, B).

Functional neurons immunostained by Calbindin-D was also increased significantly in the SN and ST of double-toxin treated mice after administration of hMSCs (C). Stereological analysis revealed that the number of Calbindin-ir cells was significantly higher in hMSCs treatment group than in the group treated with double toxin alone (n=5; * p<0.05, D). Scale bar:100mm

Fig. 6. Effect of cell therapy with hMSCs on modulation of inflammation and gliosis in animals treated with double toxins. MPTP and 3-NP treatment dramatically led to microglial activation and gliosis in the substantia nigra (SN) and striatum (ST) , however, hMSCs treatment attenuated significantly activation of microgia and gliosis in double-toxin treated SN and ST (A and C). Stereological analysis revealed that the number of activated microglia and astrocytes was significantly lower in hMSCs treatment group than in the group treated with double toxin alone (n=5; ** p<0.01,

*** p<0.001 B, D). Scale bar:100mm

Fig. 7. Effect of cell therapy with hMSCs on modulation of cell death signaling pathway. Western blot analysis performed at 4 weeks after first double-toxin injection showed that the expression of p-Akt was significantly decreased in double toxin-treated mice compared to controls, however, hMSCs administration in double toxin-toxin-treated mice increased the expression of p-Akt. hMSCs treatment decreased significantly Bax expression in double toxin-treated mice, whereas hMSCs treatment increased significantly the expression of Bcl-2 in these mice. In addition, hMSCs significantly decreased the expression of cytochrome c, which had been elevated after double-toxin treatment. (n=3,

**p<0.01, A and B).

Fig. 8. Schedule of LPS-induced animal model. male SD rats (250–270g) were anesthetized with 10% chloral hydrate for LPS injection. LPS (5 mg ⁄ 3 mL) was delivered unilaterally into the left SN (5.3 mm posterior, 2.3 mm lateral, 7.7 mm ventral from the bregma) and injected at a rate of 1 mL ⁄ 5 min using a 26-gauge Hamilton syringe attached to an automated microinjector using a stereotaxic apparatus. At 4hr after LPS injection, hMSCs were injected into the tail vain (1 ´ 10⁶ cells/ml). Evans blue (EB) dye (4% in saline, 3 mL/Kg) was intravenously injected. Fifteen minutes later the rats were anesthetized and perfused.

Fig. 9. Effect of cell therapy with hMSCs on animals treated with LPS. Immunohistochemical analysis showed that hMSCs treatment significantly increased the decline in the number of TH-ir in the substantia nigra (SN) of LPS- injected animals (A).

Stereological analysis revealed that the number of TH-ir cells was significantly higher in hMSCs treatment group than in the group treated with LPS olny (n=5; *p<0.05, B). Scale bar:100mm

Fig. 10. Effect of cell therapy with hMSCs on modulation of inflammation in animals treated with LPS. LPS treatment dramatically led to microglial activation in the substantia nigra (SN), however, hMSCs treatment attenuated significantly activation of microgia in LPS-injected SN (A). Stereological analysis revealed that the number of activated microglia was significantly lower in hMSCs treatment group than in the group treated with LPS only (n=5; ** p<0.01, *** p<0.001 B). Scale bar:100mm

Fig. 11. Effect of cell therapy with hMSCs on BBB permeability in animals treated with LPS. LPS treatment dramatically led to BBB dysfunction in the substantia nigra (SN), however, hMSCs treatment attenuated significantly inhibition of BBB permeability in LPS-injected SN (A,B). EB was examined under a confocal microscope. hMSCs treatment significantly decreased the decline in the detected EB in the SN of LPS- injected animals (A). Immunohistochemical analysis showed that hMSCs treatment significantly decreased the decline in the number of EBA-ir in the SN of LPS- injected animals (B). Stereological analysis revealed that the number of EBA-ir cells was significantly higher in hMSCs treatment group than in the group treated with LPS olny (n=5;

*p<0.05, C). Scale bar:100mm

Fig. 12. Effect of hMSCs on modulation of the densities of astrocytes at the SN of animals treated with LPS. Immunohistochemical analysis showed that hMSCs treatment significantly increased the decline in the number of GFAP-ir in the substantia nigra (SN) of LPS- injected animals (A). Astrocyte end feet around vessels displayed using GFAP/tomato lectin double staining. Astrocytes were densely located around vessels in the SN of hMSCs treatment group. Scale bar:100mm

Fig. 13. Effect of hMSCs on modulation of P-glycoprotein at the BBB of animals treated with LPS. Immunohistochemical analysis showed that hMSCs treatment significantly decreased the decline in the number of p-gp-ir in the substantia nigra (SN) of LPS- injected animals (A). Stereological analysis revealed that the number of p-gp-ir cells was significantly lower in hMSCs treatment group than in the group treated with LPS olny (n=5; *p<0.05, B). Scale bar:100mm

Fig. 14. Effect of hMSCs on MPO-1 neutrophil infiltration in the SNpc. LPS treatment dramatically led to neutrophil infiltration in the substantia nigra (SN), however, hMSCs treatment attenuated significantly neutrophil infiltration in LPS-injected SN (A). Stereological analysis revealed that the number of neutrophil was significantly lower in hMSCs treatment group than in the group treated with LPS only (n=5; ** p<0.01,

*** p<0.001 B). Scale bar:100mm

. DISC

Ⅳ USSION

A. Human mesenchymal stem cells exerts neuroprotection in an animal model of double lesion-induced multiple system atrophy-parkinsonism (MSA-P).

The present study revealed that hMSC treatment significantly protected against neuronal loss induced by MPTP and 3-NP treatment in the SN and striatum with coincident improvement in motor behavior. Neuroprotective mechanisms exerted by hMSCs may be mediated by inflammatory and cell survival and death signaling-pathway modulation as the hMSCs migrated from the peripheral circulation into the SN and striatum. These data suggest that neuroprotective strategies using hMSCs may be applicable in patients with MSA-P.

With advances in the understanding of MSA pathobiologies, it has been suggested that oligodendroglial degeneration resulting from α-synuclein inclusion formation contributes to secondary widespread neuronal degeneration. However, the initial trigger or aggravating mechanism underlying the abnormal accumulation and aggregation of α-synuclein in MSA remains unknown. In case–control epidemiological studies, occupational exposure to pesticides, insecticides, or solvents that interrupt mitochondrial electron transport is associated with increased risk of MSA (Nee et al., 1991; Vanacore et al., 2005). In animal studies, high-dose 3-NP administration also aggravated nigrostriatal and olivopontocerebellar degeneration in MSA transgenic mice using proteolipid protein promoters (Stefanova et al., 2005). Furthermore, we recently reported that 3-NP

administration in transgenic mice led to oxidation-specific modifications of α-synuclein that were concomitant with an exacerbation of behavioral deficits and widespread neuronal and oligodendrocytic pathology in a number of brain regions implicated in MSA (Ubhi et al., 2009). These data support that derangement in mitochondrial function by mitochondrial neurotoxins, such as MPTP or 3-NP used in this study may be a main mediator for progression of MSA pathology.

Our study demonstrated that hMSCs had neuroprotective properties against mitochondria- inhibiting double-toxin-induced neuronal cell loss, showing about a 20%

increase in the survival of TH- and NeuN-ir cells in the SN and striatum. A significant improvement of motor behavior after hMSC treatment was in accordance with increased survival of these neuronal cells following hMSC treatment in double-toxin-treated mice, although functional recovery was not maintained in the end of study period possibly due to the effect of spontaneous recovery in double-toxin only treated animals. The neuroprotective effects of MSCs seem to be mediated by complex mechanisms. First, our study has demonstrated that hMSCs can restore the balance between neuronal survival and apoptosis, which is disrupted by mitochondrial neurotoxins. In this study, hMSC treatment significantly increased the expression of the cell survival factor p-Akt in double-toxin treated mice. pAkt activation is modulated by growth factors and prevents apoptotic cell death signaling pathways (Saito et al., 2004). Although we did not investigate the potential factors that induced pAkt activation, MSCs are known to increase the production of various neurotrophic factors, such as NGF, BDBF, or NT-3 (Kim et al., 2010), which may modulate pAkt activation in this study. Along with upregulation of cell survival signaling pathways by

hMSCs, hMSCs also modulated expression of pro-and anti-apoptotic proteins toward suppressing apoptotic cell death signaling, and thus prevented the release of cytochrome c from mitochondria.

Second, hMSC treatment had anti-inflammatory and anti-gliotic effect, showing significantly decreased activation of microglia and astrocytes in the double-toxin-treated SN and striatum. As in PD, microglial reaction and inflammatory processes also participate in the cascade of neuronal degeneration in MSA. In human MSA, neuropathological studies suggest that the mode of microglial activation is system-specific, consistent with the known pattern or system degeneration in MSA, and is significantly correlated with the burden of GCI in the extrapyramidal motor and cerebellar input systems (Ishizawa et al., 2004). A similar pattern of microglial activation was also observed in MSA patients using [11C](R)-PK11195 positron emission tomography study (Gerhard et al., 2003). Additionally, we reported that 3-NP administration in MSA transgenic mice produced marked microglial activation and gliosis (Ubhi et al., 2009). It has been suggested that MSCs can not only inhibit nearly all cells participating in the immune response cell–cell contact-dependant mechanism, but can also release a variety of soluble factors that may be involved in the immunosuppressive activity of MSCs (Karussis et al., 2008; Krampera et al., 2006; Nauta and Fibbe, 2007). Furthermore, we recently demonstrated in vitro and in vivo evidence that hMSCs have a neuroprotective effect on dopaminergic neurons through anti-inflammatory actions, where soluble factors released from MSCs, such as IL-6, IL-10, and TGF-β may regulate the microglial response to inflammatory stimulants (Kim et al., 2009). Accordingly,

our data suggest that the neuroprotective properties of hMSCs via anti-inflammatory effects were also evident in an animal model of MSA.

MSCs characteristically migrate towards injured brain area in various animal models of ischemia and PD, possibly in response to signals that are upregulated under injury condition (Hellmann et al., 2006; Li et al., 2008). Chemokines released from damaged brain cells and their receptors, such as stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 may play an important role in migration of MSCs (Chamberlain et al., 2007; Stumm et al., 2002). SDF-1 is widely expressed in the brain, including cortex, cerebellum, basal ganglia, and SN pars compacta (Banisadr et al., 2003). Damage in the SN and striatum induced by MPTP and 3-NP may increase the expression of SDF-1 and CXCR4, leading to recruitment of MSCs to these regions. In this study, the number of surviving hMSCs in the SN and striatum 20 days after hMSC administration was approximately 2.0% and 3.8% of the total number of injected hMSCs, respectively. These migrated cells may contribute to modulate the microenvironmental cascade of the neurodegenerative process in the SN and striatum.

B. Inhibition of blood brain barrier (BBB) permeability by hMSCs.

The present study demonstrated that hMSCs treatment significantly stabilized BBB permeability in the SN of LPS-injected animals. The effects of BBB stabilization would be mediated by modulation of inflammatory, protection of astrocytic end feet, and inhibition of BBB transporter by hMSCs migrated from peripheral circulation into the SN.

Ample evidence has suggested that a microglial reaction and inflammatory processes participate in the cascade of neuronal degeneration in neurodegenerative disease through various pathomechanisms (Gao and Hong, 2008). Of those, neuroinflammation-mediated BBB breakdown is one of the main contributors to propagation of neurodegenerative changes (Desai et al., 2007). Additionally, BBB breakdown also disturbs astrocyte end feet and enhance neutrophil infiltration, thus further deteriorating BBB integrity in PD animal model (Ji et al, 2008).

Our study demonstrated that hMSCs had protective effects of BBB function against LPS-induced neutrophil infiltration. The number of EB and EBA-ir after hMSC treatment was in accordance with reduced neutrophil infiltration following hMSC treatment in LPS-induced animal models. The properties of BBB stabilization by MSCs seem to be mediated by complex mechanisms. First, our study has demonstrated that hMSCs can restore the function of astrotic end feet surrounding endotherial cells. In this study, hMSC treatment significantly increased the number of GFAP-ir in LPS-induced animal models. The lower density of astrocytes in the SNpc might be ineffective at downregulating inflammatory responses and at maintaining BBB structure (Ross et al, 1995; Tomas-Camardiel et al, 2004;

Vizuete et al, 2000). Astrocytes participate in nutritive and metabolic support of neuron.

Thus, astrocytes are in a position to disperse vascular nutrients away from the vessels in support of neural function and astrocytes can down-regulate microglial inflammatory reactions by, for example, reducing iNOS and IL-12 expression (Aloisi et al, 1997; Min et al, 2006; Pyo et al, 2003; Vincent et al, 1996). Furthermore, astrocytes play a critical role in the formation of the BBB (Kacem et al., 1998). Increase in astrocyte density would stabilize

BBB permeability by astrotic end feet surrounding endotherial cells (Ji et al, 2008). Along with increase of GFAP-ir by hMSCs, our data showed that hMSCs can also modulate astrotic end feet surrounding endotherial cells.

Second, hMSC treatment had anti-inflammatory showing significantly decreased activation of microglia in LPS-treated SN. Recently study demonstrated that BBB dysfunction occur by neuroinflammation in 6OHDA-induced animal model (Carvey et al, 2005). Previous our study demonstrated that MSCs treatment reduce microglial activation and neuroinflammation through reduction of TNF-a, pre-inflammatory factor and then MSC released anti-inflammatory factors ; IL-6, TGF-b (Kim & Park et al, 2009). A primary product of microglial activation is TNF-a, which is also known to increase BBB permeability (Tsao et al., 2001; Didier et al., 2003). Accordingly, our data suggest that hMSCs would stabilize BBB in LPS-induced animal models through anti-inflammatory effects.

Third, hMSC treatment can modulate of abnormal transporter activity, showing significantly decreased activation of p-gp in LPS-treated SN. Carvey et al, 2005 demonstrated that BBB dysfunction occur by neuroinflammation and then increase transporter, p-gp in the tight junction in 6OHDA-induced animal model, suggesting the role of p-gp in BBB stabilization.

The consequence of BBB alteration in parkinsonian diseases remains speculative. In the condition of neurodegenerative disease, the neurons or glial cells are confronted with microenvironment of decreased homeostatic reserve and thus become extremely sensitive

and vulnerable to an altered composition of the extracellular milieu (Popescu et al, 2009). In turn, increased BBB permeability may increase the probability of exposing microenvironments in the brain to environmental neurotoxins interrupting mitochondrial electron transport or elements of the peripheral immune system, and subsequently accelerate pathological changes of neuro-glial degeneration and neuroinflammation in the cascade of degenerative processes. Therefore, the strategy for BBB stabilization may act as a modifier of disease progression in neurodegenerative diseases. In this regard, our data suggest that the BBB stabilizing property of hMSCs may provide a useful therapeutic strategy for the prevention and/or treatment of PD.

. CONC

Ⅳ LUSION

In conclusion, we have shown that hMSCs treatment has a protective effect against neuronal death in the SN and striatum induced by double mitochondrial neurotoxins and protective effects of BBB function against LPS-induced neutrophil infiltration. Modulation of inflammatory actions and cell survival and death signaling pathways by hMSCs may work in the neuroprotective process, which may be applicable clinically in patients with neurodegenerative disease of parkinsonian diseases as one of candidates for neuroprotective strategies.

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