B. METHODS
12. Statistical analysis
The mean of the groups were compared using the Mann–Whitney U-test for pairs and the Kruskal–Wallis analysis for multiple comparisons. The statistical significance was indicated when p < 0.05. Statistical analyses were performed using commerciallyavailable software (version 10.0; SPSS Inc., Chicago, IL, USA).
. RESULTS
microglia drastically changed from spherical shape to process-bearing cells at 6 and 24 hr after co-culture with vehicle. However, co-culture with hMSCs (3 × 105/well, using transwell) after LPS treatment significantly decreased the number of process-bearing, activated microglia at 6 and 24 hr after co-culture with hMSCs (Fig. 1 A and B).
2. hMSCs treatment significantly decreased LPS-induced expression of inflammatory cytokine
To investigate the effect of hMSCs co-culture on LPS-induced production of inflammatory cytokines and their gene expression in microglial, microglia enriched cultures were treated with LPS for 4 hr and then co-cultured with vehicle or hMSCs using transwell. After 6, 24, 48 and 72 hr, culture supernatants and cells were collected for RT-PCR, nitrite assay and TNF-α ELISA.
LPS treatment significantly induced the mRNA expression of TNF-α and iNOS, accumulation of nitrite compared with control group, whereas co-culture with hMSCs showed significant reduction of TNF-α and iNOS mRNA expression and amount of nitrite when compared to those treated only with LPS at 24, 48, and 72 hr (Fig. 2 A - C). And co-culture of hMSCs responsively inhibited the LPS induced increase in TNF-α protein levels, the levels of TNF-α were measured by ELISA. As shown in Fig. 3 D, at 24 hr after injection hMSCs, the production of TNF-α was
dramatically increased by LPS alone injection and the level gap was maintained until 72 hr. The stimulated levels of TNF-α were significantly reduced in injection of hMSCs group.
3. hMSCs treatment significantly reduced dopaminergic neuronal death induced by LPS stimulation in mesencephalic tissue and microglia co-cultured system
Co-cultures of microglia and mesencephalic neurons were treated with LPS for 4 hr and then co-cultured with vehicle or hMSCs to determine the effect of hMSCs-induced anti-microglial activation on dopaminergic neurons. After 6 or 24 hr, cultures were immunostained with anti-TH antibody. As shown in Fig. 3, the LPS treatment resulted in a significant loss of anti-TH-ip cells, while there was significantly decreased the loss of TH-ip cells in cultures that were treated with hMSCs (Fig. 3 A). A cell counting analysis showed that co-culture with hMSCs resulted in an incremental survival of TH-ip cells at 6 and 24 hr following hMSCs treatment (Fig. 3 B).
4. hMSCs treatment significantly decreased dopaminergic neuronal loss and microglial activation induced by LPS stimulation in the SN
In animal study, hMSCs was infused via tail vein 8 hr after the injection of LPS to examine the effect of hMSCs on LPS-induced microglial activation and dopaminergic neuronal death in rat SN. LPS stimulation in the SN resulted in a considerable loss of TH-ip cells with a concomitant activation of microglia (Fig. 4 A and C). hMSCs treatment showed that the loss of TH-ip cells induced by LPS stimulation in the SN was considerably reduced (Fig. 4 A). As well, hMSCs treatment was clearly accompanied by attenuation of microglial activation, which was detected by OX-42 staining (Fig. 4 C). On stereological analysis, hMSCs treatment significantly
increased survival of TH-ip cells at 7 and 14 days following hMSCs injection, approximately two times more than the LPS-only treatment (Fig. 4 B). Additionally, cytokine assay in the SN showed that hMSCs treatment significantly down regulated the LPS-induced increase in the expression of TNF-α and iNOS mRNA at 3 day following LPS stimulation (Fig. 5 A). TNF-α and iNOS release were significantly decreased in the hMSCs group when compared to the LPS-only treatment group at 4 hr and 3 day following hMSCs injection (Fig. 5 B).
5. hMSCs reduced the protein expression of TNF-α release induced by LPS stimulation Injection of hMSCs responsively inhibited the LPS induced increase in TNF-α protein levels, the levels of TNF-α were measured by ELISA. As shown in Fig. 6, at 4 hr after injection hMSCs, the production of TNF-α was dramatically increased by LPS alone injection. The stimulated levels of TNF-α were significantly reduced in injection of hMSCs group. In 7 day after injection of vehicle or hMSCs, the amount of TNF-α present in the SN was reduced to levels comparable to the level of the control.
Fig. 1. hMSCs inhibited LPS-induced microglial activation. Rat microglia-enriched cultures 1 day after plating were treated with the hMSCs for 6 and 24 hr with 100 ng/ml LPS. The microglia were immunostained with OX-42 antibody. The morphology of microglia dramatically changed from spherical shape to process-bearing cells at 6 and 24 hr after co-culture with vehicle. However, co-co-culture with hMSCs after LPS treatment significantly decreased the number of process-bearing, activated microglia at 6 and 24 hr after co-culture with hMSCs (A and B). The data are displayed as the mean (column) ± SEM (bar) values. The results are representative of three to five in each group. **P < 0.01. ***P < 0.001. Scale bar, 100 µm.
0
Fig. 2. Effect of hMSCs co-culture on LPS induced production of proinflammatory factors and their gene expression in microglial. Microglia en-riched cultures treated with LPS for 4 hr and then co-cultured with vehicle or hMSCs (3x105 cells) using transwell. After 6, 24, 48 and 72 hr, culture supernatants and cells were collected for RT-PCR, nitrite assay and TNF-α ELISA.
LPS treatment induced the mRNA expression of TNF-α and iNOS and accumulation of nitrite compared with control group, whereas hMSCs co-culture group was significantly reduced the LPS-induced increase in the expression of TNF-α and iNOS mRNA levels (A and B) and amount of nitrite (C). And the stimulated levels of TNF-α were significantly reduced in hMSCs co-cultured group. The data are displayed as the mean (column) ± SEM (bar) values. The results are representative of three in each group. *P < 0.05. ** P < 0.01. ***P < 0.001.
Fig. 3. Neuroprotective effects of hMSCs on LPS-induced neurotoxicity on mesencephalic neuron-glia cultures. In 4-day-old in vitro cultures, the medium was replaced with a treatment medium and treated with the vehicle or hMSCs 4 hr later LPS treatment. After 6 or 24 hr, cultures were immunostained with anti-TH antibody. LPS treatment led to a loss of TH- immunopositive (TH-ip) neurons and the TH-ip neurons were unhealthy, with dramatically shortened and damaged neuritis. The hMSCs treatment blocked LPS-induced morphological changes of mesencephalic dopaminergic neurons. The data are displayed as the mean (column)
± SEM (bar) values. The results are representative of three to five in each group. *P < 0.05. **P
< 0.01. Scale bar, 100 µm.
Fig. 4. Morphological evidence of the protective effect of hMSCs against LPS induced damage to dopaminergic neurons in the SN. LPS injection (5 µg/3 µl) in the SN and 8hr later, vehicle or hMSCs (1x106 cells) were injected to tail vain. On 4hr, 3day, 7day and 14 day, rats were transcardially perfused by 4% paraformaldehyde. Frozen sections (50 µm in thickness) were cut and TH was detected by immunohistochemical staining to show dopaminergic neurons (A) and OX-42 was detected to show microglial activation (C) in the SN. The stereological estimation of the total number of TH-ip cells in the SN using the optical fractionator demonstrated that hMSCs injection significantly decreased LPS-induced dopaminergic neuron death (B) and decreased activation of microglia compared with the LPS only treatment group (C). The data are displayed as the mean (column) ± SEM (bar) values. The results are representative of three to five rats in each group. *P < 0.05.
Fig. 5. Analysis of TNF-α and iNOS gene expression was conducted via RT-PCR. At 4 hr, 3 day and 7 day after vehicle or hMSCs injection, rats were decapitated and SN was dissected and total RNA (2 µg) was extracted from dissected tissue using TRIzol. LPS elicited an increase in the mRNA expression of TNF-α and iNOS, whereas hMSCs injection group was significantly down regulated the LPS-induced increase in the expression of TNF-α and iNOS mRNA levels.
The data are displayed as the mean (column) ± SEM (bar) values. The results are representative of three to five rats in each group. *P < 0.05.
0
Concentration of TNF-αConcentration of TNF-αConcentration of TNF-αConcentration of TNF-α in SN (μg/mg protein)in SN (μg/mg protein)in SN (μg/mg protein)in SN (μg/mg protein)
con LPS only LPS + hMSCs
Concentration of TNF-αConcentration of TNF-αConcentration of TNF-αConcentration of TNF-α in SN (μg/mg protein)in SN (μg/mg protein)in SN (μg/mg protein)in SN (μg/mg protein)
con LPS only LPS + hMSCs
*
*
**
**
Fig. 6. The effect of hMSCs treatment on the TNF-α release induced by LPS stimulation.
At 4 hr, 3 day and 7 day after vehicle or hMSCs injection, rats were decapitated and SN was dissected. The concentration of TNF-α was detected by commercial ELISA kit. A significant decrease in TNF-α occurs at 4 hr and 3 day. The data are displayed as the mean (column) ± SEM (bar) values. The results are representative of three to five rats in each group. *P < 0.05,
**P < 0.01.
Ⅳ .
Ⅳ
Ⅳ Ⅳ DISCUSSION
The present study demonstrated that hMSCs has a protective effect on dopaminergic neurons through anti-inflammatory actions. First, we demonstrated that in microglia and mesencephalic neuron co-cultures, hMSCs treatment prevented dopaminergic neuronal death by reducing LPS-induced release of the pro-inflammatory cytokines. Second, we confirmed that in rats, hMSCs injection significantly reduced LPS- induced dopaminergic neuronal loss in the SN.
Besides regenerative capacity of hMSCs, it has been known that hMSCs also possess immunoregulatory properties. Although the exact mechanism responsible for hMSC-mediated immunoregulation is not fully understood, in vitro studies suggested that hMSCs can not only inhibit nearly all cells participating in the immune response cell-cell contact-dependant mechanism, but also release a variety of soluble factors, which may be implicated in the immunosuppressive activity of hMSCs (Karussis et al. 2007; Krampera et al. 2006; Nauta and Fibbe 2007). Recent animal studies in a model of experimental autoimmune encephalomyelitis reported that hMSCs treatment showed a significantly milder disease and fewer relapses compared with control mice, with decreased number of inflammatory infiltrates, reduced demyelination, and axonal loss (Gerdoni et al. 2007; Zappia et al. 2005). Additionally, Guo et al.
(Gerdoni et al. 2007) reported that MSC transplantation decreased protein production and gene expression of inflammation cytokines as well as increased functional recovery from myocardial infarct. These studies suggest that anti-inflammation action of hMSCs might be one of underlying mechanisms for the tissue protective effect.
In the present study, hMSCs significantly decreased the release of inflammatory cytokine and
dopaminergic neuronal loss induced by LPS stimulation. In co-cultures of microglia and mesencephalic neurons, these anti-inflammatory actions of hMSCs actually led to a significant decrease (up to ~40%) in dopaminergic neuronal death induced by LPS stimulation.
Furthermore, hMSCs administration dramatically decreased the dopaminergic neuronal loss in the SN induced by LPS stimulation, which was clearly accompanied by attenuation of microglial activation and a reduction in the formation of TNF-α and iNOS. In addition to a variety of pleiotrophic mechanisms of hMSCs as a trophic mediator (Caplan and Dennis 2006), our present data suggest that the neuroprotective property of hMSCs through its anti-inflammatory behavior also works in animal model of PD.
A large body of experimental evidence indicates that inhibition of the inflammatory response can prevent degeneration of nigrostriatal dopaminergic neurons. For example, sodium salicylate, COX-2 inhibitor, or minocycline have been shown to significantly reduce striatal dopaminergic depletion and dopaminergic neuronal loss induced by MPTP or LPS-models (Aubin et al. 1998;
Du et al. 2001; He et al. 2001). A large cohort study of patients has shown that the risk of developing PD in regular nonsteroidal anti-inflammatory drugs (NSAID) users was decreased by up to 45% compared with those who take NSAIDs on a non-regular basis and higher exposure to NSAID demonstrates a trend toward a greater benefit (Chen et al. 2003), supporting the neuroprotective effects of NSAID in the development or progression of PD. A very recent epidemiological study also supports that NSAIDs are protective against PD, with a particularly strong protective effect evident among regular nonaspirin NSAID users (Wahner et al. 2007).
Therefore, the evidence demonstrating the neuroprotective effect of anti-inflammatory agents on the nigrostriatal dopamine system, in an experimental system or epidemiological study, has
revitalized interest in identifying inhibition of inflammation as a possible strategy in the treatment of PD.
Recent studies indicate that human hMSCs can be induced to differentiate into neuron-like cells (Mareschi et al. 2006; Pittenger et al. 1999; Woodbury et al. 2000). Additionally, hMSCs express an expression of several specific neuronal markers and transcriptional factors, of which a large proportion of the genes was participating in the neuro-dopaminergic system, suggesting that expression of neural gene as well as gene associated with the dopaminergic system is a widespread phenomenon of hMSCs (Blondheim et al. 2006). There have been a few reports about application of hMSCs in animal model of PD. Li et al. (Li et al. 2001) and Blondheim et al. (Blondheim et al. 2006) reported that using MPTP and 6-hydroxydopamine-treated PD models, respectively, hMSCs injected intrastriatally exhibited the phenotype of dopaminergic neurons. Along with possible transdifferention potency of hMSCs into dopaminergic phenotype, neuroprotective property of hMSCs on dopaminergic neurons through anti-inflammatory actions may raise the possibility of clinical application of hMSCs as a possible strategy in the treatment of PD. In addition to the molecular and cellular benefits of hMSCs, cell therapy with hMSCs has an advantage in clinical applications. hMSCs can be easily harvested from self bone marrow, cultured in vitro, and administered to patients via various roots including intravenous, intraarterial, intrathecal, or intralesional infusion. In contrast with embryonic stem cell therapy, there is no immunological rejection, and cell therapy with hMSCs is free from ethical issues.
Importantly, regarding the safety of hMSCs application, our group recently documented that cell therapy with hMSCs in patients with multiple system atrophy and ischemic stroke is feasible and safe (Bang et al. 2005; Lee et al. 2007).
In summary, the present study demonstrated that hMSCs have a neuroprotective effect on dopaminergic neurons via an anti-inflammatory mechanism mediated by the modulation of microglial activation. Along with various trophic effect and transdifferentiational potency, anti-inflammatory mechanism of hMSCs could have major therapeutic implication in the treatment of PD.
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