Adipose-derived stem cells decreased microglia activation and protected dopaminergic loss in rat lipopolysaccharide model

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Muñoz MF, Argüelles S, Medina R, Cano M and Ayala A. Adipose-derived stem cells decreased microglia activation and protected dopaminergic loss in rat lipopolysaccharide model. J Cell Physiol. 2018; 234: 13762–13772, which has been published in final form at https://doi.org/10.1002/jcp.28055 This article may be used for noncommercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." 1 Adipose‐derived stem cells decreased microglia activation and protected dopaminergic loss in rat lipopolysaccharide model Mario F. Muñoz1, Sandro Argüelles2, Rafael Medina2, Mercedes Cano2, Antonio Ayala 1 1Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain 2Departamento de Fisiología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain Correspondence Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, C/. Tramontana s/n, 41012‐ Sevilla, Spain. Email: [email protected] Funding information Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía, Grant/Award Number: BIO‐158 Abstract Adult stem cell therapy is being used extensively to rejuvenate damaged tissue. One important tissue source to obtain these cells is adipose, which contains cells called adipose‐ derived stem cells (ADSCs). These cells have a great therapeutic potential not only for their multipotent properties as well as for immunomodulatory effects on the immune system. Parkinson’s disease is characterized as neurodegenerative disorder which etiology is undoubtedly related to neuroinflammation process. The properties of ADSCs can be used as a new tool in stem cells therapy to treat neurodegenerative disorders. However, their efficacies are still controversial. Some authors have reported neuroprotection effects, while others did not find differences or stem cells increased the damage. Our previous study showed that ADSCs can survive a long time after transplantation, suggesting to us some biological effects could need more time to be repaired. In this study, we assessed the neuroprotection 6 months after transplantation. Our results suggest ADSCs can protect the dopaminergic loss after lipopolysaccharide (LPS) injection both reducing the microglia activation and differentiating into dopaminergic cells. Keywords: adult stem cells; mesenchymal stem cells; adipose tissue stem cells, engraftment, neuroprotection, Parkinson. Abbreviations: ADSCs, MSCs, SCs, LPS, SN. 2 INTRODUCTION 1 Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive degeneration 2 of the nigrostriatal dopaminergic (DAergic) neurons in substantia nigra pars compacta (SNpc; Olanow et 3 al., 2003). This dopamine (DA) deficiency results in cardinal motor symptoms such as tremor at rest, 4 bradikinesia, muscular rigidity, stooped posture, and instability (Tolosa, Gaig, Santamaría, & Compta, 2009). 5 Age is the highest risk factor for PD and its incidence is expected to increase in the upcoming years due 6 to an increase of life expectancy (Kontis et al., 2017). Other important risk factors for PD are exposure to 7 pesticides, consumption of dairy products, history of melanoma, and neuroinflammation (Ascherio & 8 Schwarzschild, 2016; Herrera et al., 2015; Nolan, Sullivan, & Toulouse, 2013). Recent evidence suggests 9 that inflammation may play a central role in the dopaminergic neuron loss seen in PD (Calabrese et al., 2018; 10 Harms et al., 2018; Hirsch & Hunot, 2009; Tansey & Goldberg, 2010; Tansey, McCoy, & Frank‐Cannon, 11 2007). It has been extensively demonstrated in animal models that the intranigral injection of 12 lipopolysaccharide (LPS) selectively induces the death of DA neurons (Herrera, Castaño, Venero, Cano, & 13 Machado, 2000). The therapeutics options in PD have been mainly focused on the treatment of symptoms by 14 increasing the levels of DA in SN (Tuite & Riss, 2003), but in the last decade new approaches have taken 15 relevance such as neurotrophic factors, gene, and cell therapy (Arjona et al., 2003; Politis & Lindvall, 2012; 16 Stayte & Vissel, 2014). 17 The most promising novel therapy is the stem cells (SCs) transplantation aimed to replace damaged 18 dopaminergic neurons. SCs have the ability to self‐renew by symmetric divisions and the potentiality to 19 differentiate into other cell types depending on their multipotency (Teo & Vallier, 2010; Trounson, 20 Thakar, Lomax, & Gibbons, 2011). However, especially embryonic stem cells (ESCs) and induced 21 pluripotent stem cells (iPSCs) have been associated with high cost and difficulty in producing a standar22 dized product, as well as having ethical implications (Stoker, Blair, & Barker, 2017). Another resourceful 23 alternative is the use of adult SCs that can be isolated from almost all tissues but showing more limited 24 multipotentiality and self‐renewal abilities than ESCs or iPSCs (Gimble, Katz, & Bunnell, 2007; 25 Schäffler & Büchler, 2007; Trounson et al., 2011). In the case of adipose‐ derived stem cells (ADSCs), 26 which contain up to 500 times more mesenchymal stem cells (MSCs) than bone marrow (Fraser, Wulur, 27 Alfonso, & Hedrick, 2006), and can be differentiated into adipocytes, osteoblasts, chondrocytes, myocytes 28 and neuronal cells (Gimble et al., 2007; Huang et al., 2013; Zuk et al., 2002). Moreover, it has been 29 described that ADSCs show immunomodulatory abilities able to minimize the collateral harming 30 effects of inflammatory process (Dazzi, Lopes, & Weng, 2012; English & Mahon, 2011; Thakur et al., 31 2013). There are several procedures by which ADSCs can be used to repair and regenerate tissues, this 32 3 being a promising alternative, given their natural secretion of trophic factors and cytokines as well as their 33 immunosuppressive and low tumorigenic properties on transplantation into allogenic and autologous 34 hosts (Yagi et al., 2010). However, other authors showed MSCs transplanted to the intact adult brain are 35 rejected by an inflammatory response (Coyne, Marcus, Woodbury, & Black, 2006). 36 Recently, studies on functional differentiation of ADSCs into neuron cells are becoming clinically 37 important in the context of new treatment against some neurodegenerative diseases, including Parkinson’s 38 and Alzheimer’s disease. However, it is still not clear that rat ADSCs (rADSCs) can ameliorate PD 39 symptoms by autologous transplantation into the rat SN (Eliopoulos, Stagg, Lejeune, Pommey, & Galipeau, 40 2005). These controversial results can be explained because the mechanisms of neuroprotection and 41 regeneration have been investigated in short‐terms experiments. For the effective use of ADSC against 42 neurodegeneration in translational medicine, it is necessary to ensure the long‐term capacity of survival and 43 differentiation of ADSCs when they are injected in vivo. The aim of this study was to elucidate the effect 44 of rADSCs in the promotion of neuroprotection and regeneration against LPS‐induced damage in the rat 45 SN 6 months after the transplantation. 46 47 48 MATERIAL AND METHODS 49 Animals 50 All animal experiments were carried out according to the guidelines of the European Union Council 51 (Directive 2010/63/UE), in agreement with Spanish regulations (BOE 34/11370, RD 53/2013) and were also 52 approved by the University of Seville ethical committee. Male Wistar rats (300–500 g) were kept at a 53 constant temperature of 22 ± 1°C in a relative humidity of 60%, with a 12‐hr light–dark cycle and free access 54 to food and water. 55 56 Isolation, differentiation, and phenotyping of rADSCs 57 rADSCs were isolated by immunomagnetic separation from the stromal vascular fraction contained in the 58 rat adipose tissue. Cells were cultured, differentiated into adipocytes, osteoblasts, and chondroblasts, and 59 phenotyped using CD29 and CD90 as positive markers and CD11b, CD34, and CD45 as hematopoietic 60 markers, as we have previously described (Munoz et al., 2018). 61 62 63 Transfections, lentivirus production, and infections of rADSCs with a dual GFP‐Luc reporter gene 64 4 The GFP‐Luc reporter system was based on the lentivirus pHRSIN‐ DUAL‐GFP, kindly provided by Mary 65 K. Collins (Windeyer Institute, London), which expresses two GFP genes under two different promoters. 66 Transfections, lentivirus production, and infections of rADSCs with a dual GFP‐Luc reporter gene were carried 67 out as we have previously described (Munoz et al., 2018). Xenogen IVIS Lumina II imaging system and IVIS 68 Imaging 4.2 software (Caliper Life Science, Hopkinton, MA) were used to detect and analyze the 69 bioluminescence imaging (BLI) signal. The cells used in the experiments were rADSC‐GFP‐Luc+. 70 71 rADSC‐GFP‐Luc+ transplantation in SN 72 Animals were randomly divided into three groups (N = 4): control, LPS, and LPS + rADSCs. The rats were 73 anesthetized and fixed with isofluorane in O2:N2O (30%:70%). During surgery, the rats were fixed in a stereotaxic 74 instrument (Stoelting, Dublin, Ireland). The skull was exposed through a small incision and a hole was drilled 75 at the following coordinates relative to bregma: AP: −5.4; L: +1.8; DV: −8.3. Two microliters of saline (control), 76 2 µl of LPS (4 µg/µl), and 2 µl of rADSCs (25.000 cells/µl) previously resuspended in LPS (4 µg/µl) were 77 subsequently injected into the brain through a Hamilton neurosyringe. One percent Monastral blue (Sigma‐ 78 Aldrich, St. Louis, MO) was added for monitoring the injection site. 79 80 Immunofluorescence 81 Six months after the transplant, brains were post‐fixed, cryoprotected, embedded, and frozen in isopentane 82 at –80°C. Thaw‐mounted 20‐μm coronal sections of SN were cut on a cryostat at −15°C and mounted on 83 gelatine‐coated slides. Sections were rehydrated in phosphate buffered saline (PBS) for 10 min, and then 84 blocked with PBS containing 1% normal serum for 1 hr. The blocking solution was replaced with the primary 85 antibodies diluted in the blocking solution containing 0.25% Triton‐X‐100, and the slides were then 86 incubated in humid chamber overnight at 4–8°C. For the immunofluorescence, the primary antibodies were 87 rabbit or mouse anti‐luciferase (anti‐Luc; Sigma‐ Aldrich; 1:500), rabbit anti‐GFP (D5.1) XP® (Cell 88 Signaling, Danvers, MA; 1:500), rabbit anti‐Iba1 (Wako, North Chesterfield, VA; 1:200), and rabbit anti‐ 89 tyrosine hydroxylase (anti‐TH, Sigma‐Aldrich; 1:300) 90 After the incubation period, slides were incubated with secondary antibodies: AlexaFluor 488‐conjugated 91 goat anti‐rabbit (Cell Signaling; 1:200), and TexasRed‐conjugated horse anti‐mouse (Vector Laboratories, 92 Burlingame, CA; 1:200). To stain the nucleus, we used Hoechst (Sigma‐Aldrich; 1:1000). Fluorescent 93 images were acquired using a Zeiss LSM 7 DUO confocal laser scanning microscope (Carl Zeiss 94 Microscopy, Jena, Germany), and processed using the associated software package (ZEN 2010; Carl Zeiss 95 Microscopy). Colocalization fluorogram were obtained merging images from both channels in the ImageJ 96 software (NIH). The immunofluorescence controls were performed as mentioned above but without 97 primary antibodies. 98 5 99 Immunohistochemistry 100 Animals were perfused and sections were prepared as we mentioned above. Sections were treated with 0.3% 101 hydrogen peroxide in methanol and incubated with the primary antibody overnight. The primary antibodies 102 were rabbit anti‐Luciferase (Sigma‐Aldrich; 1:500), rabbit anti‐GFP (D5.1) XP® (Cell Signaling; 1:500) 103 rabbit anti‐tyrosine hydroxylase (anti‐TH; Sigma‐Aldrich; 1:300) and mouse OX‐6 (Serotec, Oxford, UK; 104 1:200). Sections were then incubated for 2 hr with biotinylated horse anti‐mouse IgG (Vector laboratories, 105 Burlingame, CA; 1:200) for OX‐6 immunostaining or biotinylated goat anti‐rabbit IgG (Vector; 1:200) for 106 TH, Luc, and GFP immunostaining. ExtrAvidin‐Peroxidase solution (Sigma‐Aldrich; 1:100) was added 107 and visualized with a standard diaminobenzidine/hydrogen peroxide reaction for 5 min. 108 109 Stereological data analysis 110 A bounded region of the SN with a length of 300 μm in the anterior– posterior axis centered at the point of 111 injection (5.5 mm with respect to bregma) was used for analysis (i.e., between 5.35 and 5.65 mm with 112 respect to bregma). Similarly, a bounded region of the striatum with a length of 300 μm in the anterior– 113 posterior axis centered at the point of injection (0.5 mm with respect to bregma) was used for analysis. For 114 the measurement of the areas expressing OX‐6 and TH immunoreactivity, we used the AnalySIS® image 115 software (Soft Imaging System GmbH, Münster, Germany) coupled to a Polaroid DMC camera (Polaroid, 116 Cambridge, MA) attached to a Leika light microscope (Leika Mikroskopie, Wetzlar, Germany). For each 117 animal, five sections (sampling fraction 1:3) were systematically sampled 118 along the anterior–posterior axis from a random starting point, following stereological criteria 119 (Gundersen et al., 1988). 120 To count cells showing OX‐6 immunoreactivity, we systematically sampled the area occupied by the 121 OX‐6‐positive cells in each section from a random starting point with a grid adjusted to count five fields per 122 section. An unbiased counting frame of a known area (40 × 25 μm= 1,000 μm2) was superimposed on the 123 tissue section image under a 100 × oil immersion lens objective. The different types of OX‐6‐positive cells 124 (displaying different shapes, depending on their activation state) were counted as a whole and expressed as 125 cells per millimeter squared. The number of TH‐positive neurons in the SN was estimated using a fractionator 126 sampling design (Gundersen et al., 1988). Counts were made at regular predetermined intervals (x = 150 μm 127 and y = 200 μm) within each section. An unbiased counting frame of known area (40 μm× 25 μm = 1000 128 μm2) was superimposed on the tissue section image under a 100 × oil immersion objective. Therefore, the area 129 sampling fraction was 1000/(150 × 200) = 0.033. The entire z‐dimension of each section was sampled; hence, 130 the section thickness sampling fraction was 1. In all animals, 20‐μm sections, each 100 μm apart, were 131 analyzed; thus, the fraction of sections sampled was 20/100 = 0.20. The number of neurons in the SN was 132 6 estimated by multiplying the number of neurons counted by the reciprocals of the area sampling fraction and 133 the fraction of section sampled. 134 135 Statistical analysis 136 Results are expressed as mean ± SEM. Means were compared by one‐ way analysis of variance followed by 137 the Tukey test for post hoc multiple range comparisons. An α level of 0.05 was used. The IBM SSPS 138 Statistics 21 package (IBM, Armonk, NY) was used for the analyses. 139 140 RESULTS 141 rADSCs characterization 142 To confirm that our cell population shows enough characteristics to be considered as MSCs, several criteria 143 established by International Society for cellular therapy (ISCT) such as adherence, differentiation, and 144 phenotyping were analyzed. First, we did not find evidence about cells in suspension after immunomagnetic 145 isolation (Figure 1a). Second, we examined the trilineage differentiation capability of rADSCs in vitro, and 146 we found that these cells can be differentiated into adipocytes, assessed by Oil‐O‐Red staining, osteoblast, 147 detected by alkaline phosphatase staining, and chondroblast determined by immunofluorescence using anti‐ 148 aggrecan antibody (Figure 1b). Third, we characterized by flow cytometry the rADSCs CD markers, and we 149 observed that up to 98% of the total of cells expressed the positive MSCs markers as CD29 and CD90, and 150 less than 3% expressed hematopoietic markers CD11b, CD34, and CD45 (Figure 1c). 151 To develop the imaging approach, rADSCs were labeled with the Luc‐GFP2 dual reporter gene by 152 lentiviral infection. Both markers were used to track the cells after the engraftment. The reporter gene was 153 driven by a constitutive SFFV promoter for Luc and UBIQ1 for GFP (Figure 1d). Both proteins were 154 expressed in rADSCs after transfection. Cells showed a strong BLI signal in culture (Figure 1e) and the 155 labeling efficiency was 68.71% based on the cytometry flow analysis of GFP‐positive cells (Figure 1f). 156 However, 6 months after transplantation, in the area of engraftment (Figure 1g) GFP‐positive cells were 157 not detectable by immunological techniques, but Luc‐positive cells were easily identified both 158 immunofluorescence and immunohistochemical staining (Figure 1h,i). We continued our tracking study 159 using Luciferase marker as tracker. 160 161 162 rADSCs suppress microglial activation 163 To determine the immunomodulatory effect of rADSCs in the immune response, we examined the 164 7 microglial activation in the engraftment after LPS treatment. As we expected, LPS led to a marked increase 165 of the number of Iba1‐positive cells, which is a marker of microglia activation (Figure 2a). However, 166 animals treated with rADSCs in combination with LPS showed considerably fewer Iba1‐ positive cells 167 (Figure 2b) along the whole engraftment (Figure 2c). The decrease of microglial activation was not the only 168 effect observed in animals treated with rADSCs in combination with LPS, we also found a colocalization 169 of Iba1 and Luc in some cells along of engraftment (Figure 2d,e). This colocalization took place into live 170 cells and not in apoptotic, as can be easily seen by the nuclear labeling staining, which did not show any 171 sign of fragmentation (Figure 2f). Moreover, the fluorogram analysis of colocalization showed a positive 172 correlation between Iba1 and Luc (Figure 2g). This colocalization between Luc and Iba1 is the result of 173 rADSCs + LPS combination, since rADSCs transplantation without LPS did not show any colocalization 174 between Luc and Iba1, even though the Iba1 expression was mainly localized in the needle‐track through 175 which cells were engrafted (Supporting Information Figure 1). 176 We observed that animals treated with rADSC in combination with LPS showed less microglia than 177 animals treated with LPS alone. Then, to confirm rADSCs are able to suppress microglia activation along 178 SN, we performed a stereological analysis using OX‐6 as microglial activation marker. 179 Immunohistochemistry of OX‐6 showed high activation of microglia as seen by a significant increase in 180 the number of OX‐6 immunopositive cells in animals treated with LPS alone, and less activation of 181 microglia in animals treated with rADSC in combination with LPS (p < 0.001). We confirmed that rADSCs 182 significantly decreased the microglial activation by 48% with respect to LPS group (Figure 3). 183 184 rADSCs prevent dopaminergic neuron loss 185 To confirm that a suppression of microglial activation mediated by rADSCs can prevent the dopaminergic 186 neurons loss, we examined how the concomitant administration of rADSCs and LPS along the engraftment 187 area could affect the dopaminergic cell population. In that area we observed cells which expressed both TH 188 and Luc (Figure 4a,b) along the engraftment. However, the TH‐positive region in middle of the engraftment 189 (Figure 4b) did not correspond with the SN (left down in Figure 4b). Moreover, some cells were able to 190 express both markers, TH and Luc (Figure 4c,d), being confirmed by the fluorogram analysis of 191 colocalization that showed a positive correlation between TH and Luc expression (Figure 4f). To discard the 192 possibility that green region in the middle of the engraftment was due to autofluorescence in the CNS tissue 193 samples, we performed an immunofluorescence and immunohistochemistry of TH in two consecutives 194 slides, respectively. As can be seen, the TH‐positives cells into the cell engraftment identified by the green 195 color (Figure 5a,b) were also shown by immunohistochemistry by DAB oxidation (Figure 5c). 196 To analyze whether rADSCs can protect the loss of dopaminergic neurons along SN, we performed in all 197 groups a stereological analysis using TH as a dopaminergic neuron marker. Immunohistochemistry of TH 198 8 showed a high absence TH‐positive cells in animals treated with LPS, although the group treated with 199 rADSCs in combination with LPS (p < 0.001) showed significantly more TH‐positive cells (Figure 6). 200 These results can demonstrate that rADSCs in combination with LPS avoid the dopaminergic loss by 28% 201 in comparison with the group treated only with LPS, which showed a dopaminergic loss of 85% with respect 202 to saline. 203 204 DISCUSSION 205 Although the therapeutic application of rADSCs in chronic or degenerative diseases should still be fully 206 elucidated, the use of these cells in translational medicine to repair and regenerate tissues is a promising 207 alternative for cell replacement therapy. To understand the biology of rADSCs, we recently studied the 208 long‐term capacity for survival and differentiation of rADSCs when they are injected in vivo. Interestingly, 209 we found that rADSCs were able to survive a long time (up to 4 months) after transplantation into healthy 210 tissue, besides showing resident cell tissue phenotype (Munoz et al., 2018). In this study, we focused on 211 determining whether rADSCs can also survive in an inflammatory environment and prevent the damage in 212 the SN induced by neurotoxic agents. We provide evidence that rADSCs can survive a long time after 213 transplantation in an inflammatory environment, suppress microglial activation and prevent dopaminergic 214 neuron loss induced by LPS in the SN at 6 months after the transplantation. 215 Our laboratory has extensive experience in LPS models of PD to induce microglia activation, 216 neuroinflammation, and death of dopaminergic neurons (Castaño, Herrera, Cano, & Machado, 1998; De 217 Pablos et al., 2014; Herrera, Cano, & Machado, 2002; Machado et al., 2011). The immunomodulatory 218 abilities and the neuronal differentiation capacity of some MSCs such as rADSCs (Dazzi et al., 2012; 219 English & Mahon, 2011; Munoz et al., 2018; Thakur et al., 2013) makes rADSCs a potential candidate to 220 treat neurodegenerative diseases. However, the use of MSCs as an effective PD treatment is still 221 controversial because MSCs can both stimulate and inhibit the immune system (Dazzi et al., 2012; English 222 & Mahon, 2011; Yagi et al., 2010). In fact, some authors have demonstrated a neuroprotective effect of 223 these cells in vitro and in vivo (Ooi, Dheen, & Tay, 2015; Schwerk et al., 2015; Stemberger et al., 2011; F. 224 Wang et al., 2010), but other authors could not demonstrate it (Camp, Loeffler, Farrah, Borneman, & 225 LeWitt, 2009; Eliopoulos et al., 2005). After positive characterization of rADSCs we confirmed that this 226 population of cells possess MSCs identity such as cell adherence, phenotyping and multipotentiality 227 (Figure 1a–c). 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Human 484 adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell, 13(12), 4279– 4295. 485 https://doi.org/10.1091/mbc.E02‐02‐0105 486 17 18 FIGURE 1. Characterization and tracking of rADSCs. Microscopic image of rADSCs adherence obtained from immunomagnetic isolation (a). Multipotential differentiation to adipocytes was shown by Oil‐O‐Red staining, osteoblast differentiation was detected by alkaline phosphatase staining and chondroblast was determinate by anti‐aggrecan antibody. Scale bar = 50 µm (b). Almost all rADSCs cells (98%) expressed typical MSCs markers CD29/CD90.1 and the majority (97%) were negative for the marker combination CD11b/CD34/CD45 for cells obtained from immunomagnetic isolation (c). Lentivirus vector scheme carrying reporter gene (GFP and Luciferase) (d). In vitro bioluminescence imaging of rADSCs‐Luc‐GFP after 3 s of exposure (e). Flow cytometry analysis of rADSCs‐Luc‐GFP with 68.71% of cells transfected (f). Overview brain region (g). Immunofluorescence (h) and Immunohistochemistry (i) of GFP and Luc in SN after 6 months of engraftment with LPS + rADSCs. Scale bar = 50 µm. rADSCs: rat adipose derived stem cells; GFP: green fluorescent protein. 19 FIGURE 2. Immunofluorescence confocal microscopy of Iba1 (green), Luc (red), and Hoechst (blue) in substantia nigra after 6 months of engraftment. Group treated with LPS (a), and group treated with LPS + rADSCs (b). Region of interest in LPS + rADSCs group separated by colors (c) and merge (d). The colocalized area (white asterisk) was enlarged in panel (e). Colocalization of Iba1 and Luc in higher magnification (f). The colocalization diagram was performed using the ImageJ software (g). White scale bar = 200 µm. Red scale bar = 20 µm. rADSCs: rat adipose derived stem cells; LPS: lipopolysaccharide. 20 FIGURE3. Quantitative score of activated microglial cells (OX‐6‐positive cells) in substantia nigra after 6 months of engraftment. Quantification was carried out as described in Section 2 (N = 4, ap < 0.01 related to control, bp < 0.01 related to LPS). White scale bar = 500 µm, black scale bar = 200 µm. rADSCs: rat adipose derived stem cells; LPS: lipopolysaccharide. 21 FIGURE 4. Immunofluorescence confocal microscopy of TH (green), Luc (red), and Hoechst (blue) in substantia nigra treated with LPS and rADSCs after 6 months of engraftment. Region of interest separated by colors (a) and merge (b). The colocalized area (white asterisk) was enlarged in (c). Colocalization of TH and Luc in higher magnification (d). The colocalization diagram was performed using the ImageJ software (e). White scale bar = 200 µm, red scale bar = 20 µm. rADSCs: rat adipose derived stem cells; LPS: lipopolysaccharide; Luc: luciferase; SN: substantia nigra; TH: tyrosine hydroxylase 22 FIGURE 5. Expression of TH‐positive cells in the engraftment area. Immunofluorescence confocal microscopy of TH (green), Luc (red), and Hoechst (blue) separated by colors (a) and merge (b). The consecutive slides were used to perform immunohistochemistry of TH (c). Scale bar = 500 µm. Luc: luciferase; TH: tyrosine hydroxylase. FIGURE 6 Quantitative score of dopaminergic cells (TH‐positive cells) in substantia nigra after 6 months of engraftment. Quantification was carried out as described in Section 2. (N = 4, ap < 0.01 related to control, bp < 0.01 related to LPS). White scale bar = 500 µm, black scale bar = 200 µm. rADSCs: rat adipose derived stem cells; LPS: lipopolysaccharide; TH: tyrosine hydroxylase.