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Anti-Inflammatory Mechanism of Neural Stem Cell Transplantation in Spinal Cord Injury

View Article: PubMed Central - PubMed

ABSTRACT

Neural stem cell (NSC) transplantation has been proposed to promote functional recovery after spinal cord injury. However, a detailed understanding of the mechanisms of how NSCs exert their therapeutic plasticity is lacking. We transplanted mouse NSCs into the injured spinal cord seven days after SCI, and the Basso Mouse Scale (BMS) score was performed to assess locomotor function. The anti-inflammatory effects of NSC transplantation was analyzed by immunofluorescence staining of neutrophil and macrophages and the detection of mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-12 (IL-12). Furthermore, bone marrow-derived macrophages (BMDMs) were co-cultured with NSCs and followed by analyzing the mRNA levels of inducible nitric oxide synthase (iNOS), TNF-α, IL-1β, IL-6 and IL-10 with quantitative real-time PCR. The production of TNF-α and IL-1β by BMDMs was examined using the enzyme-linked immunosorbent assay (ELISA). Transplanted NSCs had significantly increased BMS scores (p < 0.05). Histological results showed that the grafted NSCs migrated from the injection site toward the injured area. NSCs transplantation significantly reduced the number of neutrophils and iNOS+/Mac-2+ cells at the epicenter of the injured area (p < 0.05). Meanwhile, mRNA levels of TNF-α, IL-1β, IL-6 and IL-12 in the NSCs transplantation group were significantly decreased compared to the control group. Furthermore, NSCs inhibited the iNOS expression of BMDMs and the release of inflammatory factors by macrophages in vitro (p < 0.05). These results suggest that NSC transplantation could modulate SCI-induced inflammatory responses and enhance neurological function after SCI via reducing M1 macrophage activation and infiltrating neutrophils. Thus, this study provides a new insight into the mechanisms responsible for the anti-inflammatory effect of NSC transplantation after SCI.

No MeSH data available.


Related in: MedlinePlus

Characterization of neural stem cells (NSCs) from the GFP transgenic mouse in vitro. Mouse NSCs were cultured in growth medium supplemented with 20 ng/mL EGF and 10 ng/mL bFGF. However, to observe the differentiation ability of NSCs, bFGF and EGF were removed, and 1% fetal bovine serum was added into the medium. (A) The phase of dissociated NSCs; (B) the dissociated NSCs expressed GFP (green); (C) the phase of neurosphere; (D) neurosphere-expressed GFP (green); (E) neurosphere-expressed nestin (red); (F) the proliferation of NSCs was determined by cell labeling with 5-ethynyl-2’-deoxyuridine (EdU) (red); (G) immunofluorescent staining of the neuronal marker Tuj1 (red); (H) the astroglial marker GFAP (red); (I) the oligodendrocyte marker O4 (cyan). Nuclei in (F–I) were stained with DAPI (blue). Scale bar: 100 μm (A–F); and 20 μm (G–I).
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ijms-17-01380-f001: Characterization of neural stem cells (NSCs) from the GFP transgenic mouse in vitro. Mouse NSCs were cultured in growth medium supplemented with 20 ng/mL EGF and 10 ng/mL bFGF. However, to observe the differentiation ability of NSCs, bFGF and EGF were removed, and 1% fetal bovine serum was added into the medium. (A) The phase of dissociated NSCs; (B) the dissociated NSCs expressed GFP (green); (C) the phase of neurosphere; (D) neurosphere-expressed GFP (green); (E) neurosphere-expressed nestin (red); (F) the proliferation of NSCs was determined by cell labeling with 5-ethynyl-2’-deoxyuridine (EdU) (red); (G) immunofluorescent staining of the neuronal marker Tuj1 (red); (H) the astroglial marker GFAP (red); (I) the oligodendrocyte marker O4 (cyan). Nuclei in (F–I) were stained with DAPI (blue). Scale bar: 100 μm (A–F); and 20 μm (G–I).

Mentions: NSCs from GFP mice embryonic cerebral cortices were isolated and cultured in serum-free medium, including B27, EGF and bFGF. The cells showed strong and stable emission of the green fluorescent signal, proliferated and formed neurospheres (Figure 1A–D). Meanwhile, the neurosphere expressed nestin (Figure 1E), an intermediate filament protein present in neural stem/progenitor cells. To detect the proliferation capacity of NSCs, cells were incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 2 h, and we found that many NSCs were EdU-positive (Figure 1F). Thus, the massive incorporation of EdU showed the efficient proliferation of NSCs. Next, after seven days of differentiation in the medium without EGF and bFGF, the cells expressed the neuronal marker Tuj1 (Figure 1G), the astrocytic marker glial fibrillary acidic protein (GFAP) (Figure 1H) and the oligodendrocyte marker O4 (Figure 1I). Taken together, these results indicated that NSCs used in this study could proliferate, self-renew and exhibit the capacity to differentiate into neurons, astrocytes and oligodendrocytes. The NSCs from passage 3 were used for transplantation and co-culture with macrophages.


Anti-Inflammatory Mechanism of Neural Stem Cell Transplantation in Spinal Cord Injury
Characterization of neural stem cells (NSCs) from the GFP transgenic mouse in vitro. Mouse NSCs were cultured in growth medium supplemented with 20 ng/mL EGF and 10 ng/mL bFGF. However, to observe the differentiation ability of NSCs, bFGF and EGF were removed, and 1% fetal bovine serum was added into the medium. (A) The phase of dissociated NSCs; (B) the dissociated NSCs expressed GFP (green); (C) the phase of neurosphere; (D) neurosphere-expressed GFP (green); (E) neurosphere-expressed nestin (red); (F) the proliferation of NSCs was determined by cell labeling with 5-ethynyl-2’-deoxyuridine (EdU) (red); (G) immunofluorescent staining of the neuronal marker Tuj1 (red); (H) the astroglial marker GFAP (red); (I) the oligodendrocyte marker O4 (cyan). Nuclei in (F–I) were stained with DAPI (blue). Scale bar: 100 μm (A–F); and 20 μm (G–I).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037660&req=5

ijms-17-01380-f001: Characterization of neural stem cells (NSCs) from the GFP transgenic mouse in vitro. Mouse NSCs were cultured in growth medium supplemented with 20 ng/mL EGF and 10 ng/mL bFGF. However, to observe the differentiation ability of NSCs, bFGF and EGF were removed, and 1% fetal bovine serum was added into the medium. (A) The phase of dissociated NSCs; (B) the dissociated NSCs expressed GFP (green); (C) the phase of neurosphere; (D) neurosphere-expressed GFP (green); (E) neurosphere-expressed nestin (red); (F) the proliferation of NSCs was determined by cell labeling with 5-ethynyl-2’-deoxyuridine (EdU) (red); (G) immunofluorescent staining of the neuronal marker Tuj1 (red); (H) the astroglial marker GFAP (red); (I) the oligodendrocyte marker O4 (cyan). Nuclei in (F–I) were stained with DAPI (blue). Scale bar: 100 μm (A–F); and 20 μm (G–I).
Mentions: NSCs from GFP mice embryonic cerebral cortices were isolated and cultured in serum-free medium, including B27, EGF and bFGF. The cells showed strong and stable emission of the green fluorescent signal, proliferated and formed neurospheres (Figure 1A–D). Meanwhile, the neurosphere expressed nestin (Figure 1E), an intermediate filament protein present in neural stem/progenitor cells. To detect the proliferation capacity of NSCs, cells were incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 2 h, and we found that many NSCs were EdU-positive (Figure 1F). Thus, the massive incorporation of EdU showed the efficient proliferation of NSCs. Next, after seven days of differentiation in the medium without EGF and bFGF, the cells expressed the neuronal marker Tuj1 (Figure 1G), the astrocytic marker glial fibrillary acidic protein (GFAP) (Figure 1H) and the oligodendrocyte marker O4 (Figure 1I). Taken together, these results indicated that NSCs used in this study could proliferate, self-renew and exhibit the capacity to differentiate into neurons, astrocytes and oligodendrocytes. The NSCs from passage 3 were used for transplantation and co-culture with macrophages.

View Article: PubMed Central - PubMed

ABSTRACT

Neural stem cell (NSC) transplantation has been proposed to promote functional recovery after spinal cord injury. However, a detailed understanding of the mechanisms of how NSCs exert their therapeutic plasticity is lacking. We transplanted mouse NSCs into the injured spinal cord seven days after SCI, and the Basso Mouse Scale (BMS) score was performed to assess locomotor function. The anti-inflammatory effects of NSC transplantation was analyzed by immunofluorescence staining of neutrophil and macrophages and the detection of mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-12 (IL-12). Furthermore, bone marrow-derived macrophages (BMDMs) were co-cultured with NSCs and followed by analyzing the mRNA levels of inducible nitric oxide synthase (iNOS), TNF-α, IL-1β, IL-6 and IL-10 with quantitative real-time PCR. The production of TNF-α and IL-1β by BMDMs was examined using the enzyme-linked immunosorbent assay (ELISA). Transplanted NSCs had significantly increased BMS scores (p < 0.05). Histological results showed that the grafted NSCs migrated from the injection site toward the injured area. NSCs transplantation significantly reduced the number of neutrophils and iNOS+/Mac-2+ cells at the epicenter of the injured area (p < 0.05). Meanwhile, mRNA levels of TNF-α, IL-1β, IL-6 and IL-12 in the NSCs transplantation group were significantly decreased compared to the control group. Furthermore, NSCs inhibited the iNOS expression of BMDMs and the release of inflammatory factors by macrophages in vitro (p < 0.05). These results suggest that NSC transplantation could modulate SCI-induced inflammatory responses and enhance neurological function after SCI via reducing M1 macrophage activation and infiltrating neutrophils. Thus, this study provides a new insight into the mechanisms responsible for the anti-inflammatory effect of NSC transplantation after SCI.

No MeSH data available.


Related in: MedlinePlus