A three-dimensional matrix system containing melatonin and neural stem cells repairs damage from traumatic brain injury in rats

Abstract

Brain lesions can cause neural stem cells to activate, proliferate, differentiate, and migrate to the injured area. However, after traumatic brain injury, brain tissue defects and microenvironment changes greatly affect the survival and growth of neural stem cells; the resulting reduction in the number of neural stem cells impedes effective repair of the injured area. Melatonin can promote the survival, proliferation, and differentiation of neural stem cells under adverse conditions such as oxidative stress or hypoxia that can occur after traumatic brain injury. Therefore, we investigated the therapeutic effects of melatonin combined with neural stem cells on traumatic brain injury in rats. First, in vitro studies confirmed that melatonin promoted the survival of neural stem cells deprived of oxygen and glucose. Then, we established a three-dimensional Matrigel-based transplantation system containing melatonin and neural stem cells and then used it to treat traumatic brain injury in rats. We found that treatment with the Matrigel system containing melatonin and neural stem cells decreased brain lesion volume, increased the number of surviving neurons, and improved recovery of neurological function compared with treatment with Matrigel alone, neural stem cells alone, Matrigel and neural stem cells combined, and Matrigel and melatonin combined. Our findings suggest that the three-dimensional Matrigel-based transplantation system containing melatonin and neural stem cells is a potential treatment for traumatic brain injury.

Keywords: Matrigel; cell therapy; magnetic resonance imaging; melatonin; neural stem cells; neurological function recovery; three-dimensional transplantation; traumatic brain injury.

Figure 1

The transplantation procedure. Mixtures composed of neural stem cells (NSCs), melatonin (MEL), and Matrigel (MTX) were transplanted into lesion cavities in the M1 and M2 motor cortices using a syringe.

Figure 2

Neural stem cell identification by immunocytochemistry. The neurospheres (DAPI, blue) were positive for the neural stem cell markers nestin (CFL 488, green) and SOX2 (CFL 555, red). Scale bars: 50 μm. DAPI: 4′,6-Diamidino-2-phenylindole; SOX2: SRY-related HMG-box 2.

Figure 3

Neural stem cell differentiation was identified by immunofluorescence. Neurospheres were observed after culturing for 7 days in vitro. Neurospheres were positive for (A) doublecortin (DCX), a marker of neurons (CFL 555, red); (B) oligodendrocyte transcription factor 2 (Olig2), a marker of oligodendrocytes (CFL 488, green); (C) glial fibrillary acidic protein (GFAP), a marker of astrocytes (CFL 555, red), and (D) microtubule-associated protein 2 (MAP2), a marker of neurons (CFL 555, red). DAPI (blue) is a nuclear marker. Scale bars: 50 μm. DAPI: 4′,6-Diamidino-2-phenylindole.

Figure 4

Oxygen and glucose deprivation decreases neural stem cell viability. (A–D) Cell images under oxygen and glucose deprivation (OGD) at 0, 2, 4, and 6 hours. OGD caused cell death, and the percentage of dead cells increased over time. (E) Neural stem cell (NSC) viability was evaluated under OGD conditions. Data are expressed as mean ± SD. Each experiment was repeated four times. **P < 0.01, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparisons test).

Figure 5

Effect of melatonin on neural stem cell viability under oxygen and glucose deprivation. (A) Representative images of neural stem cells (NSCs) under oxygen and glucose deprivation and treated with 0, 6.25, 12.5, 25, 50, or 100 μM melatonin. A neuronal phenotype could be distinguished under different melatonin concentrations, and the rate of cell survival differed with different melatonin concentrations. Most cells survived at 25 μM melatonin treatment. Scale bars: 100 μm. (B) NSC viability was evaluated. Data are expressed as mean ± SD. Each experiment was repeated four times. *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparisons test). ns: Not significant.

Figure 6

Effect of the three-dimensional matrix system containing melatonin and neural stem cells on brain magnetic resonance imaging and the modified neurological severity score of rats with traumatic brain injury. (A–E) T2-weighted images of MTX (A), NSC (B), MTX + NSC (C), MTX + MEL (D), and MTX + NSC + MEL (E) groups on day 14 after treatment. The lesions in the MTX + NSC + MEL group decreased in size compared with the other groups. The lesion boundaries are marked with dotted lines. (F, G) Quantitative results of the lesion volumes (F) and modified neurological severity (mNSS) scores (G). Data are expressed as mean ± SD (n = 5). **P < 0.01, vs. MTX or NSC groups; ****P < 0.0001 (F: one-way analysis of variance followed by Tukey’s multiple comparisons test; G: two-way analysis of variance followed by Tukey’s multiple comparisons test). MEL: Melatonin; mNSS: modified neurological severity score; MTX: Matrigel.

Figure 7

Effect of the three-dimensional matrix system containing melatonin and neural stem cells (NSCs) on pathological injury in the brains of rats with traumatic brain injury on day 14 after treatment. (A–E) Hematoxylin and eosin staining reveals residual Matrigel (MTX) and surviving cells in the MTX network in the MTX + NSC and MTX+ NSC + MEL groups. Scale bars: 1 mm in original images and 100 μm in enlarged images. (F) Cell counts in the lesion, ranging from 0–200 μm about the edge of the lesion. Data are expressed as mean ± SD (n = 5). ****P < 0.0001 (one-way analysis of variance followed by Dunnett’s multiple comparisons test). MEL: Melatonin.

Figure 8

Effect of the three-dimensional matrix system containing melatonin and neural stem cells on the retention, survival, and differentiation of neural stem cells in the brains of rats with traumatic brain injury on day 14 after treatment. (A–E) Immunofluorescence staining of the lesions of rats with traumatic brain injury. Samples from the five experimental groups were stained by immunofluorescence using the nuclear stain DAPI (blue) and antibodies against nestin and GFAP. The transplanted cells were labeled with CM-Dil. Apart from D, white (Alexa Fluor® 647) indicates positive expression of nestin; CM-Dil (red) indicates exogenous neural stem cells; and green (CFL 488) indicates positive expression of GFAP. In D, white (CFL 647) indicates positive expression of GFAP; red indicates exogenous neural stem cells; and green (CFL 488) indicates positive expression of nestin. Scale bars: 500 µm in merge images and 100 µm in small images. (F) The number of CM-Dil–positive cells. Data are expressed as mean ± SD (n = 3). ****P < 0.0001 (one-way analysis of variance followed by Dunnett’s multiple comparisons test). DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; MEL: melatonin; MTX: Matrigel.