Neural stem cells over-expressing brain-derived neurotrophic factor promote neuronal survival and cytoskeletal protein expression in traumatic brain injury sites

Abstract

Cytoskeletal proteins are involved in neuronal survival. Brain-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal injury. However, the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains unclear. To examine this, we established a rat model of traumatic brain injury by controlled cortical impact. At 72 hours after injury, 2 × 10⁷ cells/mL neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells (3 mL) were injected into the injured cortex. At 1-3 weeks after transplantation, expression of neurofilament 200, microtubule-associated protein 2, actin, calmodulin, and beta-catenin were remarkably increased in the injury sites. These findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival, growth, and differentiation in the injury sites. The underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/β-catenin signaling pathway.

Keywords: brain-derived neurotrophic factor; calmodulin; Wnt/β-catenin; cytoskeleton; differentiation; microtubule-associated proteins; nerve regeneration; neural regeneration; neural stem cells; neurofilament; transfect; traumatic brain injury.

Figure 1

Effect of BDNF/NSCs on NF200 immunoreactivity and neuronal morphology in lesions following transplantation after traumatic brain injury. (A) Immunohistochemical staining for NF200 in lesioned sections. Cell bodies were larger and axons longer and thicker in BDNF/NSCs-transplanted rats compared with naive NSCs-transplanted rats. Scale bar: 50 μm. (B) Quantitative analysis of NF200-positive cell number in 400-fold field. (C) NF200 immunoreactivity in lesions. NF200 immunoreactivity was expressed as integrated optical density (× 10³). Data are expressed as the mean ± SD (n = 5), and were analyzed by one-way analysis of variance followed by independent samples t-test. *P < 0.05, **P < 0.01, ***P < 0.001, vs. NSCs group. BDNF: Brain-derived neurotrophic factor; NSCs: neural stem cells; NF200: neurofilament 200.

Figure 2

Effect of BDNF/NSCs on MAP2 expression in host cells and neuron-like cells with concomitant PKH26 at 2 and 3 weeks after BDNF/NSCs or NSCs transplantation (immunofluorescence staining). Transplanted cells were labeled using PKH26 dye (red), with MAP2 labeled by FITC (green). Merged cells show MAP2 expression in transplanted neuron-like cells with concomitant PKH26. Scale bar: 50 μm. BDNF: Brain-derived neurotrophic factor; NSCs: neural stem cells; MAP2: microtubule-associated protein 2; FITC: fluorescein isothiocyanate.

Figure 3

Effect of BDNF/NSCs on actin, CaM, and β-catenin expression in lesions at 1, 2, and 3 weeks after BDNF/NSCs or NSCs transplantation (western blot assay). (A) Representative western blot results of lesion samples from BDNF/NSCs-transplanted rats or NSCs-transplanted rats. Relative optical density of each band was standardized to optical density of the GAPDH band. Densitometric analysis showed statistically significant increased expression of β-catenin (B), actin (C), and CaM (D) in the BDNF/NSC group compared with the NSC group. Data are expressed as the mean ± SD (n = 5 in each group), and were analyzed by one-way analysis of variance followed by independent samples t-test. *P < 0.05, **P < 0.01, vs. NSCs group. BDNF: Brain-derived neurotrophic factor; NSCs: neural stem cells; CaM: calmodulin.