BAPTA, a calcium chelator, neuroprotects injured neurons in vitro and promotes motor recovery after spinal cord transection in vivo

Figures

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  FIGURE 1. BAPTA inhibits apoptosis of cells in primary stage of SCI and improve locomotor recovery. (A) Changes in modified BBB scores after SCI. (B) Change in the body weight after SCI. (C) Representative TUNEL assay images of spinal cord injury model mice. Low‐magnification (upper panels) and high‐magnification (lower panels) IHC images showed significantly different number of TUNEL/DAPI double‐positive cells; bar = 500 μm. (D) Quantification of apoptotic cells by counting TUNEL/DAPI double‐positive cells. Statistical significance is shown as follows: *P < 0.05 (Mean ± SEM, n = 5‐7)

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  FIGURE 2. BAPTA relieved the death of neurons caused by physical damage. (A) Scheme of experiments. The process of generating in vitro model of SCI and experiments for testing the function of BAPTA. (B) Quantitative analysis of cell deaths. Dead cells were collected and determined by trypan blue staining and cell count. The graphs are representative of three independent experiments. Statistical significance is shown as follows: *P<0.05 (Mean ± SEM, n = 3). (C) Bright‐field microscopy images of floating dead cells. Sham shows neurons without damage. Vehicle controls showed the neurons treated DPBS after bead damaging. BAPTA means the neuron‐treated BAPTA after bead damaging; bar = 50 μm. (D) Low‐magnification (upper panels) and high‐magnification (lower panels) SEM images of damaged neurons. White box showed magnified field. Arrows indicated general marker of apoptosis such as broken and shrunk neurites (black arrows) and blebs (white arrows); bar = 1 μm

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  FIGURE 3. BAPTA inhibits ROS generation and neuronal apoptosis. (A) TUNEL assay images of different concentration of BAPTA‐treated group after bead damaging; bar = 50 μm. (B) Quantification of apoptotic neurons by counting TUNEL/DAPI double‐positive cells. (C) Accumulated ROS level detected by DCF‐DA. (D) Changes in apoptosis‐related gene expression level depend on time and condition. All the values were normalized to the value of the sample collected immediately after 30 minutes of BAPTA (or DPBS) treatment. Statistical significance is shown as follows: *P < 0.05, **P < 0.01, ***P < 0.001 (Mean ± SEM, n = 3)

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  FIGURE 4. BAPTA relieves impairment of neuronal function after physical damaging. (A) Representative bright‐field microscopy images of neurons after scratch; bar = 50 μm. (B) Quantification of expanded damaged area for 15 days after scratching. (C) Quantification of decreasing MFR normalized by the MFR of day 0 for 15 days after scratching. Statistical significance is shown as follows: *P < 0.05, **P < 0.01, (Mean ± SEM, n = 4‐7)