Mitochondrial Transplantation Attenuates Neural Damage and Improves Locomotor Function After Traumatic Spinal Cord Injury in Rats

Abstract

Mitochondrial dysfunction is a hallmark of secondary neuroinflammatory responses and neuronal death in spinal cord injury (SCI). Even though mitochondria-based therapy is an attractive therapeutic option for SCI, the efficacy of transplantation of allogeneic mitochondria in the treatment of SCI remains unclear. Herein, we determined the therapeutic effects of mitochondrial transplantation in the traumatic SCI rats. Compressive SCI was induced by applying an aneurysm clip on the T10 spinal cord of rats. A 100-μg bolus of soleus-derived allogeneic mitochondria labeled with fluorescent tracker was transplanted into the injured spinal cords. The results showed that the transplanted mitochondria were detectable in the injured spinal cord up to 28 days after treatment. The rats which received mitochondrial transplantation exhibited better recovery of locomotor and sensory functions than those who did not. Both the expression of dynamin-related protein 1 and severity of demyelination in the injured cord were reduced in the mitochondrial transplanted groups. Mitochondrial transplantation also alleviated SCI-induced cellular apoptosis and inflammation responses. These findings suggest that transplantation of allogeneic mitochondria at the early stage of SCI reduces mitochondrial fragmentation, neuroapoptosis, neuroinflammation, and generation of oxidative stress, thus leading to improved functional recovery following traumatic SCI.

Keywords: allogenic mitochondria; mitochondrial dysfunction; mitochondrial transplantation; oxidative stress; spinal cord injury.

FIGURE 1

The spatiotemporal distribution of transplanted mitochondria in the injured spinal cord of rats with traumatic SCI. (A) Diagram of the schematic spinal cord. The red dash line indicated the injury site. The blue frame indicated the 1-cm sampling area. The asterisks denoted the sites of administration of allogeneic mitochondria. (B) Representative micrograph of MTDR signals in the injured spinal cord of rat injected with 1x PBS vehicle on PID 1. (C) Representative micrograph of MTDR signals in the injured spinal cord of rat injected with MTDR dye on PID 1. (D) Representative micrograph of MTDR signals in the injured spinal cord of rat injected with 1x PBS vehicle on PID 28. (E) Representative micrograph of MTDR signals in the injured spinal cord of rat injected with MTDR dye on PID 28. (F–K) Representative micrograph of MTDR signals which were detected on PID1, 3, 7, 10, 14, and 28 in the injured spinal cord of rats injected with MTDR-labeled mitochondria. Horizontal sections were used. Scale bar = 100 μm. N = 1 rat per panel. The asterisks indicate the points of injection.

FIGURE 2

Effects of mitochondrial transplantation on the sensory and locomotor functions and the white matter sparing in the injured spinal cord of rats with traumatic SCI. (A) Representative micrographs of SSEP traces recorded in SCI rats on PID 28. Scale = 100 μV/5 ms (B) Quantitative results of BBB scoring. **p < 0.01, ****p < 0.0001, Sidak’s multiple comparisons performed after the repeated measures two-way ANOVA. (C) Representative micrographs of LFB staining of transverse sections prepared from the injured spinal cords of rats. Scale bar = 100 μm. The red frames indicated the analyzed areas. (D) Quantitative results of percentage of LFB-positive area. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t-test. Data were expressed as mean ± standard deviation. N = 5. MT: mitochondria trans-plantation.

FIGURE 3

Effects of mitochondrial transplantation on Drp1 expression in the injured spinal cord of rats with traumatic SCI on PID 1. (A) Representative micrograph of Western blot analysis of Drp1 in sham control groups. (B) Corresponding quantitative results of relative expression of Drp1 in sham control groups. (C) Representative micrograph of Western blot analysis of Drp1 in SCI groups on PID 1. (D) Corresponding quantitative results of relative expression of Drp1 in SCI groups on PID 1. The 1-cm spinal cord specimens centered at the epicenter of the injured site were used in Western blots. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, two-tailed Student’s t-test. N = 5. MT, mitochondria transplantation.

FIGURE 4

Effects of mitochondrial transplantation on cellular apoptosis in the injured spinal cord of rats with traumatic SCI. (A) Representative micrograph of Western blots of cleaved caspase-3, Bcl-2, and BAX in sham control groups. (B–D) Corresponding quantitative results of relative expression of cleaved caspase-3, Bcl-2, and BAX in sham control groups. (E) Representative micrograph of Western blots of cleaved caspase-3, Bcl-2, and BAX in SCI groups on PID 1. (F–H) Corresponding quantitative results of relative expression of cleaved caspase-3, Bcl-2, and BAX in SCI groups on PID 1. The 1-cm spinal cord specimens centered at the epicenter of the injured site were used in Western blots. (I) Representative micrographs of TUNEL assay. The white arrowheads indicate both DAPI and TUNEL dual-positive cells. The sections used in TUNEL assay were obtained from the 1-cm spinal cord specimens centered at the epicenter of the injured site. Scale = 100 μm. (J) Quantitative results of the TUNEL assay. Data were expressed as mean ± standard deviation. *p < 0.05, ***p < 0.001 two-tailed Student’s t-test. N = 3–5. MT, mitochondria transplantation.

FIGURE 5

Effects of mitochondrial transplantation on expression of pro-inflammatory cytokines in the injured spinal cord of rats with traumatic SCI on PID 1. (A) Representative micrograph of Western blots of TNF and IL-6 in sham control groups. (B,C) Quantitative results of relative expression of TNF and IL-6 in sham control groups. (D) Representative micrograph of Western blots of TNF and IL-6 in SCI groups on PID 1. (E,F) Quantitative results of relative expression of TNF and IL-6 in SCI groups on PID 1. The 1-cm spinal cord specimens centered at the epicenter of the injured site were used in Western blots. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, two-tailed Student’s t-test. N = 3–5. MT, mitochondria transplantation.

FIGURE 6

Effects of mitochondrial transplantation on oxidative stress in the injured spinal cord of rats with traumatic SCI on PID 1. (A) Representative micrograph of Western blot of iNOS in sham control groups. (B) Quantitative results of relative expression of iNOS in sham control groups. (C) Representative micrograph of Western blot of iNOS in SCI groups on PID 1. (D) Quantitative results of relative expression of iNOS in SCI groups on PID 1. The 1-cm spinal cord specimens centered at the epicenter of the injured site were used in Western blots. (E–G) Quantitative results of the level of NO, 3-NT and malondialdehyde in SCI groups. Data were expressed as mean ± standard deviation. *p < 0.05, ***p < 0.001, two-tailed Student’s t-test. N = 4–5. MT, mitochondria transplantation.