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    Neuroprotective effects of Alda-1 mitigate spinal cord injury in mice: involvement of Alda-1-induced ALDH2 activation-mediated suppression of reactive aldehyde mechanisms
  • Figure 1|Experimental protocol. 

    The BMS was developed to assess open field locomotion deficits in spinal cord injured mice (Basso et al., 2006). This scoring system is widely used as an indicator of recovery in mouse models of SCI. Later, modifications were added to improve the scale as described (Pajoohesh-Ganji et al., 2010). BMS scoring was performed at the indicated time points, as shown in Figure 1. Two observers, blinded to the identity of groups, independently evaluated the BMS scores. 


    Figure 2|Sustained and prolonged 4-HNE load in a mouse model of SCI. 

    Reactive aldehyde load, more prominently of 4-HNE, is recognized as a pathological event in SCI. Western blot (Figure 2A) and its densitometric analysis (Figure 2B) shows a sustained accumulation of significantly high (P < 0.001) levels of 4-HNE adducts at different time points (24, 48, 72 hours, and 30 days) in SCI groups compared with sham-operated (at 30 days) group.  


    Figure 3|ALDH2 agonist Alda-1 reduces neurovascular dysfunction and inflammation in the acute phase (72 hours) of SCI in wild type mice. 

    BSCB disruption, edema and neurovascular inflammation are critical components of the acute phase of spinal cord contusion injury (Lee et al., 2018). Alda-1 treatment of SCI reduced the severity of contusion injury measured as the blood content in the injured cord (P < 0.01, Figure 3A and B), BSCB disruption (Evans’ blue extravasation, P < 0.01, Figure 3C and D), decreased edema (water content, P < 0.05, Figure 3E) and reduced expression (western blot analysis) of neuroinflammatory mediators ICAM-1 and GFAP (P < 0.01, Figure 3F and G).


    Figure 4|Alda-1 treatment decreases 4-HNE load and enhances the activity of ALDH2 in the acute phase (72 hours) of a mouse model of SCI. 

    4-HNE is one of the most reactive aldehydes, and it is structurally suitable for the formation of protein adducts due to its strong electrophilic nature. Using the two different doses (1 mg/kg vs. 10 mg/kg) of Alda-1, we determined that the effective dose was 10 mg/kg to reduce SCI-induced 4-HNE load (Figure 4A and B). The dose of 10 mg/kg body weight has been effective with significant efficacy in other animal disease models (Lu et al., 2017; He et al., 2018). Therefore, we used a 10 mg/kg dose of Alda-1 in all other experiments reported in this study. The activity of ALDH2 was significantly inhibited in the SCI group compared with the sham group (Figure 4C). Alda-1 treatment of the SCI group significantly increased the ALDH2 activity (Figure 4C). Interestingly, the expression of ALDH2 remained unchanged among the groups (Figure 4D and E).


    Figure 5|Effect of Alda-1 on locomotor function. Locomotor function was assessed using the BMS scale at indicated days.  

    Improvement in the BMS score is the gold standard endpoint determining a therapeutic agent’s efficacy in mice following SCI. On the BMS scores, SCI animals exhibited greater functional loss than the Alda-1-treated group (Figure 5). Statistical analysis showed significantly improved scores in the Alda-1-treated compared with the SCI group from day 3 onward (Figure 5).


    Figure 6|Effect of Alda-1 on pain-like behavior in a mouse model of SCI.

    Both inflammatory and neuropathic pain (caused by a lesion or disease of somatosensory function) are present in most SCI patients (Finnerup, 2013). The ALDH2/Alda-1-dependent mechanism has been reported to mitigate pain by reducing the load of reactive aldehyde in animal models of pain (Zambelli et al., 2014; Li et al., 2018). Using mechanical (paw withdrawal threshold, Figure 6A) and thermal withdrawal latency (Figure 6B), we observed that the pain threshold/latency remained almost unchanged for seven days in both the SCI and the Alda-1-treated groups. From day seven onward, the Alda-1 group had a significantly improved pain threshold that further improved with time (Figure 6). Sham animals had no change in pain sensitivity.


    Figure 7|ALDH2 agonist Alda-1 treatment reduces caspase-3 activity, apoptotic cell death, and neuronal loss in the chronic phase (30 days) of a mouse model of SCI.   

    Neuronal cell death is the critical component of a spinal cord contusion injury, and the protection against neuronal cell death is the major objective of SCI therapy. Expectedly, the expression of NeuN was drastically reduced in the SCI group (Figure 7A), which was significantly increased by Alda- 1 treatment (Figure 7A and C). In parallel to NeuN loss in the SCI group, the activity of caspase-3 was increased, as shown by western blot (Figure 7B). In contrast, the Alda-1- treated group had reduced caspase-3 activity (Figure 7B and C). Alda-1 treatment also increased number of neurons measured as the expression of NeuN (Figure 7D and E) and reduced the SCI-induced increased number of TUNEL positive cells (Figure 7F and G), indicating that Alda-1 protects against apoptotic neuronal cell death. The neuroprotective effect of Alda-1 was also supported by Nissl staining showing increased survival of neurons in the Alda-1-treated group (Figure 7H and I). These results suggest a significant neuronal loss in the chronic phase of SCI, and upregulation of ALDH2 activity by Alda-1 may reduce the loss of neuronal cells.


    Figure 8|ALDH2 agonist Alda-1 treatment of SCI decreases the activation of Iba-1 and GFAP and normalized mitochondrial oxidative stress measured as the expression of MnSOD in the chronic phase (30 days) of a mouse model of SCI. 

    Inflammation and redox alteration are essential components of SCI, which contributes to neuronal cell death and pain sensitivity, leading to a hindrance to sensory and locomotor functions’ recovery. The expression of activated microglia (Iba-1) (Figure 8A and B) and reactive astrocytes (GFAP) (Figure 8C and D) was significantly increased in the SCI compared to the sham group. Mitochondrial redox, determined as the expression of mitochondria-specific MnSOD, was also compromised considerably in the SCI compared with the sham group. Treatment of the injured animals with Ald-1 decreased the SCI-induced increased expression of Iba-1 and GFAP. The treatment with Alda-1 also improved the SCI-mediated decreased expression of MnSOD (Figure 8E and F) and the reduced levels of ATP (Figure 8G). These results indicate that Alda-1, likely by upregulating ALDH2 activity, is a potent anti-inflammatory and mitochondrial function restoring agent.


    Figure 9|ALDH2 agonist Alda-1 treatment of SCI increases the expression of BDNF, NT3, and MBP in the chronic phase (30 days) of a mouse model of SCI. 

    Stimulation of spinal cord repair is essential for optimum functional recovery following SCI (Cheng et al., 1996). Under the oxidative stress environment, as observed in SCI, neurotrophic factors including BDNF and NT3 are reduced, leading to a hindrance to the reparative process. In accordance, western blot analysis showed the reduced expression levels of both BDNF (Figure 9A and B) and NT3 (Figure 9C and D). The expression levels of MBP was also decreased in the SCI group, as indicated by immunohistochemistry (Figure 9E and F). MBP is a critical structural protein that maintains the structure of myelin via interacting with myelin lipids. Reduced MBP levels are associated with demyelination and inhibition of remyelination (Zhou et al., 2019). The treatment with Alda-1 increased the SCI-induced decreased expression levels of BDNF, NT3, and MBP (Figure 9). Taken together, these data indicate that the enhanced activity of ALDH2 and reduced levels of 4-HNE are linked to the stimulation of the expression of neurorepair mediators.


    Figure 10|Alda-1 treatment of SCI decreases 4-HNE load and enhances the activity of ALDH2 in the chronic phase (30 days) of a mouse model of SCI. 

    Like in the acute phase, Alda-1 treatment (10 mg/kg but not 1 mg/kg) significantly reduced SCI-induced 4-HNE load in the chronic phase of SCI (Figure 10A and B). While the activity of ALDH2 remained inhibited even at 30-day after the injury, the treatment with Alda-1 enhanced the activity of ALDH2 (Figure 10C). However, like in the acute phase (Figure 4D and E), the expression of ALDH2 had no change among the groups (Figure 10D and E). 


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  • 发布日期: 2021-09-22  浏览: 567
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