Fig 2: Inhibition of Cisd2 using anti-CISD2 neutralizing antibodies exacerbated the inflammatory response in the injured spinal cord and spinal cord injury (SCI)-mediated inflamed brain of mice subjected to spinal cord hemisection. (A) The spinal cord levels of Cisd2 mRNA were decreased in the 4, 12, and 36 h post-SCI groups. (B) The differences in the spinal cord levels of Tnfa mRNA between the anti-CISD2 antibody-treated and vehicle-treated groups increased by each of these time points. (C) The confirmation of Cisd2 knockdown in the brain of rats subjected to spinal cord hemisection. The brain levels of Tnfa mRNA (D) and protein (E) in the anti-CISD2-treated SCI group were higher than those in the vehicle-treated SCI group at each time point. Vertical bars indicate mean ± standard error of mean of mRNA (n = 6 for each group) and protein (for Western blot, n = 3; for enzyme-linked immunosorbent assay (ELISA), n = 6) levels. * p < 0.05, ** p < 0.01, and *** p < 0.001 (pair-wise multiple comparisons between groups were performed using the Newman–Keuls test).

Fig 3: Spinal cord injury (SCI) downregulates Cisd2 and enhances inflammatory response in the injured spinal cord of mice subjected to spinal cord hemisection. The secretion of various inflammatory mediators in the cerebrospinal fluid (CSF) of rats with SCI. (A) Experimental groups and the schedule of treatment with anti-CISD2 antibody in a mouse model of SCI. (B) The spinal cord levels of Cisd2 mRNA in the 1, 24, and 48 h post-SCI groups were significantly decreased compared with those in the sham operation group. (C) Under the same conditions as those in (B), the spinal cord levels of Tnfa mRNA were significantly increased. For B and C, vertical bars indicate mean ± standard error of mean (SEM) (n = 6 for each group). * p < 0.05, ** p < 0.01, and *** p < 0.001 (pair-wise multiple comparisons between groups were performed using the Newman–Keuls test). The spinal cord levels of the Cisd2 protein were decreased (D) and those of Nos2 were increased (E) in the 24 h post-SCI group compared with those in the sham operation group. For D and E, the upper panel indicates representative immunoblot of the protein expression of Cisd2 (15 kDa) (D) and Nos2 (135 kDa); (E) Gapdh (35 kDa) was used as an internal control. For (D) and (E), vertical bars indicate mean ± SEM of protein levels (n = 3 for each group). * p < 0.05, sham operation group vs. SCI groups (independent two-sample t-tests). (F) Detection of inflammatory mediators in the CSF using the cytokine antibody array at 4 h post-SCI in rats. Representative image of the array assay demonstrating the reactivity of pooled CSF samples (upper left panel, sham operation group; upper right panel, SCI group at 4 h post-SCI). n = 3 for each group with two replicates for each protein target. Signals were scanned with ImageQuantTM LAS 4000 (GE Healthcare Life Science, Marlborough, MA, USA). Blue box, positive controls (high intense spots); red box, negative controls (no spots); lower panel, schematic illustration of the location of positive (blue box), negative control (red box), and 34 targets. (G) Signal intensity of inflammatory mediators in the CSF of the sham operation and SCI groups was comparatively analyzed. Vertical bars indicate mean ± SEM of protein levels (n = 3 for each group). * p < 0.05, ** p < 0.01, and *** p < 0.001 (sham operation group vs. SCI groups; independent two-sample t-tests).

Fig 4: Spinal cord injury (SCI) downregulates Cisd2 and enhances inflammatory response in the brain of mice subjected to spinal cord hemisection. (A) The brain levels of Cisd2 mRNA in the 1 and 4 h post-SCI groups were significantly downregulated compared with those in the sham operation group. Additionally, the brain levels of Tnfa mRNA (B) and protein (C) (detected using enzyme-linked immunosorbent assay [ELISA]) in the 1 and 4 h post-SCI groups were significantly upregulated compared with those in the sham operation group. Vertical bars indicate mean ± standard error of mean of mRNA (n = 6 for each group) and protein (n = 6 for each group) levels. * p < 0.05, ** p < 0.01, and *** p < 0.001 (pair-wise multiple comparisons between groups were performed using the Newman–Keuls test). (D) Schematic diagrams illustrating the pivotal points in Figure 1 and Figure 2 that are indicative of the vulnerability of the central nervous system (CNS) to inflammation. After SCI, inflammatory mediators delivered through the cerebrospinal fluid (CSF) promote brain inflammation as evidenced by the upregulation of TNFA and inflammatory mediators. The expression of CISD2 is decreased in the injured spinal cord and inflamed brain, which exacerbates inflammation.

Fig 5: Illustration of Cisd2 depletion in the mouse model of spinal cord hemisection injury and CISD2 potentiation in patients with central nervous system (CNS) insults. As indicated in the left panel, spinal cord injury (SCI) decreases the expression of Cisd2 and promotes inflammation in the spinal cord and brain. Inhibition of Cisd2 using the anti-CISD2 neutralizing antibody exacerbated SCI-induced inflammation in the brain and spinal cord. Right panel shows a schematic representation of a patient with CNS insults exhibiting decreased plasma and cerebrospinal fluid (CSF) levels of CISD2 and elevated levels of inflammatory mediators. Therapeutic hypothermia potentiates CISD2 function and decreases the plasma levels of CRP and the CSF levels of IL6. The results of CISD2 inhibition and potentiation studies revealed the anti-inflammatory properties of CISD2.