Prdx6 expression is downregulated in white matter after reperfusion
Fig. 1A shows the expression of Prdx6 in WC (whole spinal cord) and in DC (dorsal column) white matter before reperfusion. Our results show that Prdx6 is expressed in both WC and white matter of DC. As expected, the DC shows the expression of GFAP (a marker for astrocytes) but not MAP2 (microtubule associated protein 2, a marker for neurons), thus, this indicates an absence of neurons in the DC white matter. Fig. 1B shows the real time gene expression of Prdx6 after 2 h and 4 h of reperfusion. Prdx6 gene expression is significantly upregulated after hypoxia as compared to sham (***p < 0.001), while downregulated after 2 h of reperfusion as compared to hypoxia (^^^p < 0.001) and sham (*p < 0.05). After 4 h of reperfusion, Prdx6 gene expression returned to normal levels compared to sham while it remained downregulated compared to the hypoxia group (^^^p < 0.001). Fig. 1C shows increase in protein expression of Prdx6 after hypoxia and this increase gradually decreases after reperfusion 2 h and 4 h as compared to hypoxia Interestingly, Prdx6 expression is upregulated significantly after 2 h (***p < 0.001) and downregulated after 4 h of reperfusion in comparison to sham (**p < 0.01).
Gene and protein expression of GFAP and NF-200 after reperfusion
The most abundant cell bodies present in white matter are glial cells and axons, thereof, we were interested to observe the expression of GFAP (a marker for astrocytes) and NF-200 (a marker for axons) after reperfusion-injury, and investigated the correlation of Prdx6 expression with GFAP and NF-200. Real time gene analysis of GFAP expression shows gradual upregulation of its expression in hypoxia after 2 h and 4 h of reperfusion-injury (Fig. 2A). However, GFAP protein shows an upregulation after hypoxia as compared to sham (***p < 0.001). Alternatively, GFAP expression shows significant downregulation after reperfusion as compared to hypoxia (^^^p < 0.001) (Fig. 2B). A similar pattern was observed with NF-200 (Fig. 2D), while gene expression analysis of NF-200 shows an upregulation in reperfusion (Fig. 2C).
Prdx6 is distinctly localized in astrocytes after hypoxia whereas it is redistributed in astrocytes and axons after reperfusion
We observed expression of GFAP (red, Fig. 3A(a)), NF-200 (green, Fig. 3A(b)), and Prdx6 (blue, Fig. 3A(c)) in the sham group. Fig. 3A(d) shows co-localization of GFAP and Prdx6, suggesting the presence of Prdx6 in astrocytes (pink color, arrowheads). In addition, Prdx6 is co-localized with GFAP in the cell body as well as in the processes, suggesting uniform distribution of Prdx6 in astrocytes (Fig. 3A(g)). Whereas Fig. 3A(e) and 3A(h) (enlarged image) shows co-localization of NF-200 (green) and Prdx6 (blue) forming a cyan color (arrow heads). Fig. 3A(i) shows superimposed images of Prdx6 (blue), GFAP (red), and NF-200 (green) in the sham group, suggesting the presence of Prdx6 in astrocytes (pink color, yellow arrows) and in axons (cyan color, white arrows) and the circles show axonal endings in astrocytes (Fig. 3A (h)).
We observed expression of GFAP (red, Fig. 3B (a)), NF-200 (green, Fig. 3B (b)) and Prdx6 (blue, Fig. 3B (c) in the hypoxia group. Interestingly, hypoxia induced the movement of Prdx6 into astrocytes as indicated by distinct co-localization of Prdx6 (blue) with GFAP (red) (pink color, yellow arrows) (Fig. 3B (i)). Unlike the sham group, we observed major co-localization of Prdx6 with GFAP in the cell body of astrocytes and limited co-localization was observed in the processes (Fig. 3B (g)). However, most of the Prdx6 is accumulated in astrocytes in the hypoxia group; we observed very low levels of co-localization of Prdx6 (blue) with NF-200 (green) (cyan color, white arrows) (Fig. 3B (h)). In addition, we observed a distinct co-localization of Prdx6, GFAP, and NF-200 where axons were making end feet to the astrocytes (white color, red arrows), suggesting possible communication of astrocytes and axons through Prdx6 (Fig. 3B (i)).
After 4 h of reperfusion, we observed co-localization of Prdx6 with GFAP (red) and NF-200 (green) suggesting a substantial movement of Prdx6 from astrocytes to axons (Fig. 3C (i)I). Like hypoxia, distinctive co-localization of Prdx6 with GFAP was observed in the cell body of astrocytes and limited co-localization was observed in astrocytes processes (Fig. 3C (g)). However, unlike the sham group, the Prdx6 was characteristically co-localized with NF-200 at the periphery of axons (Fig. 3C (h)H).
Reperfusion unable to protect hypoxia induced tissue damage and apoptosis
Next, we wanted to investigate if Prdx6 expression and its characteristic distribution in white matter after reperfusion have any correlation with apoptosis. Histopathological evaluation shows a well-organized structure and healthy looking cell bodies in the sham group (Fig. 4A (a)). Contrarily, hypoxic white matter indicated shrunken cell bodies, pronounced vacuolation, and disrupted tissue. In addition, characteristic reactive astrocytosis, indicated by abundant eosinophilic (pink) cytoplasm, was also observed in the hypoxia group (Fig. 4A (b)). An increased axonal separation (asterisk) and enhanced vacuolation was observed in reperfusion group, which suggests that reperfusion further exacerbates the hypoxia-induced tissue damage (Fig. 4A (c)).
Analysis of pro-inflammatory genes, TNFα and IL-6, showed that reperfusion further increased the hypoxia-induced inflammation in white matter (Fig. 4B and 4C). A significant continued upregulation of TNFα was observed in 2 h and 4 h of reperfusion as compared to the sham (***p < 0.001) and hypoxia group (^^^p < 0.001) (Fig. 4B). Similarly, significant upregulation of IL-6 was observed in hypoxia, 2 h, and 4 h of reperfusion as compared to sham (***p < 0.001), however, only 2 h of reperfusion shows upregulation of IL-6 in comparison to the hypoxia group (^p < 0.05) (Fig. 4C).
Apoptosis in white matter was examined by TUNEL staining, which measures the DNA fragmentation as an indication of apoptosis (Fig. 4D). Our results show that the number of TUNEL-positive cells (green) that represented fragmented DNA was markedly increased in the hypoxia group compared to the sham group. The number of TUNEL-positive cells was increased further in the reperfusion group compared to the sham as well as the hypoxia group (Fig. 4D). Altogether, these results suggest that reperfusion exacerbates the hypoxia induced inflammation, tissue damage, and apoptosis.
Nrf2 negatively regulates Prdx6 expression in hypoxia-reperfusion injury
The antioxidant genes, including Prdx6, are tightly regulated by ARE (antioxidant response elements), and Nrf2 is a major trans-activator of antioxidant genes in response to hypoxia, inflammation, and oxidative stress. It is known that Nrf2 activates the transcription of Prdx6 [20], however, its relation with Prdx6 in the white matter of spinal cord is not known. Our data shows that expression of Nrf2 is significantly upregulated in hypoxia and reperfusion groups as compared to the sham group (Fig. 5A). The gradual upregulation of Nrf2 protein expression in hypoxia and reperfusion groups is correlated with increased inflammation and oxidative stress in 2 h and 4 h of reperfusion-injury. Interestingly, expression of Prdx6 is gradually decreased with 2 h and 4 h of reperfusion, which suggests that Nrf2 negatively regulates the expression of Prdx6 in the white matter of the spinal cord.
Furthermore, upregulation of Nrf2 was observed in cytosolic and nuclear fraction of the hypoxia group as compared to the sham group, suggesting a nuclear translocation of Nrf2 in hypoxia (Fig. 5B). However, an upregulation of Nrf2 was observed only in cytosolic fraction of the reperfusion group and expression of Nrf2 remained unchanged in nuclear fraction as compared to the sham group (Fig. 5B). This suggest that the prolonged oxidative stress in the reperfusion group moves back the hypoxia-induced nuclear translocation of Nrf2 to the cytosol, therefore the cytosolic fraction majorly contributes to the increased expression of Nrf2 in the reperfusion group.
Prdx6 and Nrf2 are co-localized in hypoxia-reperfusion injury in the white matter of the spinal cord
As we have shown earlier that Prdx6 is localized with astrocytes and axons in the sham group, we observed a co-localization of Prdx6 (green) and Nrf2 (blue) with GFAP (red) (white color, yellow arrows). This suggests a possible association of Prdx6 and Nrf2 with astrocytes. In addition, most of the co-localization of Prdx6 and Nrf2 in astrocytes were observed around the nucleus (Fig. 5C, sham). In the hypoxia group, co-localization of Prdx6 (green) and Nrf2 (blue) was increased compared to the sham group and was majorly associated with reactive astrocytes (GFAP, red) (Fig. 5C, Hypx). Unlike the hypoxia group, Nrf2 expression was observed in astrocytes as well as other cell bodies after reperfusion. Compared to hypoxia, Prdx6 (green) and Nrf2 (blue) co-localization with GFAP (red) was more evenly distributed in astrocytes after reperfusion (Fig.5C, 4H-R).
Inhibition of aiPLA2 enzyme activity of Prdx6 prevents hypoxia-induced upregulation of Prdx6 and Nrf2
Hypoxia causes tissue acidification and reperfusion injury involves a “pH paradox”, where it returns from acidotic to physiologic pH that causes rapid cell death [21]. It is reported that Prdx6 exhibits maximal aiPLA2 activity at an acidic pH and maximum peroxidase activity at neutral pH [22]; therefore, we further evaluated if negative regulation of Prdx6 by Nrf2 is mediated through its aiPLA2 activity. A serine “protease” inhibitor, MJ33, inhibited the aiPLA2 activity. Our data show that expression of Prdx6 in hypoxia returned to normal levels whereas it remains upregulated in reperfusion injury after inhibition of aiPLA2 activity (Fig. 6A). Looking at the expression of Nrf2, we observed no change in expression after hypoxia and an upregulation at 2 h of reperfusion compared to the sham group (Fig. 6B). Interestingly, a significant downregulation of Nrf2 was observed after 4 h reperfusion compared to after 2 h of reperfusion (Fig. 6B). Therefore, we observed a correlation of Nrf2 expression and Prdx6 expression after MJ33 treatment, suggesting that the negative regulation of Prdx6 observed earlier by Nrf2 (Fig. 1C and Fig. 5A) is mediated through aiPLA2 activity of Prdx6.
Next, we evaluated the expression of GFAP and NF-200 after MJ33 treatment. We observed no significant change in expression of GFAP in the hypoxia group, whereas 2 h and 4 h of reperfusion showed an upregulation of GFAP compared to the sham group (Fig. 6C). Conversely, we observed significant downregulation of NF-200 in the hypoxia group and significant upregulation in the 4 h reperfusion group compared to the sham group (Fig. 6D). We observed significant upregulation of NF-200 in the 2 h as well as 4 h of reperfusion groups compared to the hypoxia group (Fig. 6D), which is an indication of increased axonal damage after inhibition of aiPLA2 activity of Prdx6. Together, these results suggest that hypoxia-induced upregulation of Prdx6, Nrf2, GFAP, and NF-200 are regulated by Prdx6 aiPLA2 activity.