Background
In recent years, it has been discovered that human umbilical cord blood (hUCB) is a valuable source of stem cells, and many studies have looked into its therapeutic potential to initiate and maintain tissue repair. This study investigated the effect of these hUCB mesenchymal stem cells on matrix metalloproteins (MMPs) in rats after spinal cord injury (SCI). MMPs are a large family of proteolytic enzymes involved in inflammation, wound healing, and other pathological processes after neurological disorders, with some being harmful and others beneficial. Although this study looked at a large number of MMPs, its main focus was on MMP-2 which is involved in glial scar formation.
Part of this study was carried out in vivo, using adult male rats of similar weights that were divided into three groups, a sham control group (no injury), a group that were subjected to spinal cord injuries with no treatment, and a third group that were also injured but were injected with hUCB treatment 21 days following the injury. Segments of spinal cord from various rats in each group were removed and analyzed at days 1, 3, 7, and 21.
The second part of this study prepared cultures of spinal neurons (collected from other rats), to test the effect of hUCB on these neurons in vitro. Cultures of spinal neurons were prepared, as well as a coculture of spinal neurons and hUCB. These were exposed to STP, a substance which induces apoptosis, as well as H2O2 which causes free radical damage, because apoptosis and free radical damage are important processes involved in secondary degeneration after SCI. The coculture of spinal neurons and hUCB was also tested with STP in the presence of MMP-2 antibody which blocks the activity of MMP-2.
Results
Real-time PCR
Fold changes of each MMP family member were evaluated in both experimental and control groups using real-time PCR, and it was found that MMP-2 showed only slight upregulation during the 21 days post injury, however when treated with hUCB the upregulation increased substantially. This did not occur in any of the other MMPs (Figure 1).
Figure 1 (click image to enlarge)MMP-2 activity & expression increased in vivo
Analysis of spinal cords using gelatin zymography showed that bands corresponding to MMP-2 activity appeared most strongly after hUCB treatment, which did not occur with other MMPs (Figure 2).
Figure 2 (click image to enlarge)The upregulation of MMP-2 by hUCB was further confirmed by immunohistochemical analysis which showed immunoreactivity for MMP-2 was much more prominent in hUCB treated rats compared to control or untreated injured rats where it was hardly detectable. Even further confirmation was provided by western blot analysis, where neurons of spinal cord sections from the hUCB treated rats, taken from an area next to the injury site showed significant expression of MMP-2 compared to injured rats. This suggests that hUCB treatment after SCI causes MMP-2 to be released from neurons next to the injury site (Figure 3).
Figure 3 (click image to enlarge)hUCB treatment reduced glial scarring
Treatment with hUCB after SCI extensively reduced the amount of glial scarring after injury, while untreated rats showed much more prominent scarring. Glial scars are formed by aggregations of reactive astrocytes in response to CNS injury, interacting with molecules (CSPGs) that arrest regrowth of injured axons across the lesion site. MMP-2 degrades CSPG’s in vitro, allowing axonal regeneration. The hUCB treatment was correlated with both an increase in MMP-2 and a decrease in CSPGs (Figure 4).
Figure 4 (click image to enlarge)MMP-2 activity & expression increased in vitro
Results of cell survival and cytotoxicity in the in vitro experiment showed that treatment of spinal neurons with either H2O2 and/or STP significantly injured the neurons. This effect of injury was reversed when the neurons were treated with hUCB. When MMP-2 antibody was present, the protection provided by hUCB treatment was significantly reduced, offering further support that MMP-2 is involved in the protection offered by hUCB. These findings were further supported by analysis with gelatin zymography and immunocytochemical analysis (Figures 5 & 6).
Figure 5 (click image to enlarge) Figure 6 (click image to enlarge)Conclusion
According to the results, it appears that hUCB plays an important role in upregulation of MMP-2 which reduces glial scar formation following spinal cord injury, creating an environment suitable for axonal regeneration. This provides evidence for the therapeutic potential of hUCB treatment for patients suffering from spinal cord injury.
My opinion
The authors of this paper succeeded in presenting a lot of information in a well organized manner that made it easy to read and understand. The experiments were conducted very carefully and analyzed by a variety of methods in order to ensure accurate results. Both experimental and control results were compared and presented clearly, and I especially found the figures very useful. As the authors suggest, future experiments testing transgenic mice or pharmacological inhibitors of specific MMPs would provide further evidence on the therapeutic efficacy of hUCB after spinal cord injury.
Reference
Veeravallia et al., 2009. Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury. Neurobiology of Disease. Volume 36: 200-212 (Link)
