This study reports a novel early up-regulation of MMP-9 in ischemic reperfusion. The acute in-vivo detection of MMP upregulation following ischemic stroke has not been reported previously. Importantly, we describe an innovative technique by which the increase in MMP-9 levels can be imaged in-situ within the first few hours of ischemia reperfusion using NIR fluorescent imaging and an activatable optical probe MMPSense750. Furthermore, we demonstrate that hypothermia attenuates the optical intensity increase in MMPSense750 and the increase in MMP-9 protein expression among the broad range of MMP’s potentially detected by MMPSense750, (e.g. MMP 2, 3, 7, 9, 12, and 13). Our data provides evidence that MMPSense750 can detect activity of MMPs in-vivo and taken together our findings support the proof of concept that NIR fluorescent imaging can be used in situ to assess the mechanisms involved in cerebral ischemia.
The expression of MMP-9 corresponded well with the spatial and temporal activation of MMPSense750. The time dependent activities of MMPs have been previously well documented [20
]. Of the two MMPs - 9 and 2 that are consistently reported to be up-regulated in ischemic stroke, we found MMP-9 to be the most strongly expressed in early ischemia reperfusion and later at 24 hours post reperfusion. The increased activity of the MMPSense750 sense probe was evident regionally and temporally in both the infarct and peri-infarct regions when compared with the regions of endogenous FITC fluorescence considered to represent areas of predominantly increased NADH within the ischemic lesion [18
]. The early increase in NIR optical intensity would suggest that MMPSense750 is being activated by increased MMP activity either at the intraluminal surface by activated leukocytes or at the basal lamina [16
]. It is important to note that when using this probe it is necessary to control for presence of unconjugated probe and potential systemic activation of the probe in vivo
, because when we added MMPSense750 to PBS fluorescent intensity immediately increased and MMPSense750 administered to sham operated mice produced immediate low level near infrared fluorescence intensity increases equally distributed over both hemispheres.
Non invasive imaging of MMP activity remains a clinically important unmet need. In a recent exploratory study, a NIR fluorescent probe with excitation emission in the 680
nm wavelength range was used to detect MMP activity in an animal model of stroke using NIRF imaging accompanied by magnetic resonance and radioisotope imaging techniques [12
]. In this study, the utility of this 680
nm probe was investigated only at 24 hours after ischemia. For optimal clinical application, it is important to have an activatable probe that can also detect early MMP upregulation following cerebral ischemia and the present results demonstrate MMPSense750 can detect earlier acute changes in MMP/protease activity.
There were recognized limitations of our study. We used gelatin zymography to detect MMP-2 and 9 in brain tissues, and the levels of these MMPs were too low to be detected by this technique suggesting the importance of our imaging method, which is sensitive enough to detect low levels of MMP activity. The fact that we were not able to detect large quantities of pro-MMP-9 would suggest that the optical changes measured included activated MMP-9. Clearly also possible is that other MMP’s contributed to the MMPSense intensity increases observed. Furthermore, our observation that trypsin activates MMPSense750 in vitro supports the conclusion that this probes activation can be induced by other proteases such as serine proteases in addition to MMPs. Another limitation was our inability to readily image NIR fluorescent changes though the skull. Non invasive detection is theoretically possible with NIR probes but considering the small magnitude of the changes observed we believe this would need substantial technical improvements with advances in non-invasive imaging systems and probe sensitivity. In addition to the small changes in signal intensity, the need for background correction for this particular probe may limit its clinical applicability.
A novel component of this study was the in vivo
demonstration that moderate hypothermia induced after ischemia reperfusion reduced both the NIR optical signal intensity and also the level of MMP-9 protein detected. Previous studies from MMP gene knockout mice and those using MMP pharmacological inhibitors suggest that the MMPs may be attractive therapeutic targets for stroke [22
]. It has been demonstrated that moderate hypothermia (32-34°C) protects the basal lamina, reduces infarct volume and hemorrhage, and reduces MMP-9 [25
]. However, emerging data now also suggests that some aspects of MMP activity during the delay of neuroinflammatory response may contribute to re-modeling in stroke recovery [27
]. Therefore, defining the time dependent relationship of MMP activation has been increasingly emphasized and timing of therapeutic strategies require detailed refinement to avoid the potential theoretical deleterious effects of MMP inhibition on stroke remodeling. In this capacity the use of experimental tools such as near NIR fluorescent probes and NIR imaging may become invaluable both in pre clinical and clinical testing.