Stroke is the third leading cause of death and the leading cause of long-term disability in adults. Several members of the matrix metalloproteinase (MMPs) family have been implicated to have detrimental roles in stroke [1, 2]. Specifically, the gelatinases MMP-2 and MMP-9 have previously been considered to specifically injure the important components of the basal lamina around the cerebral blood vessels that precede microvascular damage in cerebral ischemia . This leads to disruption of the blood brain barrier, edema, and hemorrhagic transformation in animal models of ischemia [3, 4]. Once within the CNS, MMPs continue to damage CNS tissue. In general higher MMP-9 levels are shown to correlate significantly with larger infarct volume, severity of stroke, reduced survival of neurons and worse functional outcome , and therefore, seem an appropriate target for a robust neuroprotectant such as hypothermia [6, 7].
Most current studies investigating cerebral ischemia and stroke detect MMPs in vitro using techniques such as Western Blotting , ELISA  RT-PCR or staining with anti-MMP antibodies . While these are good techniques they fall short when reporting accurate levels of the active form of MMP, are limited in their sensitivities of MMP detection, and may not be a precise depiction of MMP activity in vivo. In vivo visualization of MMP activity would provide important information regarding the spatial and temporal expression of MMP enzymatic activity with respect to the pathophysiology of the disease and to monitor response to interventions, such as therapeutic hypothermia.
NIRF emitting probes offer the advantage of increased depth of detection. Recently probes that fluoresce upon proteolytic cleavage by MMP’s have become available with the potential advantage of exhibiting low background fluorescence. One such probe, MMPSense680 (Perkin Elmer Inc (previously VisEn Medical), Boston, MA USA) with a peak excitation at approx 680 nm and emission at 700 nm, was shown to produce increased fluorescent signal considered related to MMP mediated cleavage in diseases including stroke [11, 12]. Activation of the probe is reported to occur by a broad range of MMP’s including MMP 2, 3, 9, and 13 and the manufacturer recommends imaging at 24 hr following administration.
The objective of the present study was to determine whether an alternate MMP activatable probe with a shorter optical imaging time, MMPSense™ 750 FAST (MMPSense750), could be used for visualization of MMP activity in the early stages of ischemia reperfusion in a mouse model of stroke. This probe also has fluorescent properties in the near infrared range (peak excitation and emission at approx 749 nm and 775 nm, respectively) and according to the manufacturer is sensitive to cleavage and optical activation by various MMP’s including MMP2,3,7,9,12 and MMP13. In order to investigate the responsiveness of the probe to alterations in MMP activity, the current study also examined whether the MMPSense750 probe would detect reductions in MMP’s associated with a therapeutic intervention following stroke We chose mild hypothermia for its well documented therapeutic reductions in various MMPs in a number of neurological diseases including stroke [13–16]. In parallel with in vivo studies, characteristics of the probe were investigated in vitro.