The actions of genes in the brain cells regulate most activities in the normal and diseased brain, including brain functions related to learning, response to stress, and psychiatric and neurologic disorders, as well as general medical disorders such as cardiovascular disease, stroke and traumatic injury that can change brain energy metabolism and blood flow. Our data presented herein suggest that MMP-9 activities from neurons and endothelia can adversely affect the development of cerebral metabolic disturbance after GCI. Our data indicate that, by targeting mmp-9 mRNA, short DNA has multiple applications as siDNA and reporter of mmp-9 mRNA expression in living mouse brains.
Perturbations in neurovascular functional integrity are associated with upregulation of several genes that initiate cascades of injury. Upstream signals such as oxidative stress, together with neutrophil and/or platelet interactions with activated endothelium, can upregulate the mmp-9
gene, which has been suggested to play a role in the BBB disruption associated with many acute neurological conditions. Severe disruption of the BBB diminishes its protective capabilities, and thus causes the brain to become “leaky” (Gidday et al., 2005
). In addition, attenuation of MMP-9 activity may be associated with a reduction in cell death after GCI (Lee et al., 2004
). The regional consistency observed in rADC, elevation in levels of MMP-9 antigen, and retention of SPION-mmp9 in the striatum further strengthen the suggestion that MMP-9 activities may be involved in the development of initial metabolic imbalance after GCI.
We established our GCI model in male C57black6 mice to investigate the expression of endogenous genes, cerebral oxidative stress, and brain damage. By virtue of a defect in the posterior communicating arteries of these animals, transient hypoxia/ischemia in this model induces GCI by restricting cerebral blood flow by at least 80% (Barone et al., 1993
; Fujii et al., 1997
; Murakami et al., 1998
). Because early studies reported cell death in these animals (Barone et al., 1993
; P. K. Liu et al., 1996
), many other studies have used the same mouse strain and reported apoptotic neuronal death, BBB leakage, and gliosis in the striatum, cortex, and hippocampus (Fujii et al., 1997
; Yang et al., 1997
; Kitagawa et al., 1998
; Murakami et al., 1998
; Terashima et al., 1998
; C. H. Liu et al., 2007a
). Brain injury induced by this GCI model is not characterized by distinct core and penumbral regions, as is the case in stroke models involving focal occlusion of the middle cerebral artery. Ventriculomegaly observed in MR images acquired after GCI in two mice suggests that damage includes loss of brain cells (). Some investigators believe this GCI model may simulate cerebral ischemia during cardiac arrest and cardio-pulmonary resuscitation, because cerebral blood flow is partially reduced during BCAO. Rat studies of GCI that simulates cardiac arrest show that leakage of the BBB is biphasic in nature, with stable BBB leakage at 10–48 h of reperfusion (Fujioka et al., 1994
; Mossakowski et al., 1994
; Back et al., 2004
). Our studies on VMD determined by using rADC () and our data on ex vivo
hybridization of FITC-sODN-mmp9 () support these observations. However, our method using noninvasive MRI for volumetric measurement of the threshold ADC for VMD may have an advantage over such other methods involving invasive quantification to measure BBB leakage measurements. Regions that exhibit a drop in ADC below the threshold are thought to represent brain areas of enhanced metabolic disturbance, where potential BBB leakage may occur (). We are certain that more studies will validate the potential application of noninvasive VMD to evaluate brain injury sites post-GCI.
By targeting mmp-9 mRNA, we have demonstrated the feasibility of detecting gene activities using MRI and manipulating gene action by changing VMD in the living brain after GCI. Our experiments have reliably shown that mmp-9 mRNA and protein are expressed in regions that exhibit metabolic disturbance, as detected by abnormal water content in MRI (hDWI), conventional immunohistochemistry, MMP-9 activation assay (zymography), and in vivo
hybridization with an sODN-mmp9 probe coupled with ex vivo
observation. We also demonstrated that gene knockdown by sODN-mmp9 after GCI reduces MMP-9 activities, and that this brain probe that reduces MMP-9 activities also significantly reverses striatal ADC drop, a parameter known to be associated with a reduction in brain damage (Mancuso et al., 2000
). These observation are consistent with those of gene knockdown in acute neurological disorders in rodents (Wahlestedt et al., 1993
; Zhang et al., 1999
), although in our current study, we delivered the brain probe after GCI. By measuring VMD after blocking MMP-9 activities with the sODN-mmp9 brain probe, we observed a decrease in the severity of metabolic disturbance. Having made these observations, we found evidence to support the hypothesis that MMP-9 gene expression may play a role in the formation of striatal brain damage. This conclusion is consistent with results showing lesser levels of cell death after GCI in an MMP-9 deficient mouse strain (Lee et al., 2004
An additional advantage of our method is that the probe was administered after the GCI episode, and as such more closely resembled a real clinical scenario than other methods that require delivery of brain probe before acute neurological disorder. The amount of iron needed in gene transcript MRI (120 pmol per kg body weight) has been demonstrated not to exacerbate GCI outcome (C. H. Liu et al., 2007a
). Although a dose of sODN-mmp9 much higher than the tracking dose is required for gene knockdown, the MMP-9 knockdown protocol involves less or no iron oxide contrast agent. Iron oxide concentration as high as 8 nmol in human cells is considered safe (Bulte et al., 2001
). Therefore, iron overload may not be an issue for manipulating MMP-9 activities. Indeed, we delivered repeated doses (as high as 10 nmol per kg once a week for 4 weeks via intraperitoneal route) and observed no adverse effect (C. H. Liu et al., 2008
). Unique to our sODN-mmp9 probe, as reported here, is the potential application for mechanistic investigation using gene knockdown of mmp-9 mRNA in living mouse brains. By enabling such studies of mechanism, this probe represents a great advance for neuroscience. Our technique has potential for clinical application, especially for efforts to delineate gene expression and brain injury.
Other advantages of our probe are its applications for tracking intracellular mRNA transcripts at low dose and initiating gene knockdown at high dose. Both of these applications rely on the base pairing between nucleic acids of complementary sequences, as is evident by results obtained with a control probe of random sequence. Whereas the control reports no endogenous mRNA, no effect on MMP-9 activities, or change in rADC, the sODN-mmp9 probe is shown to reduce MMP-9 activity and create specific gene knockdown in live mouse brains after GCI, without affecting the expression of actin, which is known not to be affected by GCI (Cui and Liu, 2001
). Other potential applications that have been reported include assessment of gene transcription activities, apoptotic cell death (marked by cells’ inability to take up SPION-cfos), and cell typing (cells with specific gene transcript) during brain repair by gliosis and angiogenesis in animal models of acute neurological disorders (C. H. Liu et al., 2007a
). Although the involvement of MMP-9 activities in the etiology of cell death is well established in stroke models, we know very little about brain injury after cardiac arrest. Elevated blood levels of MMP have been used to diagnose BBB disruption in humans (Clark et al., 1997
; McGirt et al., 2002
; Lynch et al., 2004
), mice (Asahi et al., 2001
; Huang et al., 2001
; Magnoni et al., 2004
) and rats (Pfefferkorn and Rosenberg, 2003
); still, an MMP assay is less likely to offer information about a regional correlation between lesion and MMP-9 activity. We show here that MMP-9 antigen is expressed in the same location where metabolic disturbance is exhibited after GCI (), and our results agree with those that have been reported in the literature.
It is reasonable to suggest that elevated VMD, as detected by in vivo
MRI, can serve as a metric of brain damage. We have observed here that GCI-induced VMD is significantly reduced when MMP-9 activity is temporarily blocked after GCI in C57black6 mice. We must conduct future studies to explore whether continuous and repeated dosing of sODN-mmp9 brings about an undesirable effect. Nevertheless, the short duration of action by antisense sODN-mmp9 may be more desirable than a longer lasting one, as MMP-9 activities may be required for angiogenesis and tissue repair (Zhao et al., 2006
; Bove et al., 2007
). Therefore, the balance of extended and repeated dosing will have to be carefully monitored as alternative approaches.
The knockdown effect of sODN-mmp9 on VMD is transient and never 100% effective; and results indicate that products of other gene transcripts may also be involved in the development of VMD or rADC. Therefore, the development of VMD after GCI is likely mediated by continuous expression of mmp-9 mRNA or multiple gene transcripts that can be investigated using the same strategies for different mRNA targets. It is important to note that sODN-mmp9 was administered in our studies after the GCI episode, eliminating the need for the preconditioning often used in therapies of cerebral ischemia. In conclusion, we have demonstrated the feasibility of using a short inhibitory nucleic acid to induce gene knockdown after vessel occlusion, and the feasibility of a novel MR contrast agent to trace sODN-mmp9 and allow MRI detection of endogenous MMP-9 activities in the striatum of living animal brains. We have further demonstrated that VMD is reduced and rADC reversed in the striatum after MMP-9 activity knockdown. The novel technique we have developed holds potential to revolutionize investigations of drug efficacy and therapeutic evaluation by alleviating reliance on methods that require postmortem tissue sampling.