The paradox of tPA in ischemic stroke: tPA knockdown following recanalization improves functional and histological outcomes.

Previous studies have demonstrated that endogenous tissue-type plasminogen activator (tPA) is upregulated in the brain after an acute ischemic stroke (AIS). While mixed results were observed in genetic models, the pharmacological inhibition of endogenous tPA showed beneficial effects. Treatment with exogenous recombinant tPA exacerbated brain damage in rodent models of stroke. Despite the detrimental effects of tPA in ischemic stroke, recombinant tPA is administered to AIS patients to recanalize the occluded blood vessels because the benefits of its administration outweigh the risks associated with tPA upregulation and increased activity. We hypothesized that tPA knockdown following recanalization would ameliorate sensorimotor deficits and reduce brain injury. Young male and female rats (2-3 months old) were subjected to transient focal cerebral ischemia by occlusion of the right middle cerebral artery. Shortly after reperfusion, rats from appropriate cohorts were administered a nanoparticle formulation containing tPA shRNA or control shRNA plasmids (1 mg/kg) intravenously via the tail vein. Infarct volume during acute and chronic phases, expression of matrix metalloproteinases (MMPs) 1, 3, and 9, enlargement of cerebral ventricle volume, and white matter damage were all reduced by shRNA-mediated gene silencing of tPA following reperfusion. Additionally, recovery of somatosensory and motor functions was improved. In conclusion, our results provide evidence that reducing endogenous tPA following recanalization improves functional outcomes and reduces post-stroke brain damage.

Therapeutic efficacy of matrix metalloproteinase-12 suppression on neurological recovery after ischemic stroke: Optimal treatment timing and duration.

We recently showed that the post-ischemic induction of matrix metalloproteinase-12 (MMP-12) in the brain degrades tight junction proteins, increases MMP-9 and TNFα expression, and contributes to the blood-brain barrier (BBB) disruption, apoptosis, demyelination, and infarct volume development. The objectives of this study were to (1) determine the effect of MMP-12 suppression by shRNA-mediated gene silencing on neurological/functional recovery, (2) establish the optimal timing of MMP-12shRNA treatment that provides maximum therapeutic benefit, (3) compare the effectiveness of acute versus chronic MMP-12 suppression, and (4) evaluate potential sex-related differences in treatment outcomes. Young male and female Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion. Cohorts of rats were administered either MMP-12shRNA or scrambled shRNA sequence (control) expressing plasmids (1 mg/kg; i.v.) formulated as nanoparticles. At designated time points after reperfusion, rats from various groups were subjected to a battery of neurological tests to assess their reflex, balance, sensory, and motor functions. Suppression of MMP-12 promoted the neurological recovery of stroke-induced male and female rats, although the effect was less apparent in females. Immediate treatment after reperfusion resulted in a better recovery of sensory and motor function than delayed treatments. Chronic MMP-12 suppression neither enhanced nor diminished the therapeutic effects of acute MMP-12 suppression, indicating that a single dose of plasmid may be sufficient. We conclude that suppressing MMP-12 after an ischemic stroke is a promising therapeutic strategy for promoting the recovery of neurological function.

The interplay between MMP-12 and t-PA in the brain after ischemic stroke.

Tissue-type plasminogen activator (t-PA) expression is known to increase following transient focal cerebral ischemia and reperfusion. Previously, we reported downregulation of t-PA upon suppression of matrix metalloproteinase-12 (MMP-12), following transient focal cerebral ischemia and reperfusion. We now present data on the temporal expression of t-PA in the brain after transient ischemia, as well as the interaction between MMP-12 and t-PA, two proteases associated with the breakdown of the blood-brain barrier (BBB) and ischemic brain damage. We hypothesized that there might be reciprocal interactions between MMP-12 and t-PA in the brain after ischemic stroke. This hypothesis was tested using shRNA-mediated gene silencing and computational modeling. Suppression of t-PA following transient ischemia and reperfusion in rats attenuated MMP-12 expression in the brain. The overall effect of t-PA shRNA administration was to attenuate the degradation of BBB tight junction protein claudin-5, diminish BBB disruption, and reduce neuroinflammation by decreasing the expression of the microglia/macrophage pro-inflammatory M1 phenotype (CD68, iNOS, IL-1ß, and TNFα). Reduced BBB disruption and subsequent lack of infiltration of macrophages (the main source of MMP-12 in the ischemic brain) could account for the decrease in MMP-12 expression after t-PA suppression. Computational modeling of in silico protein-protein interactions indicated that MMP-12 and t-PA may interact physically. Overall, our findings demonstrate that MMP-12 and t-PA interact directly or indirectly at multiple levels in the brain following an ischemic stroke. The present findings could be useful in the development of new pharmacotherapies for the treatment of stroke.

Stem cell treatment improves post stroke neurological outcomes: a comparative study in male and female rats.

BACKGROUND AND PURPOSE: The therapeutic potential of different stem cells for ischaemic stroke treatment is intriguing and somewhat controversial. Recent results from our laboratory have demonstrated the potential benefits of human umbilical cord blood-derived mesenchymal stem cells (MSC) in a rodent stroke model. We hypothesised that MSC treatment would effectively promote the recovery of sensory and motor function in both males and females, despite any apparent sex differences in post stroke brain injury. METHODS: Transient focal cerebral ischaemia was induced in adult Sprague-Dawley rats by occlusion of the middle cerebral artery. Following the procedure, male and female rats of the untreated group were euthanised 1 day after reperfusion and their brains were used to estimate the resulting infarct volume and tissue swelling. Additional groups of stroke-induced male and female rats were treated with MSC or vehicle and were subsequently subjected to a battery of standard neurological/neurobehavioral tests (Modified Neurological Severity Score assessment, adhesive tape removal, beam walk and rotarod). The tests were administered at regular intervals (at days 1, 3, 5, 7 and 14) after reperfusion to determine the time course of neurological and functional recovery after stroke. RESULTS: The infarct volume and extent of swelling of the ischaemic brain were similar in males and females. Despite similar pathological stroke lesions, the clinical manifestations of stroke were more pronounced in males than females, as indicated by the neurological scores and other tests. MSC treatment significantly improved the recovery of sensory and motor function in both sexes, and it demonstrated efficacy in both moderate stroke (females) and severe stroke (males). CONCLUSIONS: Despite sex differences in the severity of post stroke outcomes, MSC treatment promoted the recovery of sensory and motor function in male and female rats, suggesting that it may be a promising treatment for stroke.

Attenuation of the Induction of TLRs 2 and 4 Mitigates Inflammation and Promotes Neurological Recovery After Focal Cerebral Ischemia.

The intense inflammatory response triggered in the brain after focal cerebral ischemia is detrimental. Recently, we showed that the suppression of toll-like receptors (TLRs) 2 and 4 attenuates infarct size and reduces the expression of pro-inflammatory cytokines in the ischemic brain. In this study, we further examined the effect of unsuppressed induction of TLRs 2 and 4 on the expression of its downstream signaling molecules and pro-inflammatory cytokines 1 week after reperfusion. The primary purpose of this study was to investigate the effect of simultaneous knockdown of TLRs 2 and 4 on M1/M2 microglial polarization dynamics and post-stroke neurological deficits and the recovery. Transient focal cerebral ischemia was induced in young adult male Sprague-Dawley rats by the middle cerebral artery occlusion (MCAO) procedure using a monofilament suture. Appropriate cohorts of rats were treated with a nanoparticle formulation of TLR2shRNA and TLR4shRNA (T2sh+T4sh) expressing plasmids (1 mg/kg each of T2sh and T4sh) or scrambled sequence inserted vector (vehicle control) expressing plasmids (2 mg/kg) intravenously via tail vein immediately after reperfusion. Animals from various cohorts were euthanized during reperfusion, and the ischemic brain tissue was isolated and utilized for PCR followed by agarose gel electrophoresis, real-time PCR, immunoblot, and immunofluorescence analysis. Appropriate groups were subjected to a battery of standard neurological tests at regular intervals until 14 days after reperfusion. The increased expression of both TLRs 2 and 4 and their downstream signaling molecules including the pro-inflammatory cytokines was observed even at 1-week after reperfusion. T2sh+T4sh treatment immediately after reperfusion attenuated the post-ischemic inflammation, preserved the motor function, and promoted recovery of the sensory and motor functions. We conclude that the post-ischemic induction of TLRs 2 and 4 persists for at least 7 days after reperfusion, contributes to the severity of acute inflammation, and impedes neurological recovery. Unlike previous studies in TLRs 2 or 4 knockout models, results of this study in a pharmacologically relevant preclinical rodent stroke model have translational significance.

Snapshot Multispectral Imaging Is Not Inferior to SPY Laser Fluorescence Imaging When Predicting Murine Flap Necrosis.

BACKGROUND: Objective assessment of tissue viability is critical to improve outcomes of cosmetic and reconstructive procedures. A widely used method to predict tissue viability is indocyanine green angiography. The authors present an alternative method that determines the relative proportions of oxyhemoglobin to deoxyhemoglobin through multispectral reflectance imaging. This affordable, hand-held device is noninvasive and may be used in clinic settings. The authors hypothesize that multispectral reflectance imaging is not inferior to indocyanine green angiography in predicting flap necrosis in the murine model. METHODS: Reverse McFarlane skin flaps measuring 10 × 3 cm were raised on 300- to 400-g male Sprague-Dawley rats. Indocyanine green angiography and multispectral reflectance imaging was performed before surgery, immediately after surgery, and 30 minutes after surgery. Clinical outcome images acquired 72 hours after surgery were evaluated by three independent plastic surgeons. Objective data obtained immediately after surgery were compared to postsurgical clinical outcomes to determine which method more accurately predicted flap necrosis. RESULTS: Nine reverse McFarlane skin flaps were evaluated 72 hours after flap elevation. Data analysis demonstrated that the 95 percent confidence intervals for the sensitivity of postoperative multispectral reflectance imaging and indocyanine green angiography imaging to predict 72-hour tissue viability at a fixed specificity of 90 percent for predicting tissue necrosis were 86.3 to 91.0 and 79.1 to 86.9, respectively. CONCLUSIONS: In this experimental animal model, multispectral reflectance imaging does not appear to be inferior to indocyanine green angiography in detecting compromised tissue viability. With the advantages of noninvasiveness, portability, affordability, and lack of disposables, multispectral reflectance imaging has an exciting potential for widespread use in cosmetic and reconstructive procedures.

Zinc removal from basic oxygen steelmaking (BOS) filter cake by sintering.

Zinc removal from basic oxygen steelmaking filter cake was studied by tablet testing and in a millipot under different conditions. The results from tablet testing show that coke and metallic iron in filter cake play the role of reductants to remove zinc during sintering. Zinc removal increased with increasing temperature and decreasing partial pressure of oxygen. Zinc removal reached 24.6% when a tablet of filter cake without addition of coke was sintered at 1300 °C in a nitrogen atmosphere. Addition of 3 to 9 wt% of coke breeze into filter cake enhanced the zinc removal to 60.6 to 91.4%, in an atmosphere with 0.5 vol% oxygen. The results from millipot sintering show that adding filter cake in the form of tablets was effective in removing zinc, especially when additional coke was added into the tableted filter cake. Zinc vapour generated at high temperature in the reducing atmosphere diffused out of the tablets and was carried with flowing gas, and then re-oxidized and deposited on the lower layer of sintering material in the bed. Positioning the filter cake in the form of tablets at the bottom of the sinter bed is therefore suggested as a possibility for zinc removal from filter cake.

Exosomes Secreted by the Cocultures of Normal and Oxygen-Glucose-Deprived Stem Cells Improve Post-stroke Outcome.

Emerging stroke literature suggests that treatment of experimentally induced stroke with stem cells offered post-stroke neuroprotection via exosomes produced by these cells. Treatment with exosomes has great potential to overcome the limitations associated with cell-based therapies. However, in our preliminary studies, we noticed that the exosomes released from human umbilical cord blood-derived mesenchymal stem cells (MSCs) under standard culture conditions did not improve the post-stroke neurological outcome. Because of this apparent discrepancy, we hypothesized that exosome characteristics vary with the conditions of their production. Specifically, we suggest that the exosomes produced from the cocultures of regular and oxygen-glucose-deprived (OGD) MSCs in vitro would represent the exosomes produced from MSCs that are exposed to ischemic brain cells in vivo, and offer similar therapeutic benefits that the cell treatment would provide. We tested the efficacy of therapy with exosomes secreted from human umbilical cord blood (HUCB)-derived MSCs under in vitro hypoxic conditions on post-stroke brain damage and neurological outcome in a rat model of transient focal cerebral ischemia. We performed the TTC staining procedure as well as the neurological tests including the modified neurological severity scores (mNSS), the modified adhesive removal (sticky-tape), and the beam walking tests before ischemia and at regular intervals until 7 days reperfusion. Treatment with exosomes obtained from the cocultures of normal and OGD-induced MSCs reduced the infarct size and ipsilateral hemisphere swelling, preserved the neurological function, and facilitated the recovery of stroke-induced rats. Based on the results, we conclude that the treatment with exosomes secreted from MSCs at appropriate experimental conditions attenuates the post-stroke brain damage and improves the neurological outcome.

Exosomes Treatment Mitigates Ischemic Brain Damage but Does Not Improve Post-Stroke Neurological Outcome.

BACKGROUND/AIMS: Recent studies demonstrated that the treatment with mesenchymal stem cells (MSCs) obtained from the human umbilical cord blood improved survival, reduced brain damage, prevented apoptosis, suppressed inflammatory responses, downregulated the DNA damage-inducing genes, upregulated the DNA repair genes, and facilitated neurological recovery in stroke-induced animals. Emerging stroke literature supports the concept that the exosomes released from MSCs are the primary biological principles underlying the post-stroke neuroprotection offered by MSCs treatment. METHODS: Because the treatment with exosomes has a great potential to overcome the limitations associated with cell-based therapies, we tested the efficacy of exosomes secreted from HUCB-MSCs under standard culture conditions on post-stroke brain damage and neurological outcome in a rat model of ischemic stroke by performing TTC staining as well as the modified neurological severity scores, modified adhesive removal, beam-walking, and accelerating Rotarod performance tests before ischemia and at regular intervals until seven days reperfusion. RESULTS: Exosomes treatment attenuated the infarct size. Treatment with exosomes did not affect the post-stroke survival rate and body weight changes, but exacerbated the somatosensory and motor dysfunction and adversely affected the natural recovery that occurs without any treatment. CONCLUSION: Treatment with exosomes secreted from HUCB-MSCs under standard culture conditions attenuates the ischemic brain damage but does not improve the post-stroke neurological outcome.

Post-stroke mRNA expression profile of MMPs: effect of genetic deletion of MMP-12.

Background and purpose: Recent reports from our laboratory demonstrated the post-ischaemic expression profile of various matrix metalloproteinases (MMPs) in rats and the detrimental role of MMP-12 in post-stroke brain damage. We hypothesise that the post-stroke dysregulation of MMPs is similar across species and that genetic deletion of MMP-12 would not affect the post-stroke expression of other MMPs. We tested our hypothesis by determining the pre-ischaemic and post-ischaemic expression profile of MMPs in wild-type and MMP-12 knockout mice. Methods: Focal cerebral ischaemia was induced in wild-type and MMP-12 knockout mice by middle cerebral artery occlusion procedure by insertion of a monofilament suture. One hour after ischaemia, reperfusion was initiated by removing the monofilament. One day after reperfusion, ischaemic brain tissues from various groups of mice were collected, and total RNA was isolated and subjected to cDNA synthesis followed by PCR analysis. Results: Although the post-stroke expression profile of MMPs in the ischaemic brain of mice is different from rats, there is a clear species similarity in the expression of MMP-12, which was found to be predominantly upregulated in both species. Further, the post-stroke induction or inhibition of various MMPs in MMP-12 knockout mice is different from their respective expression profile in wild-type mice. Moreover, the brain mRNA expression profile of various MMPs in MMP-12 knockout mice under normal conditions is also different to their expression in wild-type mice. Conclusions: In the ischaemic brain, MMP-12 upregulates several fold higher than any other MMP. Mice derived with the genetic deletion of MMP-12 are constitutive and have altered MMP expression profile both under normal and ischaemic conditions.

Prevention of the Severity of Post-ischemic Inflammation and Brain Damage by Simultaneous Knockdown of Toll-like Receptors 2 and 4.

Toll-like receptor 2 (TLR2) and TLR4 belong to a family of highly conserved pattern recognition receptors and are well-known upstream sensors of signaling pathways of innate immunity. TLR2 and TLR4 upregulation is thought to be associated with poor outcome in stroke patients. We currently show that transient focal ischemia in adult rats induces TLR2 and TLR4 expression within hours and shRNA-mediated knockdown of TLR2 and TLR4 alone and in combination decreases the infarct size and swelling. We further show that TLR2 and TLR4 knockdown also prevented the induction of their downstream signaling molecules MyD88, IRAK1, and NFκB p65 as well as the pro-inflammatory cytokines IL-1ß, IL-6, and TNFα. This study thus shows that attenuation of the severity of TLR2- and TLR4-mediated post-stroke inflammation ameliorates ischemic brain damage.

Evaluation of chemical stability of polymers of XIENCE everolimus-eluting coronary stents in vivo by pyrolysis-gas chromatography/mass spectrometry.

The polymers poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) and poly(n-butyl methacrylate) (PBMA) are employed in manufacturing the XIENCE family of coronary stents. PBMA serves as a primer and adheres to both the stent and the drug coating. PVDF-HFP is employed in the drug matrix layer to hold the drug everolimus on the stent and control its release. Chemical stability of the polymers of XIENCE stents in the in-vivo environment was evaluated by pyrolysis-gas chromatography with mass spectrometry (Py-GC/MS) detection. For this evaluation, XIENCE stents explanted from porcine coronary arteries and from human coronary artery specimens at autopsy after 2-4 and 5-7 years of implantation, respectively, were compared to freshly manufactured XIENCE stents (controls). The comparison of pyrograms of explanted stent samples and controls showed identical fragmentation fingerprints of polymers, indicating that PVDF-HFP and PBMA maintained their chemical integrity after multiple years of XIENCE coronary stent implantation. The findings of the present study demonstrate the chemical stability of PVDF-HFP and PBMA polymers of the XIENCE family of coronary stents in the in-vivo environment, and constitute a further proof of the suitability of PVDF-HFP as a drug carrier for the drug eluting stent applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1721-1729, 2018.

MMP-12, a Promising Therapeutic Target for Neurological Diseases.

The role of matrix metalloproteinase-12 (MMP-12) in the pathogenesis of several inflammatory diseases such as chronic obstructive pulmonary disease, emphysema, and asthma is well established. Several new studies and recent reports from our laboratory and others highlighted the detrimental role of MMP-12 in the pathogenesis of several neurological diseases. In this review, we discuss in detail the pathological role of MMP-12 and the possible underlying molecular mechanisms that contribute to disease pathogenesis in the context of central nervous system diseases such as stroke, spinal cord injury, and multiple sclerosis. The available information on the specific MMP-12 inhibitors used in several preclinical and clinical studies is also reviewed. Based on the reported studies to date, MMP-12 suppression could emerge as a promising therapeutic target for several CNS diseases that were discussed in this review.

Mesenchymal Stem Cell Treatment Prevents Post-Stroke Dysregulation of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases.

BACKGROUND/AIMS: Stem cell treatment is one of the potential treatment options for ischemic stroke. We recently demonstrated a protective effect of human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) in a rat model of ischemic stroke. The treatment attenuated apoptosis and prevented DNA damage. A collection of published studies, including several from our laboratory, indicated the induction and detrimental role for several matrix metalloproteinases (MMPs) in post-stroke brain injury. We hypothesized that the HUCB-MSCs treatment after focal cerebral ischemia prevents the dysregulation of MMPs and induces the expression of endogenous tissue inhibitors of metalloproteinases (TIMPs) to neutralize the elevated activity of MMPs. METHODS: To test our hypothesis, we administered HUCB-MSCs (0.25 million cells/animal and 1 million cells/animal) intravenously via tail vein to male Sprague-Dawley rats that were subjected to a transient (two-hour) right middle cerebral artery occlusion (MCAO) and one-day reperfusion. Ischemic brain tissues obtained from various groups of rats seven days after reperfusion were subjected to real-time PCR, immunoblot, and immunofluorescence analysis. RESULTS: HUCB-MSCs treatment prevented the induction of MMPs, which were upregulated in ischemia-induced rats that received no treatment. HUCB-MSCs treatment also prevented the induction of TIMPs expression. The extent of prevention of MMPs and TIMPs induction by HUCB-MSCs treatment is similar at both the doses tested. CONCLUSION: Prevention of stroke-induced MMPs upregulation after HUCB-MSCs treatment is not mediated through TIMPs upregulation.

SPARC overexpression suppresses radiation-induced HSP27 and induces the collapse of mitochondrial Δψ in neuroblastoma cells.

Neuroblastoma is the cause of >15% of cancer-associated mortality in children in the USA. Despite aggressive treatment regimens, the long-term survival rate for these children remains at <40%. The current study demonstrates that secreted protein acidic and rich in cysteine (SPARC) suppresses radiation-induced expression of heat shock protein 27 (HSP27) in vivo and suppresses mitochondrial membrane potential (Δψ) in neuroblastoma cells. In the present study, the overexpression of SPARC in SK-N-BE(2) and NB1691 neuroblastoma cell lines suppresses radiation-induced G2M cell cycle arrest, proliferation, HSP27 expression (in vitro and in vivo) and induces the collapse of the mitochondrial Δψ. Gene ontology analysis demonstrated that the overexpression of SPARC combined with irradiation, induces the expression of dissimilar molecular function genes in SK-N-BE(2) and NB1691 cells, providing evidence of a dissimilar response signaling pathway. These results demonstrate that overexpression of SPARC suppresses radiation-induced HSP27 expression in neuroblastoma cells and the combination of SPARC and radiation induces the expression of protein 21, but suppresses neuroblastoma tumor density in in vivo mouse models. SPARC also induces mitochondrial Δψ collapse in SK-N-BE(2) and NB1691 neuroblastoma cells.

SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR­410 in human neuroblastoma cells.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N­BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the overexpression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

Matrix Metalloproteinase-12 Induces Blood-Brain Barrier Damage After Focal Cerebral Ischemia.

BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMPs) have a central role in compromising the integrity of the blood-brain barrier (BBB). The role of MMP-12 in brain damage after ischemic stroke remains unknown. The main objective of the current study is to investigate the effect of MMP-12 suppression at an early time point before reperfusion on the BBB damage in rats. METHODS: Sprague-Dawley rats were subjected to middle cerebral artery occlusion and reperfusion. MMP-12 shRNA-expressing plasmids formulated as nanoparticles were administered at a dose of 1 mg/kg body weight. The involvement of MMP-12 on BBB damage was assessed by performing various techniques, including Evans blue dye extravasation, 2,3,5-triphenyltetrazolium chloride staining, immunoblot, gelatin zymography, and immunofluorescence analysis. RESULTS: MMP-12 is upregulated ≈31-, 47-, and 66-fold in rats subjected 1-, 2-, or 4-hour ischemia, respectively, followed by 1-day reperfusion. MMP-12 suppression protected the BBB integrity by inhibiting the degradation of tight-junction proteins. Either intravenous or intra-arterial delivery of MMP-12 shRNA-expressing plasmid significantly reduced the percent Evans blue dye extravasation and infarct size. Furthermore, MMP-12 suppression reduced the endogenous levels of other proteases, such as tissue-type plasminogen activator and MMP-9, which are also known to be the key players involved in BBB damage. CONCLUSIONS: These results demonstrate the adverse role of MMP-12 in acute brain damage that occurs after ischemic stroke and, thereby, suggesting that MMP-12 suppression could be a promising therapeutic target for cerebral ischemia.

Post-transcriptional inactivation of matrix metalloproteinase-12 after focal cerebral ischemia attenuates brain damage.

This study highlights the possible pathological role of MMP-12 in the context of ischemic stroke. Male rats were subjected to a two-hour middle cerebral artery occlusion (MCAO) procedure. MMP-12 shRNA expressing plasmid formulation was administered to these rats twenty-four hours after reperfusion. The results showed a predominant upregulation of MMP-12 (approximately 47, 58, 143, and 265 folds on days 1, 3, 5, 7 post-ischemia, respectively) in MCAO subjected rats. MMP-12 expression was localized to neurons, oligodendrocytes and microglia, but not astrocytes. Transcriptional inactivation of MMP-12 significantly reduced the infarct size. The percent infarct size was reduced from 62.87±4.13 to 34.67±5.39 after MMP-12 knockdown compared to untreated MCAO subjected rats. Expression of myelin basic protein was increased, and activity of MMP-9 was reduced in ischemic rat brains after MMP-12 knockdown. Furthermore, a significant reduction in the extent of apoptosis was noticed after MMP-12 knockdown. TNFα expression in the ipsilateral regions of MCAO-subjected rats was reduced after MMP-12 knockdown in addition to the reduced protein expression of apoptotic molecules that are downstream to TNFα signaling. Specific knockdown of MMP-12 after focal cerebral ischemia offers neuroprotection that could be mediated via reduced MMP-9 activation and myelin degradation as well as inhibition of apoptosis.