Non-Invasive Multimodality Imaging Directly Shows TRPM4 Inhibition Ameliorates Stroke Reperfusion Injury
The transient receptor potential melastatin 4 (TRPM4) channel has been suggested to play a key role in the treatment of ischemic stroke. However, in vivo evaluation of TRPM4 channel, in particular by direct channel suppression, is lacking. In this study, we used multimodal imaging to assess edema formation and quantify the amount of metabolically functional brain salvaged after a rat model of stroke reperfusion. TRPM4 upregulation in endothelium emerges as early as 2 h post-stroke induction. Expression of TRPM4 channel was suppressed directly in vivo by treatment with siRNA; scrambled siRNA was used as a control. T2-weighted MRI suggests that TRPM4 inhibition successfully reduces edema by 30% and concomitantly salvages functionally active brain, measured by 18F-FDG-PET. These in vivo imaging results correlate well with post-mortem 2,3,5-triphenyltetrazolium chloride (TTC) staining which exhibits a 34.9% reduction in infarct volume after siRNA treatment. Furthermore, in a permanent stroke model, large areas of brain tissue displayed both edema and significant reductions in metabolic activity which was not shown in transient models with or without TRPM4 inhibition, indicating that tissue salvaged by TRPM4 inhibition during stroke reperfusion may survive. Evans Blue extravasation and hemoglobin quantification in the ipsilateral hemisphere were greatly reduced, suggesting that TRPM4 inhibition can improve BBB integrity after ischemic stroke reperfusion. Our results support the use of TRPM4 blocker for early stroke reperfusion.
KeywordsStroke Reperfusion MRI PET Endothelium
Acute ischemic stroke is a major cause of morbidity and mortality worldwide. Immediate restoration of blood flow can significantly improve the chances of recovery. Currently, both fibrinolytic drug tissue plasminogen activator (tPA) and mechanical thrombectomy are used to remove the blood clot and restore cerebral circulation to the affected area . The time window for successful utilization of tPA is very short after stroke onset (3 h and up to 4.5 h in certain eligible patients). Application of tPA beyond this time window leads to severe side effects, defined as ischemic reperfusion injury. Delayed reperfusion induces cerebral edema and hemorrhagic transformation, in part via disruption of the blood-brain barrier (BBB). Thus, strategies to ameliorate BBB damage are paramount. In the current study, we investigate whether inhibition of the transient receptor potential melastatin 4 (TRPM4) channel may attenuate reperfusion injury by protecting vascular endothelial cells.
TRPM4 is a voltage-dependent, non-selective monovalent cation channel which is activated by elevated cytosolic Ca2+ and modulated by ATP . TRPM4 upregulation in vascular endothelium has previously been reported in animal models of spinal cord injury  and stroke , as well as in human stroke post-mortem brains . The pathological role of TRPM4 has been well defined . As a non-selective monovalent cation channel , TRPM4 activation leads to cell depolarization and importantly excessive Na+ influx causing oncotic cell death . In our previous study using a permanent stroke model, TRPM4 blockade has been shown to temporarily improve motor functions . However, much less is known regarding the suppression of TRPM4 expression in live animals in stroke, in particular, after ischemia reperfusion. In this study, we employed multimodality imaging techniques in a single examination to allow acquisition of co-registered complementary data to evaluate BBB integrity and subsequent reperfusion injury with a focus on stroke reperfusion in live animals.
Materials and Methods
Animal Model and Treatment
Animal Exclusion Criteria and Proper Animal Practice
Based on preliminary study, we expect to detect a 30% change in infarct reduction with a standard deviation of 5%. The animal number was calculated on the prediction of a significance of 0.05 and a power of 0.8. During the operation, nine animals died (three from siRNA-treated group, four from scrambled siRNA-treated group, and two from permanent group). For the animals that survived the operation, 8 are excluded from the study. The exclusion criteria are as follows: (1) six rats (three from siRNA group and three from scrambled siRNA group) showed no infarct formation identified by TTC staining at day 1; (2) two rats (one from siRNA group and one from scrambled siRNA group) exhibited an enhanced motor function (> 100% as compared with baseline) at day 1 post-surgery.
For better laboratory practice, treatment groups (siRNA, scrambled siRNA, and sham operation) were determined by rolling a dice and participants in the experiments were blinded to treatment. Animal operation was performed by BC and GN. Behavioral studies were carried out by SWL and YG. TTC staining and western blot was performed by SWL. Immunostaining experiments were done by GN and YG and analyzed by ES and CT. Imaging study was performed by JG, BR, and SS and analyzed by JG.
TTC Staining, Evans Blue Extravasation, and Hemoglobin Quantification
2, 3, 5-triphenyltetrazolium chloride (TTC) staining was performed at day 1 following operation to evaluate infarct formation. After the animals were euthanized, the brains were collected, and the cerebellum and overlying membranes were removed. The brains were sectioned into 2-mm-thick coronal slices using a brain-sectioning block. The sections were stained with 0.1% TTC (Sigma, USA) solution at 37 °C for 30 min and then preserved in 4% formalin solution. The sections were scanned and the infarct size was analyzed using an image analyzer system (Scion image, Microsoft). Calculation of edema-corrected lesion was performed as described previously .
Blood-brain barrier permeability was assessed by measuring Evans blue extravasations 1 day after reperfusion. Evans blue (E2129; Sigma-Aldrich) of 2% concentration was injected into the jugular vein at a dose of 4 mL/kg of body weight. Six hours later, the rats were transcardially perfused with phosphate-buffered saline (PBS). Ipsilateral and contralateral hemispheres were dissected, weighted, and homogenized in 1:3 weight (mg)/volume (μl) ratios of 50% trichloroacetic acid (TCA) (T9159; Sigma-Aldrich) in saline. After centrifugation at 12,000×g for 20 min, supernatant was collected and thoroughly mixed with 95% ethanol (1:3) by pipetting for fluorescence spectroscopy (620 /680 nm) using the Tecan infinite plate reader. The results were quantified according to a standard curve and presented as microgram of Evans Blue per gram of tissue.
The hemoglobin volume was measured by a spectrophotometric assay. Briefly, both contralateral and ipsilateral hemispheres were dissected out after transcardial perfusion. The brain tissues were homogenized in 3 ml PBS and centrifuged at 15,000×g for 30 min. The supernatants were collected and incubated with Drabkin reagent (D5941; Sigma-Aldrich) for 15 min at room temperature. The optical density was quantified at 540 nm with a spectrophotometer. Hemoglobin volumes (μl) of ipsilateral and contralateral hemispheres were calculated from a standard curve obtained by adding incremental volumes of whole blood (0, 2.8, 5.6, 11.2, 22.4, 44.8 μl) to a control brain hemisphere.
Immunofluorescent Staining and Western Blot
Motor function after MCAO was evaluated using a rotarod apparatus for rat (Ugo Basile, Italy). The performance of the rats was measured by observing the latency with which the rats fell off the rotarod. Before operation, the rats received three training trials with 15-min intervals for 5 days. The accelerating rotarod was set from 4 to 80 rpm within 10 min. The mean duration of time that the animals remained on the device was recorded 1 day before MCAO as an internal baseline control. At different time points following surgery, the mean duration of latency was recorded and compared to the internal baseline control.
HBMECs and Scratch Assay
Human brain microvascular endothelial cells (HBMECs) was purchased from Lonza, UK. A detailed description on cell culture and treatment can be found in the supplementary material.
One day after transient MCAO, cerebral injuries in live rats were evaluated by magnetic resonance imaging (MRI) and positron emission tomography (PET) scans (nsiRNA = 8, nscram = 8, and npMCAO = 5). MRI imaging was performed with a 9.4 T Biospec horizontal bore magnet equipped with actively shielded magnetic field gradient coils and a linear volume coil (72 mm bore diameter; Bruker, Ettlingen, Germany). PET was performed on an Inveon PET/CT system (Siemens Inc., Washington DC). The detailed procedure can be found in the supplementary material.
One day after stroke reperfusion, the brains were harvested and the tissues surrounding the infarct (~ 2 mm) were collected for RNA extraction. Raw cel files were processed using standard procedures as recommended in affy packages . The detailed procedure can be found in the supplementary material.
Quantitative Real-Time PCR
Expression of claudin-1 and claudin-2 was quantified using real-time PCR as a validation of microarray results. Brain tissues surrounding the infarct area (~ 2 mm) were dissected from TRPM4 siRNA and scrambled siRNA-treated rats (MCAO 2 h, reperfusion 1 day). The detailed procedure can be found in the supplementary material.
All of the results are presented as the mean ± S.E.M. Student’s t test was used to compare two means and one-way ANOVA followed by Bonferroni’s post hoc analysis used to compare the means of data from three groups. Two-way ANOVA followed by Bonferroni post hoc test was used for Evans Blue extravasation. Repeated ANOVA with Bonferroni’s post hoc analysis was applied for behavioral studies. The results were considered significant if p < 0.05.
TRPM4 Blockade in an In Vitro Model of Ischemia Reperfusion
TRPM4 Expression in the Hyperacute Phase of Stroke
TRPM4 Inhibition Reduced Infarction and Improved Motor Function in Transient MCAO
TRPM4 Blockade Improves Cerebral Vascular Morphology
TRPM4 Inhibition Protected Blood-Brain Barrier Following Stroke Reperfusion and Reduced Cerebral Edema
Together with T2-weighted imaging, positron emission tomography (PET) imaging with [18F]FDG was performed to measure metabolically active brain tissue following ischemia reperfusion (Fig. 6a lower panel). Total [18F]FDG brain tissue uptake volume was increased from 1554 ± 40.6 mm3 in scrambled siRNA-treated animals to 1681 ± 37.2 mm3 in animals with TRPM4 siRNA treatment. Brain areas with severely reduced [18F]FDG were considered ischemic/infarcted. The infarct volume in TRPM4 siRNA-treated animals (220 ± 46.6 mm3) was significantly lower than in scrambled siRNA-treated rats (338.8 ± 12.3 mm3) (Fig. 6c), representing a 34.9% reduction. This result again correlates well with the 34% reduction as determined by TTC staining (Fig. 4).
To further characterize BBB disruption, we assessed Evans blue extravasation 24 h after reperfusion. In contralateral hemispheres, very small amount of Evans blue was detected. However, in ipsilateral hemisphere, Evans blue extravasation, corrected for the baseline reading, was significantly attenuated by TRPM4 siRNA treatment (Fig. 6f, g). As hemorrhagic transformation is an important feature of BBB disruption following reperfusion, we quantified hemoglobin volume in the ipsilateral hemisphere. Again, TRPM4 siRNA treatment significantly reduced hemorrhage at 1 day following stroke reperfusion (Fig. 6h).
TRPM4 Inhibition Enhanced Claudin-1 and Claudin-2 Expression
TRPM4 Blockade Accelerated Vascular Recovery
Reperfusion therapy was first used in managing ischemic stroke patient within 3 h of stroke onset . After years of examination of its efficacy and safety, the time window has been extended to 4.5 h in certain eligible patients . Even though the global outcome was improved with thrombolysis, the incidence of intracranial hemorrhage caused by vascular injury was still higher in patients with thrombolysis than with placebo [11, 12]. Therefore, the need for a novel way of vascular protection, even in patients with early stroke onset, remains urgent. In the present study, we identified that TRPM4 channel was upregulated as early as 2 h post-stroke induction. The differential expression of TRPM4 in endothelium and neurons at early stage of stroke suggests that the effect of hypoxia is heterogeneous among various cells and locations.
Using a 2-h MCAO reperfusion model, we evaluate the effect of TRPM4 suppression on edema formation and tissue infarction in live animals for the first time using multimodality imaging technique. A reduction in edema formation was identified in rats receiving TRPM4 siRNA, accompanied by an increase of metabolically active tissue compared to scrambled siRNA-treated animals. Attenuated edema is indicative of improved BBB integrity during ischemia reperfusion. The reduction in tissue damage, measured by TTC staining, T2-weighted MRI, and [18F]FDG-PET imaging was very consistent (around 30%), strongly suggesting that TRPM4 inhibition in this transient stroke model could salvage significant amounts of brain tissue. The protection of BBB integrity by TRPM4 inhibition was further supported by the results showing that none of the TRPM4 siRNA-treated animals exhibited areas of hyperintensity within the infarct core, a sign of severe BBB leakage which occurred in 50% of control animals. Imaging of the tissues surrounding the infarct core revealed interesting differences between the permanent and transient models. In permanent stroke, [18F]FDG was taken up by substantial amounts of edematous tissue surrounding the infarct core. Surprisingly, in the transient stroke model, only small areas of tissue surrounding the infarct core showed the signs of both edema and [18F]FDG uptake. These results indicate that the brain tissues salvaged by TRPM4 inhibition after stroke reperfusion are well protected, likely due to the blood resupply.
In addition to imaging live animals, multiple lines of evidence from both in vitro and in vivo experiments support the use of TRPM4 blockers in vascular protection. In cultured HBMECs, TRPM4 inhibition enhanced endothelial survival and migration which is in line with previous study showing that TRPM4 blockade prevented lipopolysaccharide-induced endothelial cell death . Furthermore, a reduction of Evans blue extravasation and hemoglobin quantification was observed within the ipsilateral hemispheres, suggesting that TRPM4 inhibition could alleviate reperfusion-associated BBB disruption. Morphological examination of the vasculature revealed more exciting results. In our previous study using a permanent stroke model, although the vessels with TRPM4 siRNA treatment were longer and smooth, the diameter of the capillaries was similar to that observed in control animals  with no visible lumen, indicating a lack of functional blood flow in these vessels, whereas in the current transient stroke model, the capillary diameter in TRPM4 siRNA-treated animals was much larger and displayed clear lumen in most blood vessels. Indeed, the diameter of capillaries after TRPM4 siRNA treatment (8.3 ± 1 μm) is more representative of normal perfused capillaries (~ 4–8 μm) , suggesting that these blood vessels could conduct functional blood flow. Interestingly, TRPM4 inhibition could also accelerate vascular recovery. vWF, an indicator of hypoxia-induced endothelial stimulation , was found downregulated by TRPM4 siRNA treatment at 14 days post-operation. Furthermore, as TRPM4 is found upregulated in neurons and astrocytes , TRPM4 inhibition could contribute to the survival of these cells. We identified an upregulation of TRPM4 in cortical neurons close to the infarct, but not in the contralateral hemisphere. Interestingly, the TRPM4 expression pattern was heterogeneous. About 80% of the neurons express TRPM4, suggesting that hypoxic impact varies among neurons close to the infarct core. Therefore, blocking TRPM4 channel may help improve the survival of these neurons.
This study identified for the first time a novel mechanism of TRPM4 suppression in improving BBB integrity. We observed that TRPM4 inhibition could enhance the expression of TJ components claudin-1 and claudin-2. In control animals, the expression of claudin-1 and claudin-2 is extremely low which is consistent with previous studies showing that claudin-3, claudin-5, and claudin-12, rather than claudin-1 and claudin-2, are major components of TJ in BBB . In brain, claudin-1 and claudin-2 are primarily expressed in epithelial cells, including choroid plexus. Claudin-2 has not been found in endothelial TJ and claudin-1 expression in the TJ is controversial . It is unlikely that the induction of claudin-1 and claudin-2 is due to hypoxia as their expression was low in scrambled siRNA-treated animals, and previous studies failed to show a link between hypoxia and the induction of claudin-1 expression in HBMEC . Therefore, our data indicate that the expression of claudin-1 and claudin-2 may be a result from TRPM4 suppression after stroke. De novo expression of claudin-1 and claudin-2 in vascular endothelium is beneficial to BBB integrity. It has been reported that in contrast to occludin, claudin-1 or claudin-2 were able to reconstitute de novo TJ strands in TJ-free mouse fibroblasts . Furthermore, ectopic expression of claudin-1 has been shown to seal BBB TJs in experimental autoimmune encephalomyelitis . Thus, in ischemia reperfusion, induction of claudin-1 and claudin-2 may strengthen BBB integrity by forming additional TJs, which could be a novel therapeutic mechanism of TRPM4 inhibition. Further experiments are needed to verify the exact underlying mechanisms.
To suppress TRPM4 expression in the hyperacute stage of stroke, TRPM4 siRNA was delivered at the start of the operation in this study. Ideally, TRPM4 blockers should be administered before recanalization. TRPM4 blocker 9-phenanthrol is a metabolized product of highly toxic polycyclic aromatic hydrocarbon (PAH) phenanthrene and thus is unlikely to be used in clinical practice as it has the potential to be concentrated in tissues . Currently, sulfonylureas such as glibenclamide have been shown to be promising TRPM4 blockers. TRPM4 has been reported to form a channel complex with sulfonylurea receptor-1 (SUR1), an auxiliary subunit of KATP channel , and SUR1 blocker sulfonylureas have been used to treat brain diseases such as stroke . However, there is controversy regarding the regulatory role of SUR1 on TRPM4 and the effect of sulfonylureas in stroke treatment [21, 22, 23, 24]. A recent review has summarized the preclinical findings on glibenclamide for the treatment of stroke in various stroke models . In clinical practice, SUR1 blocker sulfonylureas are widely used to manage diabetes mellitus. Multiple studies on stroke patients with or without diabetes mellitus revealed that use of sulfonylureas before or after stroke onset could reduce hemorrhage transformation, attenuate cerebral edema, and improve neurological outcome [20, 22]. In contrast, some studies on diabetic patients with stroke showed that application of sulfonylureas achieved no better outcome than other anti-diabetic treatments [26, 27]. Such controversies may arise from differences in patient inclusion criteria, dose of sulfonylureas, or the severity of diabetes mellitus.
Interestingly, a recent study showed that application of sulfonylurea glimepiride achieved neuroprotection against stroke only in normal mice but not in type 2 diabetic mice , suggesting that the presence of diabetes mellitus could be a confounding factor for the use of sulfonylureas to manage stroke. To avoid the effect of sulfonylureas on diabetes mellitus, antagonists that act directly on TRPM4 channel is another good option.
Ischemia reperfusion injury is a major confounding factor for reperfusion therapy in stroke. Delayed reperfusion induces cerebral edema and hemorrhagic transformation, in part via disruption of the BBB. This study provides robust evidence in support of using TRPM4 blockers to ameliorate reperfusion injury. Mitigating edema formation will surely improve stroke outcome in patients receiving reperfusion therapy. Next critical experiment is to examine whether TRPM4 inhibition could extend current reperfusion time window, which is limited by the severe reperfusion injury during delayed recanalization. As TRPM4 inhibition can protect vasculature and improve BBB integrity following stroke reperfusion, TRPM4 blockers may potentially extend current time window for reperfusion therapy when applied in together with tPA or other recanalization treatments, thereby offering great therapeutic management for stroke patients who arrive at hospitals late.
The current study indicates that TRPM4 inhibition improves BBB integrity and ameliorates reperfusion injury. TRPM4 is an ideal target for stroke treatment as it is not expressed in brain tissue; thus, blockade of TRPM4 is unlikely to affect normal brain functions. Furthermore, possibilities of severe side effects on other tissues or organs are also low. Therefore, injection of TRPM4 blockers before recanalization may constitute a new strategy to mitigate reperfusion injury.
We thank Eong Rui Xuan and Jamie Quek for the technical support.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no competing interest.
All procedures in studies involving animals were approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the National Neuroscience Institute, Singapore.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.