Transient Receptor Potential Canonical 3 Inhibitor Pyr3 Improves Outcomes and Attenuates Astrogliosis After Intracerebral Hemorrhage in Mice
Background and Purpose—Intracerebral hemorrhage (ICH) stems from the rupture of blood vessels in the brain, with the subsequent accumulation of blood in the parenchyma. Increasing evidence suggests that blood-derived factors induce excessive inflammatory responses that are involved in the progression of ICH-induced brain injury. Thrombin, a major blood-derived factor, leaks into the brain parenchyma on blood–brain barrier disruption and induces brain injury and astrogliosis. Furthermore, thrombin dynamically upregulates transient receptor potential canonical 3 channel, which contributes to pathological astrogliosis through a feed-forward upregulation of its own expression. The present study investigated whether Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), a specific transient receptor potential canonical 3 inhibitor, can improve functional outcomes and attenuate astrogliosis after ICH in mice.
Methods—Male C57BL6 mice received an intracerebral infusion of collagenase or autologous blood to induce ICH. Pyr3 was given both intracerebroventricularly and intraperitoneally after ICH induction. ICH-induced brain injury was evaluated by quantitative assessment of neurological deficits, brain swelling, and injury volume after ICH. Astrocyte activation was evaluated by immunohistochemical assessment of changes in S100 protein expression.
Results—Neurological deficits, neuronal injury, brain edema, and astrocyte activation were all significantly improved by administration of Pyr3. Moreover, delayed administration of Pyr3 at 6 hours or 1 day after blood or collagenase infusion, respectively, also improved the symptoms.
Conclusions—Pyr3, a specific inhibitor of transient receptor potential canonical 3, reduced the perihematomal accumulation of astrocytes and ameliorated ICH–induced brain injury. Therefore, transient receptor potential canonical 3 provides a new therapeutic target for the treatment of hemorrhagic brain injury.
Intracerebral hemorrhage (ICH) is a subtype of stroke with high morbidity and mortality.1 Although several therapeutic strategies for management of ICH are currently in clinical practice,1 virtually none are aimed at neuroprotection. Basic research has demonstrated that various drugs that possess antioxidative, anti-inflammatory, or neurotrophic/neuroprotective properties produce therapeutic effects on ICH animal models2,3; however, effective drug therapies for ICH are yet unavailable.
Modulation of inflammatory processes mediated by astrocytes, neutrophils, etc, may provide an opportunity for restricting the expansion of ICH-induced tissue damage.3,4 Astrocytes accumulate in the perihematomal region5 and induce toxic edema, provoke inflammation, release cytotoxins, and form scars after ICH.6 Moreover, neutrophils, macrophages, and microglia are major central nervous system sources of cytokines, chemokines, and other immunomolecules4 and are thought to provoke secondary brain damage after ICH.
Transient receptor potential (TRP) channels are formed by homomeric or heteromeric tetramers of 6 subtypes of TRP proteins that constitute 6 subfamilies: the TRP-ankyrin transmembrane protein 1, TRP-canonical (TRPC), TRP-melastatin, TRP-mucolipin, TRP-polycystin, and TRP-vanilloid subfamilies.7 There are 7 TRPC channel isoforms that function as homotetrameric or heterotetrameric cation channels. At least 1 isoform is expressed in almost every tissue, where it facilitates voltage-independent Ca2+ entry in response to calcium store depletion induced by receptor stimulation.8 TRPC3 channels are directly activated by diacylglycerol9 and underlie the store-operated channels observed in many cell types. We previously showed that thrombin, a predominant blood-derived factor, induced functional activation of astrocytes via opening of the TRPC3 channel in human astrocytoma cell lines10 and rat-cultured astrocytes.11 Although systemic administration of argatroban, a direct thrombin inhibitor, significantly reduced brain edema after ICH,12 the effect of TRPC3 inhibition, per se, has not yet been clarified in an in vivo model of ICH.
In the present study, we investigated whether Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), a selective TRPC3 inhibitor, would attenuate brain injury and inflammatory response in the collagenase/autologous blood infusion mouse models of ICH.13 It is clearly demonstrated that Pyr3 directly binds TRPC3 channel protein, selectively inhibits TRPC3 channel activity, and can improve TRPC3-related diseases, such as cardiac hypertrophy.14 In addition, Pyr3 reduced Ca2+ responses in wild-type acini to the same extent as deletion of Trpc3 and had no effect on the Ca2+ signal in Trpc3−/− acini,15 suggesting that Pyr3 is a useful tool for clarification of TRPC3 functions and for treatments of TRPC3-mediated diseases. Our data suggest that TRPC3 is involved in the development of brain injury after ICH, implicating TRPC3 as a new therapeutic target for the prevention of secondary brain injury and neurological deficits after stroke.
Materials and Methods
Induction of ICH and Pyr3 Treatment
All experiments were performed in accordance with the ethical guidelines of the Kyoto University Animal Research Committee. Male C57BL/6J mice (8–13 weeks of age) weighing 21 to 28 g were used to produce the collagenase3 and autologous blood infusion ICH models.16 Animals were maintained at constant ambient temperature (22°C±1°C) under a 12-hour light/dark cycle. After intraperitoneal injection of 50 mg/kg pentobarbital, mice were placed in a stereotaxic frame. A 30-gauge needle was inserted through a burr hole on the skull into the striatum (stereotaxic coordinates: 2.3 mm lateral to the midline, 0.2 mm anterior to the bregma, and 3.5 mm below the skull). In the collagenase infusion model, ICH was induced by microinfusion pump–mediated injection of 0.025 U collagenase type VII (Sigma) in 0.5 μL PBS at a constant rate of 0.20 μL/min. In the autologous blood infusion model, ICH was induced by injection of 5 μL autologous blood from the tail vein at a constant rate of 2 μL/min. The injection was terminated and 2.5 minutes later, 10 μL autologous blood was injected at a constant rate of 2 μL/min. Body temperature was measured with a rectal probe and maintained at 37°C after surgery.
Pyr3 was suspended in 10% PEG-60 Hydrogenated Castor Oil 60 at a concentration of 4 mmol/L and intracerebroventricularly administered just once at a concentration of 1, 10, or 20 nmol/5 μL at 5 minutes after the induction of ICH. Thereafter, Pyr3 was intraperitoneally administered at a concentration of 2, 20, or 40 mg/kg BID unless otherwise noted. In post-treatment studies, intracerebroventricular administration of Pyr3 was performed 24 or 48 hours after collagenase injection or 6 hours after blood injection, followed by intraperitoneal administration. The mice were randomly assigned to receive vehicle alone or varying doses of Pyr3. All behavioral and histological experiments and their subsequent evaluation were performed by an experimenter blinded to the identity of the treatment groups.
Neurological and sensorimotor functions were evaluated via the neurological deficit scoring (NDS) system, rotarod test, and rope grip test at 1, 3, and 7 days after surgery.
In the NDS system, mice were scored by using a 28-point NDS system.17 The tests included body symmetry, gait, climbing, circling behavior, front limb symmetry, compulsory circling, and whisker response. Each point was graded from 0 to 4. Maximum deficit score was 28.
In the rotarod test, mice were placed on a rotarod cylinder, and the duration for which the mouse remained on the rotarod was recorded. The rotation speed was slowly increased from 4 to 40 revolutions/min within a period of 3 minutes. The trial was ended if the animal fell off the rotarod or gripped the device and spun around for ≥2 consecutive revolutions. Animals were trained in advance before induction of ICH.
In the rope grip test, mice were placed midway on a string between 2 supports and rated as follows: 0, falls off; 1, hangs onto string by one or both forepaws; 2, same as for 1, but attempts to climb onto string; 3, hangs onto string by one or both forepaws plus one or both hindpaws; 4, hangs onto string by forepaws and hindpaws plus tail wrapped around string; and 5, escapes to the supports.18 The final score was the average of 5 trials.
Hemorrhagic Injury and Hemispheric Enlargement Analysis
Twenty-micrometer coronal sections were cut with a cryostat and stored at −80°C before staining with Nissl. Sections were digitized and analyzed with the use of Image J software. The hemorrhagic injury area was calculated by quantifying the Nissl staining–negative area in each section, and the hemorrhagic injury volume was computed by summation of the Nissl staining–negative areas multiplied by the interslice distance (200 μm). Brain edema was measured on the basis of hemispheric enlargement at the bregma, which was calculated according to the following formula: ([ipsilateral hemisphere volume−contralateral hemisphere volume]/contralateral hemisphere volume)×100%.
Three days after ICH, mice were anesthetized with pentobarbital and perfused transcardially with PBS (10 mL), followed by 4% paraformaldehyde (10 mL). Brains were isolated and fixed in 4% paraformaldehyde for 3 hours and then soaked in 15% sucrose overnight at 4°C. After freezing, brains were cut into 20-μm thick sections, and 5 sections around the injection site were collected every 200 μm and mounted onto slides. After rinsing with PBS containing 0.1% Triton X-100 (tPBS), specimens were treated with tPBS-containing blocking serum for 1 hour at room temperature and then incubated with rabbit anti-S100 antibody (1:400; Dako, Tokyo, Japan), rabbit anti-Iba1 antibody (1:500; Wako, Osaka, Japan), and rat anti-Gr1/Ly6G antibody (1:300; R&D Systems, MN) overnight at 4°C. After rinsing with PBS, specimens were incubated with Alexa Fluor 594 donkey anti-rabbit IgG (1:200; Invitrogen; California) or Alexa Fluor 594 goat anti-rat IgG (1:200; Invitrogen) for 1 hour at room temperature. The average number of S100- and Iba1-positive cells at the perihematomal area and Gr1-positive cells in the hematoma per 640×640 μm2 was counted for ≥2 independent sections. Confocal images were obtained by using a Fluoview FV10i system (Olympus; Tokyo, Japan).
Data are presented as the mean±SD. For comparisons among multiple groups, 1-way or 2-way analysis of variance followed by a post hoc Bonferroni test was used to determine significant differences. Differences between 2 groups were assessed with the Student t test. Statistical significance was set at P<0.05.
Effect of Pyr3 on Hematoma Formation and Neurological Deficits in the Collagenase Infusion ICH Model
ICH is accompanied by hematoma formation and characteristic behavioral deficits. To assess whether Pyr3 affects hematoma formation, we first compared the size of hematomas in vehicle- and Pyr3-treated mice at 6 hours after the induction of ICH in the striatum by collagenase injection. No difference was observed in hematoma formation between groups (Figure 1A). To evaluate whether Pyr3 influenced recovery from neurological deficits, various behavioral experiments were performed. As a result, the NDS of vehicle-treated mice was found to increase substantially at 1 to 7 days after collagenase injection. Pyr3 administration substantially improved the NDS at all time points examined (Figure 1B).
In the rotarod test, induction of ICH resulted in substantial performance deficits in vehicle-treated mice, as indicated by a decrease in the latency to fall. Pyr3 treatment significantly prevented the decrease at 3 days after ICH (Figure 1C). A decrease in rope climbing performance was also evident after ICH in the rope grip test, but Pyr3 treatment had a tendency to improve the test score (Figure 1D). Pyr3 administration alone did not cause any neurological adverse effect, including gait abnormality (data not shown), unlike the previous data in TRPC3-deficient mice.19
Effect of Pyr3 on ICH-Induced Neuronal Loss and Brain Edema
The effect of Pyr3 was evaluated in relation to the hemorrhagic injury volume and hemispheric enlargement of the injured brain, which both increased after ICH because of neuronal loss and brain edema, respectively. Pyr3 prevented the increase in hemorrhagic injury volume (Figure 2A–2C) and hemispheric enlargement (Figure 2D) at 3 days after injury.
Intracerebroventricular Versus Intraperitoneal Administration of Pyr3
Next, we evaluated the efficacy of intraperitoneal versus intracerebroventricular administration of Pyr3 for treatment of ICH. Intracerebroventricular administration alone had a tendency to improve motor function in the rotarod or rope grip test but not intraperitoneal alone (Figure IB and IC in the online-only Data Supplement). However, both the NDS and hemorrhagic injury volume were slightly, but significantly, improved at day 3 after ICH (Figure IA and ID in the online-only Data Supplement). However, the best protection was afforded by the combined intraperitoneal/intracerebroventricular route. Therefore, this method of Pyr3 administration was used for the remainder of the study.
Concentration-Dependent Actions of Pyr3
The concentration dependence of Pyr3 was next examined after combined intracerebroventricular/intraperitoneal administration of the drug (1, 10, or 20 nmol/5 µL ICV, and 2, 20, or 40 mg/kg IP). The lowest drug dose (1 nmol/5 µL ICV and 2mg/kg IP) yielded no significant differences between vehicle- and Pyr3-treated mice (Figure 3). However, the hemorrhagic injury volume, NDS, running performance in the rotarod test, and rope grip score were all improved in a concentration-dependent manner.
Delayed Administration of Pyr3
To assess the therapeutic time window for Pyr3 administration, Pyr3 was administered to mice 1 or 2 days after ICH induction. Drug administration 2 days after ICH did not protect mice against ICH-induced injury; however, drug administration 1 day after ICH significantly decreased hemorrhagic injury volume and the NDS and had a tendency to improve motor function in the rotarod and rope grip test (Figure 4).
Reduction in Perihematomal Accumulation of Astrocytes in Pyr3-Treated Mice
We have previously shown that TRPC3 contributes to pathological activation of astrocytes. To investigate the influence of TRPC3 inhibition on astrocyte behavior in vivo, we examined the perihematomal accumulation of activated astrocytes 1, 3, and 7 days after ICH via immunohistochemical staining for S100 protein. The number of S100-positive astrocytes in the perihematomal area was increased 1 day after ICH and remained elevated throughout the course of the experiment, whereas Pyr3 partially prevented the increase (Figure 5A and 5B). Moreover, we assessed the accumulation of microglia/macrophages and neutrophils using anti-Iba1 and anti-Gr1 antibodies, respectively, 1, 3, and 7 days after ICH. The number of Iba1-positive cells in the perihematomal area was increased after ICH and peaked at 3 to 7 days after ICH, which was significantly inhibited in that of Pyr3-treated mice (Figure II in the online-only Data Supplement). The number of Gr1-positive cells in the hematoma was increased after ICH and peaked 1 day after ICH, whereas they did not appear at 7 days. There was no difference in the number of Gr1-positive cells between the vehicle- and Pyr3-treated groups (Figure III in the online-only Data Supplement).
Effect of Pyr3 in the Autologous Blood Infusion ICH Model
We next investigated the actions of Pyr3 in the autologous blood infusion ICH model. Hematoma formation was comparable between vehicle- and Pyr3-treated mice at 6 hours after ICH (Figure 6A). The NDS increased in vehicle-treated mice after autologous blood infusion, but Pyr3 treatment significantly prevented the increase (Figure 6B). In the rotarod and rope grip tests, induction of ICH in vehicle-treated mice resulted in a substantial decline in performance (Figure 6B). This decline was also prevented by Pyr3 (Figure 6B).
Furthermore, the therapeutic time window for Pyr3 administration after autologous blood infusion was assessed by administration of the drug 6 hours after ICH. Even such delayed administration of Pyr3 significantly decreased the NDS at 24 and 72 hours after ICH and significantly increased the rope grip test score at 24 hours (Figure 6C).
Moreover, the distribution of activated astrocytes in the perihematomal area was examined via immunohistochemistry 1, 3, and 7 days after ICH. The number of S100-positive cells increased at day 1 and remained elevated for 7 days in vehicle-treated mice, whereas Pyr3 administration significantly prevented the increase on days 1 and 3 (Figure IV in the online-only Data Supplement).
This study demonstrates for the first time that Pyr3, a selective TRPC3 inhibitor, ameliorated reactive astrogliosis and neurological deficits resulting from collagenase and autologous blood infusion–induced ICH, whereas the drug did not affect hematoma formation. In addition, delayed administration of Pyr3 after the onset of brain injury was effective in both ICH models. These results suggest that Pyr3 can improve neuropathological outcomes after ICH in mice.
Emerging evidence suggests that activated astrocytes contribute to brain tissue damage after ICH. For example, matrix metalloproteinase 920 and aquaporin 4 and 921 are upregulated in activated astrocytes and stimulate the formation of edema after ICH. Moreover, activated astrocytes release S100B into the serum, which is significantly correlated with brain edema formation and hematoma volume in ICH22 and hemorrhagic transformation in ischemia.23 Furthermore, ICH-associated astrocyte reactivity is increased in aged compared with young rats,5 whereas arundic acid, a specific inhibitor of S100B synthesis, mitigates delayed infarct expansion and neurological deficits.24 Thus, excessive activation of astrocytes is likely to influence clinical outcomes after stroke.
Our previous investigations demonstrated that thrombin-induced stimulation of proteinase-activated receptor-1 in astrocytes resulted in the opening of the TRPC3 channel as detected by Ca2+ imaging, the influxed Ca2+-dependent activation of specific signaling molecules, de novo TRPC3 protein synthesis, feed-forward amplification of TRPC3 expression and, finally, functional astrogliosis in vitro.11 These results imply that thrombin leaked from ruptured blood vessels can activate astrocytes through TRPC3 channels in vivo. This hypothesis is strongly supported by our present results, demonstrating that the selective TRPC3 inhibitor Pyr3 suppressed reactive astrogliosis and ameliorated functional recovery in mouse ICH model. Hence, suppression of astrocyte activation may ameliorate secondary injury after ICH.
However, it is necessary to consider that Pyr3 treatment inhibits TRPC3 channels expressed in multiple cell types, including neurons, immune cells, and endothelial cells. Accordingly, the inhibitory effect of Pyr3 against ICH might be attributed to a specific blockade of not only astrocyte but also monocyte/macrophage or neutrophil TRPC3 because these cells are mainly involved in the progression of secondary injury after ICH.4
It has been reported that TRPC3 mediates ATP-induced vascular cell adhesion molecule 1 expression and resultant monocyte recruitment to the endothelium,25 and that minocycline, an inhibitor of microglial/macrophage activation, can reduce the blood–brain barrier (BBB) damage and edema after ICH,26 implying that Pyr3-mediated suppression of monocytes and endothelial cell functions via TRPC3 might contribute to the amelioration of ICH-induced brain injury. However, stimulation of phagocytosis in microglia/macrophage leads to the promotion of hematoma resolution, thereby the suppression of neuronal damage after ICH,27 and macrophage TRPC3 contributes to survival signaling and efferocytic properties,28 raising the fear that Pyr3 can exacerbate secondary injury after ICH to disrupt the physiological function of monocyte/macrophage. Furthermore, astrocytic accumulation was comparatively early and peaked at day 1 after ICH, whereas microglial/macrophage accumulation was gradual and peaked at days 3 to 7, indicating that activation of astrocytes seems to precede that of microglial/macrophage in this ICH model. Taking into consideration that activated astrocytes can facilitate distant microglial activation in central nervous system disease,29 the observed suppression of microglia/macrophages might be just a result of Pyr3-mediated suppression of astrocytes.
It has been reported that TRPC3 is also involved in the regulation of nicotinamide adenine dinucleotide phosphate oxidase in the human neutrophil-like cell line,30 and that neutrophil depletion reduces the BBB breakdown, axon injury, and astrocytic and microglia/macrophage responses after ICH,31 raising the possibility that the inhibitory effect of Pyr3 may be because of a blockade of neutrophil TRPC3. Because our present data show that Pyr3 does not affect the infiltration of neutrophils after ICH, the observed effects of Pyr3 seem to be independent of neutrophils.
Meanwhile, TRPC3 is required to promote cerebellar granule neuron survival32 and for metabotropic glutamate receptor–dependent synaptic transmission and motor coordination.19 Therefore, the blockade of TRPC3 in neurons by Pyr3 may negatively impact neurological recovery after ICH. Although Pyr3 alone did not show any neurological adverse effect in this study, development of a more selective, cell type–specific TRPC3 inhibitor may preserve the inhibitory actions against astrocytes, while sparing neurons, further improving outcomes after ICH.
In this study, Pyr3 was administered through a combined intracerebroventricular/intraperitoneal route. Although intraperitoneal administration alone weakly, but significantly, improved ICH-induced injury, the intracerebroventricular route was more effective, and the combined route was the most efficacious of all. These results are reminiscent of experiments with minocycline33 and suggest that Pyr3 does not readily cross the BBB.
In the collagenase infusion ICH model (but not the autologous blood infusion model), the BBB was disrupted, and bleeding continued for ≤24 hours.34 Although hematoma formation was comparable in vehicle- versus Pyr3-treated mice subsequent to collagenase treatment, Pyr3 substantially improved collagenase-induced behavioral deficits and attenuated reactive astrogliosis. These results suggest that Pyr3 can ameliorate secondary brain injury stemming from ICH without affecting the enzyme activity of collagenase or the integrity of the BBB.
In conclusion, the present findings indicate that thrombin released into the brain after ICH activates astrocytes via TRPC3, setting off a chain of events that ultimately induces neuronal death and neurological deficits. Thus, cell type–specific inhibitors of TRPC3 may constitute a novel class of therapeutic drugs for ICH.
Sources of Funding
This work was financially supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by grants from Ono Pharmaceutical Co, Ltd; the Research Foundation for Pharmaceutical Sciences; and the Suzuken Memorial Foundation.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.679332/-/DC1.
- Received October 4, 2012.
- Accepted April 11, 2013.
- © 2013 American Heart Association, Inc.
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