Protease-Activated Receptor 1 and 4 Signal Inhibition Reduces Preterm Neonatal Hemorrhagic Brain Injury
Background and Purpose—This study examines the role of thrombin’s protease-activated receptor (PAR)-1, PAR-4 in mediating cyclooxygenase-2 and mammalian target of rapamycin after germinal matrix hemorrhage.
Methods—Germinal matrix hemorrhage was induced by intraparenchymal infusion of bacterial collagenase into the right ganglionic eminence of P7 rat pups. Animals were treated with PAR-1, PAR-4, cyclooxygenase-2, or mammalian target of rapamycin inhibitors by 1 hour, and ≤5 days.
Results—We found increased thrombin activity 6 to 24 hours after germinal matrix hemorrhage, and PAR-1, PAR-4, inhibition normalized cyclooxygenase-2, and mammalian target of rapamycin by 72 hours. Early treatment with NS398 or rapamycin substantially improved long-term outcomes in juvenile animals.
Conclusions—Suppressing early PAR signal transduction, and postnatal NS398 or rapamycin treatment, may help reduce germinal matrix hemorrhage severity in susceptible preterm infants.
Germinal matrix hemorrhage (GMH) is the leading cause of mortality and morbidity from prematurity because this brain region is selectively vulnerable to spontaneous bleeding within the first 72 hours of preterm life.1 Cerebroventricular expansion contributes to long-term injury through mechanical compression of surrounding brain tissues.2 Neurological outcomes include hydrocephalus, mental retardation, and cerebral palsy.1,3 Current neonatal intensive care treatments are ineffective at preventing GMH, and neurosurgical shunts are prone to devastating complications.4
Importantly, the blood constituent thrombin has been identified as a causative factor in hydrocephalus formation.5 Thrombin activates a subfamily of G-protein-coupled receptors, named proteinase-activated receptors (PARs; specifically PAR-1 and PAR-4),6 leading to phosphorylation and activation of mammalian target of rapamycin (mTOR)7 and increased cyclooxygenase (COX)-2 expression.8
Therefore, we hypothesized that modulation of the thrombin-(PAR)-1,-4–(COX)-2/mTOR pathway could be a promising strategy to improve outcomes after GMH.
Materials and Methods
All studies, protocols, and procedures were approved by the Loma Linda University IACUC. One hundred fifty-seven P7 rat pups (comparable with human 30–32 gestational weeks9; 14–19 g; Harlan Laboratories, Indianapolis, IN) were randomly subjected to either GMH or sham operations. GMH was induced by a stereotactically guided infusion (using bacterial collagenase, denatured collagenase, blood, or thrombin) to mimic preterm right-sided ganglionic eminence bleeds.9 Details are in the online-only Data Supplement.
Tissue Processing and Analysis
Rats were euthanized at 72 hours (for Western blot), at various time points between 6 hours and 21 days (for thrombin assay), and after 28 days (for neuropathological analysis) post GMH, and analyzed by blinded experts.9–11 Details are in the online-only Data Supplement.
Animal Treatments and Experimental Groups
For the 72-hour (short term; n=49) Western blot study, GMH animals received intraperitoneal coinjections of PAR-1 (SCH79797) and PAR-4 (P4pal-10) antagonists (1, 3, 7, 10, or 15 mg/kg given 1, 24, and 48 hours post GMH). For thrombin assay time-course study (n=49, n=7 per time point), GMH animals were euthanized at 0 hour, 6 hours, 1 day, 5 days, 7 days, 10 days, and 21 days after GMH. For the 28-day (long term; n=26) study, COX-2 (NS398) or mTOR (rapamycin) treatment consisted of 6 intraperitoneal injections (1, 6, 24, 36, 48, and 60 hours after GMH). Inhibitors were prepared using distilled water containing 5% dimethyl sulfoxide (DMSO) solvent. Controls received the vehicle (5% DMSO). All drugs were purchased from Sigma-Aldrich (St. Louis, MO).10,11
Assessment of Neurological Deficits
Cognitive (T-maze, Water-maze) and sensorimotor (Rotarod, Foot Fault) testing from 21 to 28 days post GMH was performed by experienced blinded investigators as described.9–11 Details are in the online-only Data Supplement.
P values of <0.05 were considered statistically significant. Neurobehavioral data were analyzed using 1-way ANOVA on ranks with Student–Newman–Keuls post hoc test. All other data were analyzed by 1-way ANOVA with Tukey post hoc test. Data are expressed as mean±SEM.
Hemorrhage progression and hydrocephalus formation after GMH are shown in Figure I in the online-only Data Supplement, and changes in body weight after GMH are shown in Figure II in the online-only Data Supplement.
Thrombin activity increased at 6 and 24 hours after collagenase infusion compared with sham (P<0.05), and then normalized over 5 to 21 days (Figure 1B).
Molecular Mediators of Posthemorrhagic Hydrocephalus
Posthemorrhagic hydrocephalus at 28 days was greatest in the group receiving direct intraparenchymal infusion of collagenase into the ganglionic eminence (P<0.05; Figure 1C and 1D) compared with intracerebroventricular injections of collagenase, heat-deactivated collagenase, donor blood, or thrombin.
This study investigated the effectiveness of modulating thrombin-PAR-1 and PAR-4 in reversing COX-2 and phospho-mTOR (p-mTOR) upregulation, as well as the effect of direct COX-2 and p-mTOR inhibition on posthemorrhagic hydrocephalus, and neurological deficits. Previous studies hypothesized the mechanism of hydrocephalus involved increased production of infiltrating extracellular matrix proteins throughout the cerebroventricular system, leading to the disruption of cerebrospinal fluid outflow.1,2,9,11–13 Our results suggest that thrombin-induced PAR-1, PAR-4 stimulation upregulates detrimental signaling: exacerbating inflammatory (ie, COX-2 mediated) and proliferative responses (ie, p-mTOR mediated) that are potentially upstream of extracellular matrix protein dysregulation.1,5,7–9,11,14 Multiple parallels (especially thrombin)1,2,7–11 exist between our study and the pathophysiology of adult intracerebral hemorrhage.4,5,12–14 Thus, in extension, our findings may have a much broader therapeutic implication in terms of further adult stroke mechanistic study (these fall outside the scope of this article15).
To address the question of molecular mediators of GMH, our first aim demonstrated that intraparenchymal infusion of collagenase generated the majority of hydrocephalus. This is likely the sum contribution of blood products14 (eg, red blood cell lysis and inflammation) and thrombin. In fact, thrombin demonstrated greatest activity in the acute phase, between 6 and 24 hours post ictus, with tendency to remain elevated ≤10 days and normalized by 21 days.
We next hypothesized that thrombin binds to PAR-1, PAR-4 and consequently upregulates COX-2 and p-mTOR. Because thrombin is most active in the acute phase, we examined levels at 72 hours post ictus, and determined COX-2 and p-mTOR were significantly greater in vehicle-treated animals than in sham. Furthermore, inhibiting PAR-1, PAR-4 using SCH79797 (PAR-1 antagonist) and p4pal10 (PAR-4 antagonist) significantly normalized COX-2 and p-mTOR levels at 72 hours.
Then, we asked whether directly inhibiting COX-2 or p-mTOR after GMH could circumvent long-term posthemorrhagic ventricular dilation, cortical cell loss, and improve sensorimotor and neurocognitive outcomes. Our findings demonstrated that vehicle-treated animals had significantly worsened outcomes compared with shams, and treating with either NS398 (COX-2 inhibitor) or rapamycin (mTOR inhibitor) significantly improved brain neuropathology and neurological ability. Thus, by attenuating early inflammatory (ie, COX-2) and proliferative (ie, p-mTOR) signaling pathways, we improved long-term outcome in the juvenile animals.
In summary, this study is the first to show thrombin-PAR-1, thrombin-PAR-4 signal inhibition normalizing early COX-2 and p-mTOR expression levels, and this in turn, improving long-term neurological outcomes after GMH.
Sources of Funding
This research was supported by the National Institutes of Health grant RO1 NS078755 to Dr Zhang.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.007889/-/DC1.
- Received October 25, 2014.
- Revision received February 27, 2015.
- Accepted March 23, 2015.
- © 2015 American Heart Association, Inc.
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