Inhibition of PAR1 attenuates meningeal macrophage activation and vascular inflammation in a rat model of thrombin-induced hydrocephalus

Background and Purpose— Our previous studies demonstrated that intraventricular injection of thrombin could induce hydrocephalus. The inflammation of subarachnoid space plays a key role in hydrocephalus. As thrombin, inducing coagulation, could contribute to inflammation, its effects on subarachnoid space have not been well studied. Macrophagic dysfunction may contribute to this course. However, the mechanisms that how thrombin affects macrophage in subarachnoid space have not been illustrated. Our aim was to explore the possible role that macrophage played in thrombin-induced meningeal inflammation, and to furtherly understand its contribution during thrombin-induced hydrocephalus. Methods— There were two parts in this study. Firstly, rats had an intraventricular injection of saline or thrombin. Secondly, rats received thrombin injection with vehicle or PAR1 antagonist treatment. Immunofluorescence staining was applied to observe the activation of meningeal macrophage and the expression of NeuN in the cortex. Meanwhile, the expression of intercellular adhesion molecule 1 (ICAM1) in meningeal vessels were tested to detect the vascular inflammation. Western blot was applied to measure the secretion of pro-inflammatory cytokines (IL-1β and IFNγ). Results— Our results demonstrated that intraventricular injection of thrombin caused significant activation of meningeal macrophages, vascular inflammation, and neuron loss. Inhibition of PAR1 pathway attenuated the M1 polarization of meningeal macrophage, reduced the inflammatory infiltrations and prevented the neuron loss, as well as hydrocephalus after thrombin injection. Conclusions— Clinically available PAR1 antagonists may offer a novel therapeutic approach candidate for the prevention or the management of inflammation in hemorrhage-induced hydrocephalus. means Student t-tests compare data between two We first observed the accumulation of inflammatory cells in subarachnoid space. Our findings suggested that inflammation is widespread throughout the subarachnoid space after intraventricular injection of thrombin. In this study, Iba1 was used to label meningeal macrophages, CD68 was used as a marker of M1 macrophages and CD206 was used as a marker of M2 phenotype. CD68 or CD206 positive inflammatory cells were counted and the results were expressed as positive cells per millimeter of meninges. In areas of subarachnoid space, there was abundant CD68 positive meningeal macrophages in the thrombin group compared with markedly lower numbers in the saline group (115.4±28.41 versus 37.7±17.19 positive cells/mm in the saline group, p<0.01). It indicated that thrombin induced the M1 polarization of meningeal macrophage in subarachnoid space. While there was a potential increase tendency in the distribution of CD206 positive macrophages between thrombin and saline group (122.4±60.76 versus 63.57±14.72 positive cells/mm in the saline group, p = 0.1088). These results demonstrated that thrombin remarkably promoted M1 polarized meningeal macrophage rather than M2 phenotypic macrophage. These results demonstrated that meningeal macrophages were activated by intraventricular thrombin injection and it also exaggerated neuron loss and peri-vascular inflammation in the subarachnoid space. The clinically available PAR1 antagonists could reverse the meningeal injury and inflammation, thus might offer a new therapeutic approach for management in ventricular hemorrhage-induced hydrocephalus.


Abstract
Background and Purpose-Our previous studies demonstrated that intraventricular injection of thrombin could induce hydrocephalus. The inflammation of subarachnoid space plays a key role in hydrocephalus. As thrombin, inducing coagulation, could contribute to inflammation, its effects on subarachnoid space have not been well studied. Macrophagic dysfunction may contribute to this course. However, the mechanisms that how thrombin affects macrophage in subarachnoid space have not been illustrated. Our aim was to explore the possible role that macrophage played in thrombin-induced meningeal inflammation, and to furtherly understand its contribution during thrombin-induced hydrocephalus.
Methods-There were two parts in this study. Firstly, rats had an intraventricular injection of saline or thrombin. Secondly, rats received thrombin injection with vehicle or PAR1 antagonist treatment. Immunofluorescence staining was applied to observe the activation of meningeal macrophage and the expression of NeuN in the cortex. Meanwhile, the expression of intercellular adhesion molecule 1 (ICAM1) in meningeal vessels were tested to detect the vascular inflammation. Western blot was applied to measure the secretion of pro-inflammatory cytokines (IL-1β and IFNγ).
Results-Our results demonstrated that intraventricular injection of thrombin caused significant activation of meningeal macrophages, vascular inflammation, and neuron loss.
Inhibition of PAR1 pathway attenuated the M1 polarization of meningeal macrophage, reduced the inflammatory infiltrations and prevented the neuron loss, as well as hydrocephalus after thrombin injection.
Conclusions-Clinically available PAR1 antagonists may offer a novel therapeutic approach candidate for the prevention or the management of inflammation in hemorrhageinduced hydrocephalus.

Background
Hydrocephalus is an independent risk factor of hemorrhage-associated morbidity [1]. Our previous study found that thrombin played a critical role in hydrocephalus [2,3].
Cerebrospinal fluid (CSF) is mainly produced in choroid plexus and absorbed in subarachnoid space, and the key point of hydrocephalus should be the changes in formation or absorption of CSF [4,5]. Thrombin is produced immediately after hemorrhage stroke and plays an essential role in brain damage after intracerebral hemorrhage (ICH) [2,3,6,7]. Thrombin could disrupt blood-cerebrospinal fluid (CSF) barrier and generate hydrocephalus via proteinase-activated receptor 1 (PAR1) pathway, which leads to vascular endothelial-cadherin (VE-cadherin) downregulation in choroid plexus [2,3].
Inflammation in the ventricular system is strongly associated with the blood-CSF barrier and hydrocephalus formation [8][9][10][11][12]. Subarachnoid space plays a critical role in cerebrospinal fluid absorption, the dysfunction of which would contribute to hydrocephalus [5,10,13]. Acute inflammatory cell infiltration in subarachnoid space caused the transformation of pial cells into macrophages, promoted fibroblasts and extracellular matrix deposition leading to subarachnoid fibrosis and hydrocephalus [10,13,14]. The meningeal macrophage is one type of non-parenchymal macrophages that mediate immune responses at brain boundaries [15,16]. The relationship between thrombin and meningeal inflammation is still unknown. In this study, the thrombin-induced inflammation in subarachnoid space was investigated.
The high-affinity thrombin receptor, PAR1, has been implicated in mediating the interaction between coagulation and inflammation [17]. The role of inflammation and PAR1 in thrombin-induced hydrocephalus needs to be further elucidated. In this study, we aimed to explore the role of thrombin in brain injury and further illustrate the mechanism of hemorrhage-associated hydrocephalus.

Animals model
Animal protocols were approved by and conducted in accordance with the ethical guidelines of the Zhejiang University Animal Experimentation Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. A total of 32 male Sprague-Dawley rats (3-month old, Zhejiang University Laboratories), at the weight of 280-320 g, were used in this study. Animals were anesthetized with pentobarbital (50 mg/kg intraperitoneally (IP)) and were positioned in a stereotaxic frame. A cranial burr hole (1 mm) was drilled and a 26-gauge needle was inserted stereotaxically into the right lateral ventricle (coordinates: 0.6 mm posterior, 4.5 mm ventral, and 1.6 mm lateral to the bregma). Saline or thrombin (3U in 50μl) was infused using a microinfusion pump (World Precision Instruments). The needle was removed after injection, the burr hole was filled with bone wax, and the skin incision was sutured.

Experimental groups
In brief, the rats were randomly divided into two parts. First, rats received an intracerebroventricular (ICV) injection of 3 units rat thrombin (Sigma-Aldrich, St Louis, MO, USA) in saline or saline alone (50 µl) in 7 minutes and were euthanized for intracardiac perfusion and the brains were used for Western blot (n = 4 for each group), brain histology (n = 4 for each group) at 24 hours. Second, rats were treated with vehicle (1% dimethyl sulfoxide in saline) or PAR1 inhibitor (SCH79797, Abcam, USA, 25μg/kg) immediately while thrombin infusion. Rats were treated with SCH79797 at a dosage accordingly [3,7]. The rats were euthanized at 24 hours after injection and the brains were used for western blot (n = 4 for each group) and brain histology (n = 4 for each group).

Immunohistochemistry and HE staining
Rats were anesthetized with pentobarbital (100 mg/kg, IP) and perfused with 4% paraformaldehyde in 0.1mol/L phosphate-buffered saline (pH 7.4). The brains were removed and kept in 4% paraformaldehyde for 24 hours and then immersed in 30% sucrose for 2 to 3 days at 4℃. Brains were embedded in optimal cutting temperature compound and 18-µm thick slices cut using a cryostat (LEICA, CM3050S).
Immunohistochemistry and HE staining were performed as previously described. The

Statistical analysis
Values are given as means ± SD. Student t-tests were used to compare the data between two groups. Differences were considered significant at p<0.05.

Results
Thrombin promoted the activation of meningeal macrophage We first observed the accumulation of inflammatory cells in subarachnoid space. Our findings suggested that inflammation is widespread throughout the subarachnoid space after intraventricular injection of thrombin. In this study, Iba1 was used to label meningeal macrophages, CD68 was used as a marker of M1 macrophages and CD206 was used as a

Thrombin induced vascular inflammation in subarachnoid space
The adherence and migration of leukocytes through the endothelium of blood vessels in subarachnoid space is an important event which triggers the early inflammatory response [18]. In subarachnoid space, prominent ICAM1 staining of endothelial cells in the thrombin group was observed. A significant rise in the expression of ICAM1 in endothelial cells and meningeal macrophages after thrombin injection was observed. At 24 hours after thrombin injection, the higher levels of proinflammatory cytokines (IL-1β, IFNγ) were also detected compared to the saline group.
Thrombin caused hydrocephalus and neuron loss Thrombin is an important component of the coagulation cascade and plays an essential role in hydrocephalus [2,3,6]. Our previous study found that thrombin disrupted the blood-CSF barrier via VE-cadherin downregulation and led to hydrocephalus [3]. And the inflammation response was previously proved to be closely associated with the formation of hydrocephalus [9,10,19,20]. Systemic inflammation is a therapeutic target in acute ischemic stroke [21,22]. Also, it was indicated that anti-inflammation treatment could alleviate spontaneous hydrocephalus [23]. In the current study, the role of inflammation in thrombin-induced hydrocephalus was investigated.
Non-parenchymal brain macrophages are mononuclear phagocytes that are increasingly recognized to be critical players in the diseases of the central nervous system [23][24][25][26].
There are three types of central nervous system macrophages between parenchyma and circulation-namely, meningeal, perivascular and choroid plexus macrophages [15,16,26].
Meningeal inflammation has been identified as a key feature of hydrocephalus [10,13,19]and may contribute to the extensive cortical pathology that accompanies progressive disease [27][28][29][30]. Meningeal cells are involved in cortical development, fibrotic scar formation, brain inflammation, and neurodegenerative disorders such as Multiple Sclerosis (MS) and Alzheimer's disease (AD) and other brain disorders [29][30][31]. Here, our study aimed to explore whether meningeal macrophages were involved in the inflammation of subarachnoid space and the formation of hydrocephalus [10,13]. Two macrophage populations were found in the central nervous system, corresponding to the proinflammatory M1 and "alternatively activated" anti-inflammatory M2 classes [16,25].
The M1 macrophages are characterized by the high expressions of oxygen intermediates and proinflammatory cytokines which are closely associated with brain injury [23,32].
While the M2 phenotype is considered to be involved in the promotion of tissue remodeling [16,32]. In our study, a significant increase in M1 polarization was observed in meningeal macrophages and many of them are CD68 positive after thrombin injection.
Interestingly, there was no significant difference in M2 macrophages which were CD206 positive between saline and thrombin group.
One of the most important features of proinflammatory macrophage is the secretion of pro-inflammatory cytokines [32]. In this study, activated endothelial cells and inflammatory infiltrations were observed after thrombin injection, pro-inflammatory cytokines were also significantly increased. Cytokines (like IL-1β, IFNγ) were shown to decrease VE-cadherin protein levels and induce endothelial barrier dysfunction [33,34].
Intercellular adhesion molecule 1 (ICAM1), induced by IL-1β, IFNγ [35], is best known for its role in leukocyte adhesion and typically expressed on endothelial cells and cells of the immune system [18,36]. Additionally, ICAM1 could mediate Src activation [37,38], which is involved in the regulation of VE-cadherin [3,7]. In this study, the increased expression of ICAM1, IL-1β, and IFNγ after thrombin injection were observed, which highlighted the role of the inflammatory response in thrombin-induced hydrocephalus. Furthermore, a growing body of evidence demonstrated that leukocyte-endothelial adhesion interaction could lead to posthemorrhagic vasospasm which also could exacerbate the neuron loss [39][40][41].
To investigate the relationship between inflammation and thrombin-induced brain injury, the ventricular volume and the neuron loss in the cortex were analyzed. Interestingly, the level of infiltrating CD68 positive macrophages in meninges, correlated modestly with the severity of hydrocephalus and neuron loss. Rats with increased meningeal inflammation had a more severe brain injury. These results suggested that diffuse meningeal inflammation may contribute to the pathological mechanisms driving the progression of hydrocephalus and cortical neuronal pathology. These findings are consistent with other observations in meningeal inflammation [27][28][29][30].
The cellular signaling effects of thrombin are mediated by protease-activated receptors (PARs) [2,3,42]. PAR1 plays a critical role in the detrimental thrombin signaling [7,17,43]. The activation of PAR1 disrupts vascular integrity [3,7], results in neurotoxicity [44,45] and increases secondary brain injury after stroke [2,6,43]. In our study, inhibition of PAR1 reduced the meningeal inflammation, neuron loss, and hydrocephalus. It is possible that anti-inflammation will provide novel therapeutic targets for hydrocephalus management in the future.

Conclusion
These results demonstrated that meningeal macrophages were activated by intraventricular thrombin injection and it also exaggerated neuron loss and peri-vascular inflammation in the subarachnoid space. The clinically available PAR1 antagonists could reverse the meningeal injury and inflammation, thus might offer a new therapeutic approach for management in ventricular hemorrhage-induced hydrocephalus.