The Effect of Fibulin-5 on Hydrocephalus After Subarachnoid Hemorrhage in Mice

The Effect of Fibulin-5 on Hydrocephalus After Subarachnoid Hemorrhage in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Effect of Fibulin-5 on Hydrocephalus After Subarachnoid Hemorrhage in Mice Yume Suzuki, Mai Nampei, Fumihiro Kawakita, Hiroki Oinaka, Hideki Nakajima, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6322105/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chronic hydrocephalus following aneurysmal subarachnoid hemorrhage (SAH) is a complication that can lead to deterioration in neurological status and cognitive impairment. Our recent clinical study reported that a high concentration of plasma fibulin-5 (FBLN5), one of matricellular proteins, was associated with the occurrence of chronic hydrocephalus after SAH. This study aimed to investigate whether and how FBLN5 was associated with hydrocephalus during acute to later phases of SAH in mice. C57BL/6 male mice underwent sham or filament perforation SAH modeling, and vehicle or two dosages (0.01 and 0.1µg) of short or long recombinant FBLN5 (rFBLN5) were randomly administrated by an intracerebroventricular injection. Neurobehavioral tests, measurements of the degree of ventricular enlargement, Western blotting, and immunohistochemical staining were performed to evaluate hydrocephalus 24 and 48 hours after SAH. After SAH, ventricular dilatation did not occur at 24 hours but developed at 48 hours, and both doses of long rFBLN5 with an arginine-glycine-aspartic acid domain suppressed ventricular dilatation at 48 hours after SAH. Long rFBLN5 also decreased phosphorylated p38 in the brain parenchyma and prevented post-SAH increases in perivascular macrophages as well as microglia activation in the brain parenchyma at 48 hours after SAH. Although further research is required to clarify the detailed mechanism, this study demonstrated for the first time that exogenous administration of FBLN5 may have a protective effect against ventricular dilatation after experimental SAH. Hydrocephalus Extracellular matrix protein Perivascular macrophage Fibulin Subarachnoid hemorrhage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Aneurysmal subarachnoid hemorrhage (SAH) accounts for 80% of all cases of SAH [ 1 ], representing a condition with poor outcomes and a high mortality rate of 35% [ 2 ]. Chronic hydrocephalus following aneurysmal SAH is a complication that can lead to deterioration in neurological status and cognitive impairment, occurring in 9–64% of cases [ 3 – 10 ]. The causes of hydrocephalus include changes in cerebrospinal fluid (CSF) dynamics, obstruction of arachnoid granulations by blood components, and adhesion of the ventricular system [ 11 – 13 ]. Research indicates that perivascular macrophage (PVM) and leptomeningeal macrophage regulate CSF dynamics [ 14 ]. Fibulin-5 (FBLN5) is a matricellular protein (MCP) that is part of the extracellular matrix (ECM) components [ 15 ]. It contains six calcium-binding epidermal growth factor (EGF)-like motifs and an arginine-glycine-aspartic acid (RGD) motif, with a molecular weight of 66-kDa [ 15 ]. FBLN5 expression decreases during growth but can re-increase in injured tissues [ 16 , 17 ]. Although prior experimental reports have not established the relationship between SAH and FBLN5, our recent clinical study suggested that elevated plasma levels of FBLN5 in a subacute phase were associated with subsequent development of chronic hydrocephalus after aneurysmal SAH [ 18 ]. As PVM influence CSF dynamics [ 14 ], post-SAH changes in PVM may contribute to hydrocephalus development. However, only a few studies have explored the relationship between FBLN5 and macrophages: it was reported that FBLN5 modulates the inflammatory microenvironment including macrophages in the dermis [ 19 ] and that tumor-associated macrophages degrade FBLN5 in epithelial ovarian cancer [ 20 ]. This study aimed to investigate whether the administration of recombinant FBLN5 (rFBLN5) influences the development of hydrocephalus following SAH in mice and whether this effect is associated with changes in PVMs. Materials and methods All procedures were approved by the Animal Ethics Review Committee of Mie University and were carried out according to the institution’s and the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines. Study Protocols (Fig. 1 ) First, to investigate the effect of FBLN5 on ventricular enlargement in an acute phase after SAH, 59 mice were randomly divided into six groups: sham + vehicle (phosphate-buffered saline [PBS]) (n = 12), SAH + vehicle (n = 16), SAH + low-dose (0.01µg) short rFBLN5 (SrFBLN5) (n = 8), SAH + high-dose (0.1µg) SrFBLN5 (n = 7), SAH + low-dose long rFBLN5 (LrFBLN5) (n = 9), and SAH + high-dose LrFBLN5 (n = 7) groups (Figs. 1 a and 2 a). Based on our preliminary study, two doses of SrFBLN5 or LrFBLN5 and the vehicle were administered intracerebroventricularly at 30 minutes post-modeling. After neurobehavioral function was assessed 24 hours after modeling, mice were euthanized to evaluate SAH grades, the width of ventricle-to-brain ratio (wVBR) and the area of ventricle-to-brain ratio (aVBR) as Fig. 2 b, which were separately compared among the vehicle-treated and two doses of SrFBLN5-treated groups, and among the vehicle-treated and two doses of LrFBLN5-treated groups. Second, to investigate the effect of FBLN5 on ventricular enlargement in a later phase after SAH, 100 mice were randomly divided into six groups: sham + vehicle (n = 16), SAH + vehicle (n = 39), SAH + low-dose SrFBLN5 (n = 15), SAH + high-dose SrFBLN5 (n = 14), SAH + low-dose LrFBLN5 (n = 7) and SAH + high-dose LrFBLN5 (n = 9) groups (Fig. 1 b). Drugs were administered via intracerebroventricular infusion (ICV) as described above. After neurobehavioral test was performed 24 and 48 hours after modeling, mice were euthanized, and SAH grade, wVBR and aVBR were measured. Comparisons were made separately among the vehicle-treated and 2 doses of SrFBLN5-treated groups and among the vehicle-treated and 2 doses of LrFBLN5-treated groups. Third, to elucidate the mechanisms of LrFBLN5’s inhibitory effects on ventricular enlargement, 29 mice were randomly divided into four groups: sham + vehicle (n = 6), SAH + vehicle (n = 8), SAH + low-dose LrFBLN5 (n = 6), and SAH + high-dose LrFBLN5 (n = 9) groups (Fig. 1 c). Drug administration was performed via ICV as described above. After neurobehavioral test was assessed 24 and 48 hours after modeling, mice were euthanized, and SAH grading and Western blotting were performed. Fourth, to assess the effect of LrFBLN5 on macrophages in the perivascular space, 37 mice were randomly divided into four groups: sham + vehicle (n = 6), SAH + vehicle (n = 15), SAH + low-dose LrFBLN5 (n = 7), and SAH + high-dose LrFBLN5 (n = 9) groups (Fig. 1 d). Drug administration was the same as described above. After neurobehavioral test was assessed 24 and 48 hours after modeling, mice were euthanized to perform SAH grading and immunohistochemical staining. rFBLN5 (Fig. 2a) To clarify the functional site of FBLN5, two different lengths of rFBLN5 were administrated intracerebroventricularly: a SrFBLN5 consisting of only the third and fourth calcium-binding EGF-like motifs (RPD153Mu02, Cloud-Clone Corp, Houston, TX, USA) and a nearly full-length rFBLN5 containing the RGD motif (LrFBLN5; 9006-FB, R&D System, Minneapolis, MN, USA). SAH Modeling C57BL/6 male adult mice (age 10–12 weeks, 25–30 g; SLC, Hamamatsu, Japan) were used for this study. As this study was intended to clarify the pathophysiology rather than therapeutic intent, we used only male mice. As previously described, mice underwent endovascular perforation SAH or sham modeling [ 21 ]. Mice were anesthetized with an intraperitoneal injection of mixed 3-type anesthetic agents (0.75mg/kg of medetomidine hydrochloride, 4mg/kg of midazolam, and 5mg/kg of butorphanol tartrate). After the anesthesia, mice were placed in a supine position, and a skin incision was made at the midline of the neck to expose the left carotid arteries. A 4 − 0 nylon monofilament with a sharpened tip was inserted from the left external carotid artery stump into the left internal carotid artery about 15mm to perforate the bifurcation of the left anterior cerebral artery and the left middle cerebral artery. Then, the filament was withdrawn, and the stump of external carotid artery was coagulated. The wound was sutured. The sham mice underwent the same procedure as described above, except that the artery was not perforated. During the operation, blood pressure and heart rate were monitored via the tail, and body temperature was kept at 37 ℃. ICV At 30 minutes post-modeling, surviving mice underwent ICV as previously described [ 21 , 22 ]. Mice were placed in a stereotactic head holder, and a skin incision was made at the midline of the head. The needle of a 2µL Hamilton syringe (Hamilton Company, Reno, Nev., USA) was inserted via the burr hole perforated on the skull into the left lateral ventricle using the following coordinates relative to the bregma: 0.2mm posterior, 1.0mm lateral, and 2.25mm below the horizontal plane of the bregma. Sterile 2µL vehicle (PBS, regulated to pH 7.2 − 7.4) with and without SrFBLN5 or LrFBLN5 (0.01 or 0.1µg) was injected at a rate of 1µL/min. The needle was gently removed 5 minutes after an injection, and the wound was quickly sutured. After surgery, mice were returned to clean cages and allowed free access to food and water, and the room temperature was kept constant at 25 ± 1°C. Neurobehavioral Test Neurobehavior functions were blindly assessed using the modified Garcia’s neurological score system as previously described [ 22 , 23 ]. The evaluation consisted of six tests scored 0 to 3 or 1 to 3. The six tests included spontaneous activity, spontaneous movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to whisker stimulation. Mice were given a score of 2 to 18 in 1-number steps, and higher scores indicated better function. SAH Grade and Exclusion Criteria The severity of SAH was blindly evaluated using high-resolution pictures of the base of the brain taken at each sacrifice. Two evaluators (Y.S. and M.N.) scored each model and the average of these scores was calculated. The SAH grading system was as follows. The basal cistern was divided into six segments, and each segment was allotted a grade from 0 to 3 depending on the amount of subarachnoid blood clot in the segment: grade 0, no subarachnoid blood; grade 1, minimal subarachnoid blood; grade 2, moderate blood clot with recognizable arteries; and grade 3, blood clot obliterating all arteries within the segment [ 23 ]. The mice received a total score ranging from 0 to 18 after adding the scores from all six segments. Mice with SAH grading scores ≤ 7 at 24 hours and ≤ 4 at 48 hours were excluded because they had no significant brain injury according to our preliminary study. Ventricle-to-brain Ratio (VBR) (Fig. 2b) The degree of ventricular enlargement was morphologically evaluated using the coronal section 0.5mm anterior to bregma as previously described [ 24 ]. Mice were deeply anesthetized and transcardially perfused with 30 mL PBS followed by 15 minutes of 10% neutral buffered formalin at 60–80 mmHg. Brains were fixed in 10% neutral buffered formalin for approximately 12 hours and embedded in paraffin. Four-micrometer-thick coronal sections at 0.5mm anterior to the bregma were cut and mounted on the slide. The width and area were quantified by densitometric analyses using Image J software (NIH, Bethesda, Maryland, USA). The percentage of VBR was calculated in two ways according to the following formula: wVBR = (maximum width of the right lateral ventricle + maximum width of the left lateral ventricle) / maximum width of the brain; and aVBR = (area of the right lateral ventricle + area of the left lateral ventricle) / area of the brain). Western Blotting (WB) WB was performed as previously described [ 22 ]. The left cerebral hemisphere was used for analyses. Equal amounts of protein samples were separately loaded on SDS-PAGE gels, electrophoresed, and transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% bovine serum albumin or 5% weight/volume nonfat dry milk followed by incubation overnight at 4℃ with the following primary antibodies: mouse monoclonal transforming growth factor (TGF)-β1 (1:200, sc-52893; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-Smad 2/3 (1:1000, ab202445; Abcam, Cambridge, UK), rabbit monoclonal anti-phosphorylated Smad 2/3 (1:1000, ab254407; Abcam, Cambridge, UK), rabbit monoclonal anti-phosphorylated p38 (1:1000, #4511; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-phosphorylated c-Jun N-terminal kinase (JNK; 1:1000, sc-6254; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-phosphorylated extracellular signal-related kinase (ERK) 1/2 (1:1000, #4370; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-ERK1/2 (1:1000, #4695; Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-tenascin-C (TNC) (1:1000, ab108930; Abcam, Cambridge, UK) antibodies. Then, the membrane was incubated with goat anti-rabbit secondary antibodies (PI-1000; Vector, Burlingame, CA, USA) or anti-mouse secondary antibodies (PI-2000; Vector, Burlingame, CA, USA) for 1 hour at room temperature. A chemiluminescence reagent kit (ECL Prime; Amersham Bioscience, Arlington Heights, IL, USA) was used to detect immunoreactive bands. The bands were quantified by densitometric analyses using Image J software (NIH, Bethesda, Maryland, USA). β-tubulin (1:2000, #2146; Cell Signaling Technology, Danvers, MA, USA) was used as a loading control. Immunohistochemical Staining Immunohistochemical staining was performed as previously described [ 22 ]. Mice were deeply anesthetized and transcardially perfused with 30 mL PBS followed by 15 minutes of 10% neutral buffered formalin at 60–80 mmHg. Brains were fixed in 10% neutral buffered formalin for approximately 12 hours and embedded in paraffin. Four-micrometer-thick coronal sections at 0.5mm anterior to the bregma were cut and mounted on the slide. After the sections were dewaxed and dehydrated, antigen retrieval was performed in 1 mmol/L ethylenediaminetetraacetic acid (pH 8.0) at 80℃ for 20 minutes. To quench any endogenous peroxidase activity, the sections were incubated in 3% hydrogen peroxide for 10 minutes, followed by being blocked with normal serum for 60 minutes at room temperature. Then, the sections were incubated with rabbit monoclonal anti-ionized calcium-binding adaptor molecule 1 (Iba1; 1:2000, ab178847; Abcam, Cambridge, UK) antibody as the primary antibody overnight at 4℃, followed by incubation with biotinylated goat anti-rabbit polyclonal immunoglobulin G (1:200; Vector Laboratories, Burlingame, CA, USA) as the secondary antibody for 30 minutes at room temperature. Sections were then incubated with an avidin-biotin-horseradish peroxide complex (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. The sections were visualized by diaminobenzidine/hydrogen peroxide solution and counterstained with hematoxylin for light microscopic examination. To evaluate the expression of Iba1, four continuous pictures of the left (perforation side) secondary somatosensory cortex [ 25 , 26 ] at ×200 magnification were photographed under a light microscope. The relative quantity of Iba1 per picture was measured by integrated optical density using Image Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA), and the average value in the four continuous pictures was calculated. Statistical Analysis All statistical analyses were performed with SPSS software, version 30.0 (IBM, Armonk, New York, USA). Following tests of normality with Shapiro-Wilk tests, continuous variables were described as a mean ± standard error of the mean, or a median ± 25–75 percentile, and were compared among more than three groups using Kruskal-Wallis tests followed by post hoc Steel-Dwass multiple comparisons or one-way analysis of variance (ANOVA) followed by post hoc Tukey multiple comparisons. Mortality was analyzed using Fisher’s exact test. A significant level was set at a p value < 0.05. Results Effects of FBLN5 on Ventricular Enlargement at an Acute Phase In the first experiment, SAH grade and mortality were similar among the SAH groups (Supplementary Table S1 , Supplementary Figs. S1a and S1c). Both doses of SrFBLN5 failed to influence post-SAH deterioration of neurological scores (Supplementary Fig. S1 b), but 0.01µg of LrFBLN5 administrations improved neurological scores to a degree that was not different from those in the sham group at 24 hours after SAH (Supplementary Fig. S1 d). However, neither SAH nor rFBLN5 administration had significant effects on VBR, although higher doses of LrFBLN5 tended to suppress VBR (Supplementary Fig. S2 ). Inhibitory Effects of FBLN5 on Ventricular Enlargement at a Later Phase In the second experiment, ventricular enlargement was evaluated at 48 hours after SAH, because it was impossible to evaluate ventricular enlargement at a chronic phase due to a high mortality of SAH mice (data not shown). The SAH + vehicle group had a significantly higher mortality rate than the rFBLN5-treated SAH groups, especially the LrFBLN5-treated SAH groups (Supplementary Table S2 ). The SAH + 0.1µg SrFBLN5 group showed worse neurological scores at 24 and 48 hours, but this was thought to reflect its more severe SAH grades (Supplementary Figs. S3a-c). LrFBLN5 administrations did not significantly affect SAH grades and neurological scores, but a higher dose of LrFBLN5 tended to improve neurological scores at 48 hours (Supplementary Figs. S3d-f). As to ventricular enlargement at 48 hours, both wVBR and aVBR were significantly higher in the SAH + vehicle group than the sham + vehicle group, and LrFBLN5 administration improved them to a level not significantly different from those in the sham group, especially in terms of wVBR (Fig. 3 ). Exploring Mechanisms for LrFBLN5 to Prevent Post-SAH Ventricular Dilatation WB analyses were performed to investigate the possible mechanisms for LrFBLN5 to inhibit ventricular dilatation and therefore changes in expressions of proteins potentially affected by LrFBLN5. There were no significant differences in the mortality and SAH grades among the SAH groups (Supplementary Table S3 and Fig. S4a). Neurological scores were similar to the findings in experiment 2 (Supplementary Figs. S4b and S4c). WB showed that SAH did not significantly increase any of the proteins tested including TGF-β1, Smad, mitogen-activated protein kinases (MAPKs) and TNC. However, phosphorylated p38 was markedly decreased in the SAH + LrFBLN5 groups compared with the SAH + vehicle group (Fig. 4 ). Effects of LrFBLN5 on Iba1-positive cells in the Brain Parenchyma and Perivascular Space Next, immunohistochemical staining of Iba1 was performed to assess the effect of LrFBLN5 on PVMs after SAH. The SAH grade was unexpectedly more severe in the higher dose group, but the mortality and neurological scores were similar to those in experiments 2 and 3 (Figs. 5 a-c and Supplementary Table S4). Iba1-positive cells were significantly increased in both the brain parenchyma and the perivascular space in the SAH + vehicle group compared to the sham + vehicle group and were suppressed by the administration of LrFBLN5 (Figs. 5 d and 5 e). Discussion The novel findings in this study were as follows: 1) administration of LrFBLN5 tended to improve neurobehavioral functions, although not necessarily in a dose-dependent manner; 2) ventricular dilatation did not occur at 24 hours, but developed at 48 hours after SAH; 3) both doses of LrFBLN5 suppressed ventricular dilatation at 48 hours after SAH; and 4) LrFBLN5 decreased phosphorylated p38 in the brain parenchyma and prevented post-SAH increases in activated microglia in the brain parenchyma and PVMs at 48 hours after SAH. The causes of hydrocephalus include changes in CSF dynamics, fibrosis of the leptomeninges and arachnoid granulations due to blood components, and adhesion in the ventricular system [ 11 – 13 , 27 , 28 ]. Previous research indicated that PVMs and leptomeningeal macrophages regulate CSF dynamics [ 14 ]. PVMs are classified as border-associated macrophages (BAMs), which also encompass meningeal macrophages and choroid plexus macrophages [ 29 ]. BAMs and microglia play critical roles in the brain’s innate immunity and inflammation [ 29 ]. The functions of PVM include perivascular drainage, cerebrovascular flexibility, phagocytic activity, antigen presentation, inflammatory responses, and maintenance of blood-brain barrier integrity [ 30 ]. Under normal conditions, PVM function as a scavenger and surveillance cells; however, in pathological states, they can have detrimental effects [ 29 ]. Following SAH, PVM interacts with erythrocytes and other blood components, increasing perivascular inflammation and contributing to microvascular thrombus formation [ 31 – 33 ]. PVM also participates in neuronal apoptosis and perivascular gliosis after SAH [ 33 ]. Experimental models showed that depleting of PVM with clodronate could suppress perivascular inflammation, neuronal apoptosis, and perivascular gliosis, improving the outcomes after SAH [ 33 ]. PVMs are integral to the glymphatic system [ 34 ]. CSF enters the perivascular space, penetrating the arteries and flowing into the interstitial space [ 34 ]. PVM functions as gatekeepers, preventing the accumulation of large particles in the perivascular space and reducing the inhibition of glymphatic flux [ 33 , 35 ]. They also regulate the flow rate of CSF by inducing contraction and relaxation in vascular smooth muscle cells [ 36 ]. However, under SAH, PVMs are thought to cause perivascular inflammation, thereby restricting arteriolar pulsation and compromising CSF flow [ 31 – 33 , 35 ]; therefore, it is possible that the clearance of CSF may improve via PVM depletion from the glymphatic system after SAH. FBLN5 is a 66-kDa MCP that plays a crucial role in organizing elastic fibers and mediating various cellular functions essential for tissue development and homeostasis [ 15 , 37 , 38 ]. It consists of six calcium-binding EGF-like motifs and fibulin modules, including one motif containing the RGD sequence [ 15 ]. FBLN5 expression decreases during growth but increases again in injured tissues [ 16 , 17 ]. Our previous research suggested that in clinical settings, the elevated plasma levels of FBLN5 during the subacute phase of aneurysmal SAH may contribute to the development of chronic hydrocephalus [ 18 ]. Only a few studies have explored the relationship between FBLN5 and macrophages. FBLN5 is involved in regulating the inflammatory microenvironment in the dermis of Snail-transgenic mice and influences the proliferation and reduction of macrophages [ 19 ]. In epithelial ovarian cancer, FBLN5 is degraded by proteases prevalent in the tumor microenvironment macrophages, with the degraded form facilitating ovarian cancer cell adhesion and local metastasis [ 20 ]. Given these findings, a potential interaction between FBLN5 and PVM is also considered. Our experimental results suggested that PVM, which may adversely affect CSF dynamics in the perivascular space following SAH, were depleted by LrFBLN5 administration, leading to the improvement of post-SAH ventricular enlargement at 48 hours. The observed improvement in neurological outcomes was not necessarily consistent with the inhibitory effect related to ventricular enlargement. This discrepancy can be explained by the fact that LrFBLN5 was also protective against early brain injury after experimental SAH (unpublished data). Following SAH, TGF-β1 levels in the CSF increased, suggesting its involvement in subarachnoid fibrosis and chronic hydrocephalus [ 39 – 42 ]. Additionally, it is proposed that Smad proteins and MAPKs, which relate to this pathway, may also play a role in fibrosis [ 43 – 47 ]. TNC, a type of MCP, is implicated in neuronal apoptosis, breakdown of the blood-brain barrier, and vasospasm following SAH [ 48 , 49 ]. TNC can also promote tissue fibrosis by enhancing leptomeningeal collagen synthesis, leading to ventricular enlargement and the development of chronic hydrocephalus [ 49 , 50 ]. Similar to TGF-β1, TNC is associated with various inflammatory cytokines such as interleukins [ 51 – 53 ]. In this study, it is unclear why these proteins did not increase significantly after SAH in the WB. The possible explanations include: 1) although they increased at the meningeal level, their expression changes were not detected well in the evaluation of the entire brain; and 2) because this study was conducted 48 hours after SAH, it was too early to detect changes in their proteins’ expression, which could have been captured more clearly if they had been evaluated in the chronic phase. However, this study revealed that suppression of p38 activation by LrFBLN5 may have been involved in its preventive effects against activation of PVM and microglia, as well as ventricular enlargement after SAH, although further detailed investigation is required. The present study has several limitations. First, experiments were conducted only within 48 hours of SAH. While this limitation arose from the difficulty in achieving long-term survival in SAH + vehicle mice, evaluations during the chronic phase are needed. Second, no experiments verified the mechanism by which LrFBLN5 affected PVM and ventricular enlargement, although the difference in the effects of LrFBLN5 and SrFBLN5 suggested the involvement of the RGD motif. Third, WB analysis was performed on the whole brains, and therefore may not have been possible to accurately detect changes in locally expressed proteins. However, this is the first study to experimentally show the relationship among FBLN5, PVM and hydrocephalus. Conclusion This study first revealed that exogenous administration of FBLN5 may have a protective effect against ventricular dilatation after experimental SAH. Abbreviations ANOVA , analysis of variance; ARRIVE , Animals in Research: Reporting In Vivo Experiments; aVBR , area of ventricle-to-brain ratio; BAM , border-associated macrophage; CSF , cerebrospinal fluid; ECM , extracellular matrix; EGF , epidermal growth factor; ERK , extracellular signal-related kinase; FBLN5 , fibulin-5; Iba1 , ionized calcium-binding adaptor molecule 1; ICV , intracerebroventricular infusion; JNK , c-Jun N-terminal kinase; LrFBLN5 , long recombinant fibulin-5; MAPK , mitogen-activated protein kinase; MCP , matricellular protein; PBS , phosphate-buffered saline; PVM , perivascular macrophage; rFBLN5 , recombinant fibulin-5; RGD , arginine-glycine-aspartic acid; SAH , subarachnoid hemorrhage; SrFBLN5 , short recombinant fibulin-5; TGF , transforming growth factor; TNC , tenascin-C; VBR , ventricle-to-brain ratio; WB , Western blotting; wVBR , width of ventricle-to-brain ratio. Declarations Acknowledgments The authors wish to thank Ms. Chiduru Nakamura, Department of Neurosurgery, Mie University Graduate School of Medicine, for providing technical assistance. Funding information This work was funded by Sanikai Foundation (Grant Number, N/A) to Dr. Nakajima. Compliance with Ethical Standards Conflicts of Interest Dr. H. Suzuki reported personal fees from Eisai, Kowa, Otsuka and Nxera Pharma, and a research fund from Japan Blood Products Organization and Nxera Pharma outside the submitted work. The other authors declare that they have no conflict of interest. Authors’ Contributions Conceptualization and Design, Yume Suzuki; Methodology, Material Preparation, and Data Collection, Yume Suzuki, Mai Nampei; Data Analyses, Yume Suzuki; Writing – Original Draft Preparation, Yume Suzuki; Writing – Review & Editing, Mai Nampei, Fumihiro Kawakita, Hiroki Oinaka, Hideki Nakajima, and Hidenori Suzuki. 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Transl Stroke Res 3: 25–38. https://doi.org/10.1007/s12975-012-0182-9 Uchikawa H, Uekawa K, Hasegawa Y (2024) Perivascular macrophages in cerebrovascular diseases. Exp Neurol; https://doi.org/10.1016/j.expneurol.2024.114680 Yang T, Guo R, Zhang F (2019) Brain perivascular macrophages: recent advances and implications in health and diseases. CNS Neurosci Ther 25: 1318–1328. https://doi.org/10.1111/cns.13263 Vergouwen MD, Vermeulen M, Coert BA, Stroes ES, Roos YB (2008) Microthrombosis after aneurysmal subarachnoid hemorrhage: an additional explanation for delayed cerebral ischemia. J Cereb Blood Flow Metab 28: 1761–1770. https://doi.org/10.1038/jcbfm.2008.74 McBride DW, Blackburn SL, Peeyush KT, Matsumura K, Zhang JH (2017) The role of thromboinflammation in delayed cerebral ischemia after subarachnoid hemorrhage. 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Biomed Rep 2: 787–792. https://doi.org/10.3892/br.2014.323 Lodyga M, Hinz B (2020) TGF-β1—a truly transforming growth factor in fibrosis and immunity. Semin Cell Dev Biol 101: 123–139. https://doi.org/10.1016/j.semcdb.2019.12.010 Runyan CE, Schnaper HW, Poncelet AC (2003) Smad3 and PKCdelta mediate TGF-beta1-induced collagen I expression in human mesangial cells. Am J Physiol Ren Physiol 285: F413–F422. https://doi.org/10.1152/ajprenal.00082.2003 Frangogiannis N (2020) Transforming growth factor-β in tissue fibrosis. J Exp Med 217: e20190103. https://doi.org/10.1084/jem.20190103 Suzuki H, Kanamaru K, Shiba M, Fujimoto M, Imanaka-Yoshida K, Yoshida T, Taki W (2011) Cerebrospinal fluid tenascin-C in cerebral vasospasm after aneurysmal subarachnoid hemorrhage. J Neurosurg Anesthesiol 23: 310–317. https://doi.org/10.1097/ANA.0b013e31822aa1f2 Suzuki H, Kanamaru K, Suzuki Y, Aimi Y, Matsubara N, Araki T, Takayasu M, Kinoshita N, Imanaka-Yoshida K, Yoshida T, Taki W (2010) Tenascin-C is induced in cerebral vasospasm after subarachnoid hemorrhage in rats and humans: a pilot study. Neurol Res 32: 179–184. https://doi.org/10.1179/174313208X355495 Chiquet-Ehrismann R, Chiquet M (2003) Tenascins: regulation and putative functions during pathological stress. J Pathol 200: 488–499. https://doi.org/10.1002/path.1415 Chiovaro F, Chiquet-Ehrismann R, Chiquet M (2015) Transcriptional regulation of tenascin genes. Cell Adhes Migr 9: 34–47. https://doi.org/10.1080/19336918.2015.1008333 Borel CO, McKee A, Parra A, Haglund MM, Solan A, Prabhakar V, Sheng H, Warner DS, Niklason L (2003) Possible role for vascular cell proliferation in cerebral vasospasm after subarachnoid hemorrhage. Stroke 34: 427–433 Kitazawa K, Tada T (1994) Elevation of transforming growth factor-beta 1 level in cerebrospinal fluid of patients with communicating hydrocephalus after subarachnoid hemorrhage. Stroke 25: 1400–1404. https://doi.org/10.1161/01.str.25.7.1400 Additional Declarations Competing interest reported. Dr. H. Suzuki reported personal fees from Eisai, Kowa, Otsuka and Nxera Pharma, and a research fund from Japan Blood Products Organization and Nxera Pharma outside the submitted work. The other authors declare that they have no conflict of interest. Supplementary Files SupplementaryMaterialsMolNeurobiol250327.docx WBGelsandBlots.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6322105","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449397614,"identity":"20f7e4ff-cf97-488d-85a9-35f38655e14a","order_by":0,"name":"Yume Suzuki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACAyBm/GBgw8PYcPgAA2MDVFiCgBZmiYo0OebGYwnEa2HgOXPYmL35jAFCCz5gzt5jwCDZlpbY23bmm8TPHTZyDOxAF1ruwK3FsgfonsI2m8SZPWe3SfaeSTNm4ElLYJA8g8dhN5IPgG3ZOOPsNgnetsOJDRI8IHvxaUlsYACp3H//zTPJv8RpAdoC8j5jwxk2aaJsAfkFHMiMDceMrWXb0ozZgH45gM8voBCDReXDm2/bbOT42Q8ffCyJJ8SAgP0HlMECjkA2ID4s2YBXCxwwf4CxGD8SqWUUjIJRMApGBAAA3nBUw3TiJ7EAAAAASUVORK5CYII=","orcid":"","institution":"Mie University","correspondingAuthor":true,"prefix":"","firstName":"Yume","middleName":"","lastName":"Suzuki","suffix":""},{"id":449397615,"identity":"252affb4-ce57-4e16-b820-d869e96987c9","order_by":1,"name":"Mai Nampei","email":"","orcid":"","institution":"Mie University","correspondingAuthor":false,"prefix":"","firstName":"Mai","middleName":"","lastName":"Nampei","suffix":""},{"id":449397616,"identity":"af8bb2bd-7326-41a1-a185-e133ee132af3","order_by":2,"name":"Fumihiro Kawakita","email":"","orcid":"","institution":"Mie University","correspondingAuthor":false,"prefix":"","firstName":"Fumihiro","middleName":"","lastName":"Kawakita","suffix":""},{"id":449397617,"identity":"0e31eb41-a82f-4d1b-9639-70b75d35e8bf","order_by":3,"name":"Hiroki Oinaka","email":"","orcid":"","institution":"Mie University","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Oinaka","suffix":""},{"id":449397618,"identity":"55c22aa7-f120-49b8-a337-091493a95508","order_by":4,"name":"Hideki Nakajima","email":"","orcid":"","institution":"Mie University","correspondingAuthor":false,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Nakajima","suffix":""},{"id":449397619,"identity":"6be0e966-3700-43f3-a979-e280b0ae6d3b","order_by":5,"name":"Hidenori Suzuki","email":"","orcid":"","institution":"Mie University","correspondingAuthor":false,"prefix":"","firstName":"Hidenori","middleName":"","lastName":"Suzuki","suffix":""}],"badges":[],"createdAt":"2025-03-27 16:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6322105/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6322105/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81702549,"identity":"7e83824b-7751-4690-97ed-ec3c399e851a","added_by":"auto","created_at":"2025-04-30 13:10:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":338908,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental designs. Experiment 1 to evaluate effects of fibulin-5 (FBLN5) against ventricular dilatation 24 hours after subarachnoid hemorrhage (SAH) (\u003cstrong\u003ea\u003c/strong\u003e); experiment 2 to evaluate effects of FBLN5 against ventricular dilatation 48 hours after SAH (\u003cstrong\u003eb\u003c/strong\u003e); experiment 3 to elucidate the mechanisms of FBLN5’s effect on hydrocephalus 48 hours after SAH (\u003cstrong\u003ec\u003c/strong\u003e); and experiment 4 to evaluate effects of FBLN5 against activation of microglia and macrophage in the brain parenchyma 48 hours after SAH (\u003cstrong\u003ed\u003c/strong\u003e). ICV, intracerebroventricular; PBS, phosphate-buffered saline; SrFBLN5, short recombinant FBLN5; LrFBLN5, long recombinant FBLN5; VBR, ventricular-to-brain ratio; WB, Western blot; IHC, immunohistochemistry\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6322105/v1/fda16b26e137bb61621bfa51.png"},{"id":81702402,"identity":"c187bb59-b1f4-4469-838c-b4d489c2baeb","added_by":"auto","created_at":"2025-04-30 13:09:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":219397,"visible":true,"origin":"","legend":"\u003cp\u003eThe structure schemas of fibulin-5 (FBLN5) (\u003cstrong\u003ea\u003c/strong\u003e) and the calculation method of the ventricular dilatation (\u003cstrong\u003eb\u003c/strong\u003e). Full-length FBLN5 (upper), the short type of recombinant FBLN5 (middle), and the long type of recombinant FBLN5 (lower). CB EGF, calcium-binding epidermal growth factor; RGD, arginine-glycine-aspartic acid\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6322105/v1/6f0764f2b5856f24c4aaf293.png"},{"id":81702441,"identity":"b83f2e78-0d8a-4716-83bb-b38b3f511e48","added_by":"auto","created_at":"2025-04-30 13:09:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":521999,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of two dosages (0.01 or 0.1μg) of administered short or long recombinant fibulin-5 (rFBLN5) on ventricle-to-brain ratio at 48 hours after subarachnoid hemorrhage (SAH). *p\u0026lt;0.05 vs Sham phosphate-buffered saline (PBS) group, and **p\u0026lt;0.01 vs Sham PBS group; Kruskal-Wallis test (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, and \u003cstrong\u003ed\u003c/strong\u003e) or one-way ANOVA (\u003cstrong\u003ec\u003c/strong\u003e). aVBR, area of ventricle-to-brain ratio; wVBR, width of ventricle-to-brain ratio\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6322105/v1/a5a04ef0e1682731da8275f5.png"},{"id":81702343,"identity":"076b1ce7-aa26-4563-bd77-0652b91bee37","added_by":"auto","created_at":"2025-04-30 13:09:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":363380,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of two dosages (0.01 or 0.1μg) of administered long recombinant fibulin-5 on expressions of transforming growth factor (TGF)-β1, Smad 2/3, p38, c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK) 1/2, and tenascin-C (TNC) in the left cerebral hemisphere 48 hours after subarachnoid hemorrhage (SAH). *p\u0026lt;0.05 vs sham + phosphate-buffered saline (PBS) group; one-way ANOVA. p-, phosphorylated\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6322105/v1/910796ccf85736ea0a02ec29.png"},{"id":81702407,"identity":"28bb09ed-984c-40b1-addf-afce47cf33e0","added_by":"auto","created_at":"2025-04-30 13:09:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":797560,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of 2 dosages (0.01 or 0.1μg) of long recombinant fibulin-5 (rFBLN5) administration on subarachnoid hemorrhage (SAH) grade (\u003cstrong\u003ea\u003c/strong\u003e), neurological score at 24 hours (\u003cstrong\u003eb\u003c/strong\u003e) and 48 hours (\u003cstrong\u003ec\u003c/strong\u003e), and the ionized calcium binding adaptor molecule 1 staining in brain parenchyma (integrated optical density [\u003cstrong\u003ed\u003c/strong\u003e] and representative images [\u003cstrong\u003ee\u003c/strong\u003e]) at 48 hours after SAH. *p\u0026lt;0.05 vs Sham phosphate-buffered saline (PBS) group, **p\u0026lt;0.01 vs Sham PBS group, and \u003csup\u003e††\u003c/sup\u003ep\u0026lt;0.01 vs SAH PBS group; ANOVA (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, and \u003cstrong\u003ed\u003c/strong\u003e) or Kruskal-Wallis test (\u003cstrong\u003eb\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6322105/v1/6ca8beb12570136cac872faf.png"},{"id":83251582,"identity":"7b3ca2dd-9089-429e-9602-ca9651bb9bff","added_by":"auto","created_at":"2025-05-21 20:16:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2978565,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6322105/v1/27aab996-43bc-4256-a092-3d6f575b3f25.pdf"},{"id":81702342,"identity":"38db44c2-ec23-4af4-9810-f2f8d2ed0291","added_by":"auto","created_at":"2025-04-30 13:09:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1336629,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsMolNeurobiol250327.docx","url":"https://assets-eu.researchsquare.com/files/rs-6322105/v1/3a5daba0410fca6085fd4be5.docx"},{"id":81702562,"identity":"7aa7b1ac-0b88-4e33-b98f-632e6d8ca9ff","added_by":"auto","created_at":"2025-04-30 13:10:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":687962,"visible":true,"origin":"","legend":"","description":"","filename":"WBGelsandBlots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6322105/v1/3e86576b7857c16bd6db5194.pdf"}],"financialInterests":"Competing interest reported. Dr. H. Suzuki reported personal fees from Eisai, Kowa, Otsuka and Nxera Pharma, and a research fund from Japan Blood Products Organization and Nxera Pharma outside the submitted work. The other authors declare that they have no conflict of interest.","formattedTitle":"The Effect of Fibulin-5 on Hydrocephalus After Subarachnoid Hemorrhage in Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAneurysmal subarachnoid hemorrhage (SAH) accounts for 80% of all cases of SAH [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], representing a condition with poor outcomes and a high mortality rate of 35% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Chronic hydrocephalus following aneurysmal SAH is a complication that can lead to deterioration in neurological status and cognitive impairment, occurring in 9\u0026ndash;64% of cases [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The causes of hydrocephalus include changes in cerebrospinal fluid (CSF) dynamics, obstruction of arachnoid granulations by blood components, and adhesion of the ventricular system [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Research indicates that perivascular macrophage (PVM) and leptomeningeal macrophage regulate CSF dynamics [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFibulin-5 (FBLN5) is a matricellular protein (MCP) that is part of the extracellular matrix (ECM) components [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It contains six calcium-binding epidermal growth factor (EGF)-like motifs and an arginine-glycine-aspartic acid (RGD) motif, with a molecular weight of 66-kDa [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. FBLN5 expression decreases during growth but can re-increase in injured tissues [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although prior experimental reports have not established the relationship between SAH and FBLN5, our recent clinical study suggested that elevated plasma levels of FBLN5 in a subacute phase were associated with subsequent development of chronic hydrocephalus after aneurysmal SAH [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As PVM influence CSF dynamics [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], post-SAH changes in PVM may contribute to hydrocephalus development. However, only a few studies have explored the relationship between FBLN5 and macrophages: it was reported that FBLN5 modulates the inflammatory microenvironment including macrophages in the dermis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and that tumor-associated macrophages degrade FBLN5 in epithelial ovarian cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This study aimed to investigate whether the administration of recombinant FBLN5 (rFBLN5) influences the development of hydrocephalus following SAH in mice and whether this effect is associated with changes in PVMs.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e All procedures were approved by the Animal Ethics Review Committee of Mie University and were carried out according to the institution\u0026rsquo;s and the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Protocols (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, to investigate the effect of FBLN5 on ventricular enlargement in an acute phase after SAH, 59 mice were randomly divided into six groups: sham\u0026thinsp;+\u0026thinsp;vehicle (phosphate-buffered saline [PBS]) (n\u0026thinsp;=\u0026thinsp;12), SAH\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;16), SAH\u0026thinsp;+\u0026thinsp;low-dose (0.01\u0026micro;g) short rFBLN5 (SrFBLN5) (n\u0026thinsp;=\u0026thinsp;8), SAH\u0026thinsp;+\u0026thinsp;high-dose (0.1\u0026micro;g) SrFBLN5 (n\u0026thinsp;=\u0026thinsp;7), SAH\u0026thinsp;+\u0026thinsp;low-dose long rFBLN5 (LrFBLN5) (n\u0026thinsp;=\u0026thinsp;9), and SAH\u0026thinsp;+\u0026thinsp;high-dose LrFBLN5 (n\u0026thinsp;=\u0026thinsp;7) groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Based on our preliminary study, two doses of SrFBLN5 or LrFBLN5 and the vehicle were administered intracerebroventricularly at 30 minutes post-modeling. After neurobehavioral function was assessed 24 hours after modeling, mice were euthanized to evaluate SAH grades, the width of ventricle-to-brain ratio (wVBR) and the area of ventricle-to-brain ratio (aVBR) as Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, which were separately compared among the vehicle-treated and two doses of SrFBLN5-treated groups, and among the vehicle-treated and two doses of LrFBLN5-treated groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecond, to investigate the effect of FBLN5 on ventricular enlargement in a later phase after SAH, 100 mice were randomly divided into six groups: sham\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;16), SAH\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;39), SAH\u0026thinsp;+\u0026thinsp;low-dose SrFBLN5 (n\u0026thinsp;=\u0026thinsp;15), SAH\u0026thinsp;+\u0026thinsp;high-dose SrFBLN5 (n\u0026thinsp;=\u0026thinsp;14), SAH\u0026thinsp;+\u0026thinsp;low-dose LrFBLN5 (n\u0026thinsp;=\u0026thinsp;7) and SAH\u0026thinsp;+\u0026thinsp;high-dose LrFBLN5 (n\u0026thinsp;=\u0026thinsp;9) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Drugs were administered via intracerebroventricular infusion (ICV) as described above. After neurobehavioral test was performed 24 and 48 hours after modeling, mice were euthanized, and SAH grade, wVBR and aVBR were measured. Comparisons were made separately among the vehicle-treated and 2 doses of SrFBLN5-treated groups and among the vehicle-treated and 2 doses of LrFBLN5-treated groups.\u003c/p\u003e \u003cp\u003eThird, to elucidate the mechanisms of LrFBLN5\u0026rsquo;s inhibitory effects on ventricular enlargement, 29 mice were randomly divided into four groups: sham\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;6), SAH\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;8), SAH\u0026thinsp;+\u0026thinsp;low-dose LrFBLN5 (n\u0026thinsp;=\u0026thinsp;6), and SAH\u0026thinsp;+\u0026thinsp;high-dose LrFBLN5 (n\u0026thinsp;=\u0026thinsp;9) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Drug administration was performed via ICV as described above. After neurobehavioral test was assessed 24 and 48 hours after modeling, mice were euthanized, and SAH grading and Western blotting were performed.\u003c/p\u003e \u003cp\u003eFourth, to assess the effect of LrFBLN5 on macrophages in the perivascular space, 37 mice were randomly divided into four groups: sham\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;6), SAH\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;15), SAH\u0026thinsp;+\u0026thinsp;low-dose LrFBLN5 (n\u0026thinsp;=\u0026thinsp;7), and SAH\u0026thinsp;+\u0026thinsp;high-dose LrFBLN5 (n\u0026thinsp;=\u0026thinsp;9) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Drug administration was the same as described above. After neurobehavioral test was assessed 24 and 48 hours after modeling, mice were euthanized to perform SAH grading and immunohistochemical staining.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003erFBLN5 (Fig. 2a)\u003c/h3\u003e\n\u003cp\u003eTo clarify the functional site of FBLN5, two different lengths of rFBLN5 were administrated intracerebroventricularly: a SrFBLN5 consisting of only the third and fourth calcium-binding EGF-like motifs (RPD153Mu02, Cloud-Clone Corp, Houston, TX, USA) and a nearly full-length rFBLN5 containing the RGD motif (LrFBLN5; 9006-FB, R\u0026amp;D System, Minneapolis, MN, USA).\u003c/p\u003e\n\u003ch3\u003eSAH Modeling\u003c/h3\u003e\n\u003cp\u003eC57BL/6 male adult mice (age 10\u0026ndash;12 weeks, 25\u0026ndash;30 g; SLC, Hamamatsu, Japan) were used for this study. As this study was intended to clarify the pathophysiology rather than therapeutic intent, we used only male mice. As previously described, mice underwent endovascular perforation SAH or sham modeling [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Mice were anesthetized with an intraperitoneal injection of mixed 3-type anesthetic agents (0.75mg/kg of medetomidine hydrochloride, 4mg/kg of midazolam, and 5mg/kg of butorphanol tartrate). After the anesthesia, mice were placed in a supine position, and a skin incision was made at the midline of the neck to expose the left carotid arteries. A 4\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon monofilament with a sharpened tip was inserted from the left external carotid artery stump into the left internal carotid artery about 15mm to perforate the bifurcation of the left anterior cerebral artery and the left middle cerebral artery. Then, the filament was withdrawn, and the stump of external carotid artery was coagulated. The wound was sutured. The sham mice underwent the same procedure as described above, except that the artery was not perforated. During the operation, blood pressure and heart rate were monitored via the tail, and body temperature was kept at 37 ℃.\u003c/p\u003e\n\u003ch3\u003eICV\u003c/h3\u003e\n\u003cp\u003eAt 30 minutes post-modeling, surviving mice underwent ICV as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Mice were placed in a stereotactic head holder, and a skin incision was made at the midline of the head. The needle of a 2\u0026micro;L Hamilton syringe (Hamilton Company, Reno, Nev., USA) was inserted via the burr hole perforated on the skull into the left lateral ventricle using the following coordinates relative to the bregma: 0.2mm posterior, 1.0mm lateral, and 2.25mm below the horizontal plane of the bregma. Sterile 2\u0026micro;L vehicle (PBS, regulated to pH 7.2\u0026thinsp;\u0026minus;\u0026thinsp;7.4) with and without SrFBLN5 or LrFBLN5 (0.01 or 0.1\u0026micro;g) was injected at a rate of 1\u0026micro;L/min. The needle was gently removed 5 minutes after an injection, and the wound was quickly sutured. After surgery, mice were returned to clean cages and allowed free access to food and water, and the room temperature was kept constant at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eNeurobehavioral Test\u003c/h3\u003e\n\u003cp\u003eNeurobehavior functions were blindly assessed using the modified Garcia\u0026rsquo;s neurological score system as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The evaluation consisted of six tests scored 0 to 3 or 1 to 3. The six tests included spontaneous activity, spontaneous movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to whisker stimulation. Mice were given a score of 2 to 18 in 1-number steps, and higher scores indicated better function.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSAH Grade and Exclusion Criteria\u003c/h2\u003e \u003cp\u003eThe severity of SAH was blindly evaluated using high-resolution pictures of the base of the brain taken at each sacrifice. Two evaluators (Y.S. and M.N.) scored each model and the average of these scores was calculated. The SAH grading system was as follows. The basal cistern was divided into six segments, and each segment was allotted a grade from 0 to 3 depending on the amount of subarachnoid blood clot in the segment: grade 0, no subarachnoid blood; grade 1, minimal subarachnoid blood; grade 2, moderate blood clot with recognizable arteries; and grade 3, blood clot obliterating all arteries within the segment [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The mice received a total score ranging from 0 to 18 after adding the scores from all six segments. Mice with SAH grading scores\u0026thinsp;\u0026le;\u0026thinsp;7 at 24 hours and \u0026le;\u0026thinsp;4 at 48 hours were excluded because they had no significant brain injury according to our preliminary study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVentricle-to-brain Ratio (VBR) (Fig. 2b)\u003c/h3\u003e\n\u003cp\u003eThe degree of ventricular enlargement was morphologically evaluated using the coronal section 0.5mm anterior to bregma as previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Mice were deeply anesthetized and transcardially perfused with 30 mL PBS followed by 15 minutes of 10% neutral buffered formalin at 60\u0026ndash;80 mmHg. Brains were fixed in 10% neutral buffered formalin for approximately 12 hours and embedded in paraffin. Four-micrometer-thick coronal sections at 0.5mm anterior to the bregma were cut and mounted on the slide. The width and area were quantified by densitometric analyses using Image J software (NIH, Bethesda, Maryland, USA). The percentage of VBR was calculated in two ways according to the following formula: wVBR = (maximum width of the right lateral ventricle\u0026thinsp;+\u0026thinsp;maximum width of the left lateral ventricle) / maximum width of the brain; and aVBR = (area of the right lateral ventricle\u0026thinsp;+\u0026thinsp;area of the left lateral ventricle) / area of the brain).\u003c/p\u003e\n\u003ch3\u003eWestern Blotting (WB)\u003c/h3\u003e\n\u003cp\u003eWB was performed as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The left cerebral hemisphere was used for analyses. Equal amounts of protein samples were separately loaded on SDS-PAGE gels, electrophoresed, and transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% bovine serum albumin or 5% weight/volume nonfat dry milk followed by incubation overnight at 4℃ with the following primary antibodies: mouse monoclonal transforming growth factor (TGF)-β1 (1:200, sc-52893; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-Smad 2/3 (1:1000, ab202445; Abcam, Cambridge, UK), rabbit monoclonal anti-phosphorylated Smad 2/3 (1:1000, ab254407; Abcam, Cambridge, UK), rabbit monoclonal anti-phosphorylated p38 (1:1000, #4511; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-phosphorylated c-Jun N-terminal kinase (JNK; 1:1000, sc-6254; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-phosphorylated extracellular signal-related kinase (ERK) 1/2 (1:1000, #4370; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-ERK1/2 (1:1000, #4695; Cell Signaling Technology, Danvers, MA, USA), and rabbit monoclonal anti-tenascin-C (TNC) (1:1000, ab108930; Abcam, Cambridge, UK) antibodies. Then, the membrane was incubated with goat anti-rabbit secondary antibodies (PI-1000; Vector, Burlingame, CA, USA) or anti-mouse secondary antibodies (PI-2000; Vector, Burlingame, CA, USA) for 1 hour at room temperature. A chemiluminescence reagent kit (ECL Prime; Amersham Bioscience, Arlington Heights, IL, USA) was used to detect immunoreactive bands. The bands were quantified by densitometric analyses using Image J software (NIH, Bethesda, Maryland, USA). β-tubulin (1:2000, #2146; Cell Signaling Technology, Danvers, MA, USA) was used as a loading control.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical Staining\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining was performed as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Mice were deeply anesthetized and transcardially perfused with 30 mL PBS followed by 15 minutes of 10% neutral buffered formalin at 60\u0026ndash;80 mmHg. Brains were fixed in 10% neutral buffered formalin for approximately 12 hours and embedded in paraffin. Four-micrometer-thick coronal sections at 0.5mm anterior to the bregma were cut and mounted on the slide. After the sections were dewaxed and dehydrated, antigen retrieval was performed in 1 mmol/L ethylenediaminetetraacetic acid (pH 8.0) at 80℃ for 20 minutes. To quench any endogenous peroxidase activity, the sections were incubated in 3% hydrogen peroxide for 10 minutes, followed by being blocked with normal serum for 60 minutes at room temperature. Then, the sections were incubated with rabbit monoclonal anti-ionized calcium-binding adaptor molecule 1 (Iba1; 1:2000, ab178847; Abcam, Cambridge, UK) antibody as the primary antibody overnight at 4℃, followed by incubation with biotinylated goat anti-rabbit polyclonal immunoglobulin G (1:200; Vector Laboratories, Burlingame, CA, USA) as the secondary antibody for 30 minutes at room temperature. Sections were then incubated with an avidin-biotin-horseradish peroxide complex (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. The sections were visualized by diaminobenzidine/hydrogen peroxide solution and counterstained with hematoxylin for light microscopic examination.\u003c/p\u003e \u003cp\u003eTo evaluate the expression of Iba1, four continuous pictures of the left (perforation side) secondary somatosensory cortex [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] at \u0026times;200 magnification were photographed under a light microscope. The relative quantity of Iba1 per picture was measured by integrated optical density using Image Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA), and the average value in the four continuous pictures was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed with SPSS software, version 30.0 (IBM, Armonk, New York, USA). Following tests of normality with Shapiro-Wilk tests, continuous variables were described as a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean, or a median\u0026thinsp;\u0026plusmn;\u0026thinsp;25\u0026ndash;75 percentile, and were compared among more than three groups using Kruskal-Wallis tests followed by post hoc Steel-Dwass multiple comparisons or one-way analysis of variance (ANOVA) followed by post hoc Tukey multiple comparisons. Mortality was analyzed using Fisher\u0026rsquo;s exact test. A significant level was set at a p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of FBLN5 on Ventricular Enlargement at an Acute Phase\u003c/h2\u003e \u003cp\u003eIn the first experiment, SAH grade and mortality were similar among the SAH groups (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Supplementary Figs. S1a and S1c). Both doses of SrFBLN5 failed to influence post-SAH deterioration of neurological scores (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb), but 0.01\u0026micro;g of LrFBLN5 administrations improved neurological scores to a degree that was not different from those in the sham group at 24 hours after SAH (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed). However, neither SAH nor rFBLN5 administration had significant effects on VBR, although higher doses of LrFBLN5 tended to suppress VBR (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInhibitory Effects of FBLN5 on Ventricular Enlargement at a Later Phase\u003c/h2\u003e \u003cp\u003eIn the second experiment, ventricular enlargement was evaluated at 48 hours after SAH, because it was impossible to evaluate ventricular enlargement at a chronic phase due to a high mortality of SAH mice (data not shown). The SAH\u0026thinsp;+\u0026thinsp;vehicle group had a significantly higher mortality rate than the rFBLN5-treated SAH groups, especially the LrFBLN5-treated SAH groups (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The SAH\u0026thinsp;+\u0026thinsp;0.1\u0026micro;g SrFBLN5 group showed worse neurological scores at 24 and 48 hours, but this was thought to reflect its more severe SAH grades (Supplementary Figs. S3a-c). LrFBLN5 administrations did not significantly affect SAH grades and neurological scores, but a higher dose of LrFBLN5 tended to improve neurological scores at 48 hours (Supplementary Figs. S3d-f). As to ventricular enlargement at 48 hours, both wVBR and aVBR were significantly higher in the SAH\u0026thinsp;+\u0026thinsp;vehicle group than the sham\u0026thinsp;+\u0026thinsp;vehicle group, and LrFBLN5 administration improved them to a level not significantly different from those in the sham group, especially in terms of wVBR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExploring Mechanisms for LrFBLN5 to Prevent Post-SAH Ventricular Dilatation\u003c/h2\u003e \u003cp\u003eWB analyses were performed to investigate the possible mechanisms for LrFBLN5 to inhibit ventricular dilatation and therefore changes in expressions of proteins potentially affected by LrFBLN5. There were no significant differences in the mortality and SAH grades among the SAH groups (Supplementary Table S3 and Fig. S4a). Neurological scores were similar to the findings in experiment 2 (Supplementary Figs. S4b and S4c). WB showed that SAH did not significantly increase any of the proteins tested including TGF-β1, Smad, mitogen-activated protein kinases (MAPKs) and TNC. However, phosphorylated p38 was markedly decreased in the SAH\u0026thinsp;+\u0026thinsp;LrFBLN5 groups compared with the SAH\u0026thinsp;+\u0026thinsp;vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffects of LrFBLN5 on Iba1-positive cells in the Brain Parenchyma and Perivascular Space\u003c/h2\u003e \u003cp\u003eNext, immunohistochemical staining of Iba1 was performed to assess the effect of LrFBLN5 on PVMs after SAH. The SAH grade was unexpectedly more severe in the higher dose group, but the mortality and neurological scores were similar to those in experiments 2 and 3 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c and Supplementary Table S4). Iba1-positive cells were significantly increased in both the brain parenchyma and the perivascular space in the SAH\u0026thinsp;+\u0026thinsp;vehicle group compared to the sham\u0026thinsp;+\u0026thinsp;vehicle group and were suppressed by the administration of LrFBLN5 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe novel findings in this study were as follows: 1) administration of LrFBLN5 tended to improve neurobehavioral functions, although not necessarily in a dose-dependent manner; 2) ventricular dilatation did not occur at 24 hours, but developed at 48 hours after SAH; 3) both doses of LrFBLN5 suppressed ventricular dilatation at 48 hours after SAH; and 4) LrFBLN5 decreased phosphorylated p38 in the brain parenchyma and prevented post-SAH increases in activated microglia in the brain parenchyma and PVMs at 48 hours after SAH.\u003c/p\u003e \u003cp\u003eThe causes of hydrocephalus include changes in CSF dynamics, fibrosis of the leptomeninges and arachnoid granulations due to blood components, and adhesion in the ventricular system [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Previous research indicated that PVMs and leptomeningeal macrophages regulate CSF dynamics [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePVMs are classified as border-associated macrophages (BAMs), which also encompass meningeal macrophages and choroid plexus macrophages [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. BAMs and microglia play critical roles in the brain\u0026rsquo;s innate immunity and inflammation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The functions of PVM include perivascular drainage, cerebrovascular flexibility, phagocytic activity, antigen presentation, inflammatory responses, and maintenance of blood-brain barrier integrity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Under normal conditions, PVM function as a scavenger and surveillance cells; however, in pathological states, they can have detrimental effects [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Following SAH, PVM interacts with erythrocytes and other blood components, increasing perivascular inflammation and contributing to microvascular thrombus formation [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. PVM also participates in neuronal apoptosis and perivascular gliosis after SAH [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Experimental models showed that depleting of PVM with clodronate could suppress perivascular inflammation, neuronal apoptosis, and perivascular gliosis, improving the outcomes after SAH [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePVMs are integral to the glymphatic system [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. CSF enters the perivascular space, penetrating the arteries and flowing into the interstitial space [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. PVM functions as gatekeepers, preventing the accumulation of large particles in the perivascular space and reducing the inhibition of glymphatic flux [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. They also regulate the flow rate of CSF by inducing contraction and relaxation in vascular smooth muscle cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, under SAH, PVMs are thought to cause perivascular inflammation, thereby restricting arteriolar pulsation and compromising CSF flow [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]; therefore, it is possible that the clearance of CSF may improve via PVM depletion from the glymphatic system after SAH.\u003c/p\u003e \u003cp\u003eFBLN5 is a 66-kDa MCP that plays a crucial role in organizing elastic fibers and mediating various cellular functions essential for tissue development and homeostasis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. It consists of six calcium-binding EGF-like motifs and fibulin modules, including one motif containing the RGD sequence [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. FBLN5 expression decreases during growth but increases again in injured tissues [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our previous research suggested that in clinical settings, the elevated plasma levels of FBLN5 during the subacute phase of aneurysmal SAH may contribute to the development of chronic hydrocephalus [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOnly a few studies have explored the relationship between FBLN5 and macrophages. FBLN5 is involved in regulating the inflammatory microenvironment in the dermis of Snail-transgenic mice and influences the proliferation and reduction of macrophages [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In epithelial ovarian cancer, FBLN5 is degraded by proteases prevalent in the tumor microenvironment macrophages, with the degraded form facilitating ovarian cancer cell adhesion and local metastasis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Given these findings, a potential interaction between FBLN5 and PVM is also considered. Our experimental results suggested that PVM, which may adversely affect CSF dynamics in the perivascular space following SAH, were depleted by LrFBLN5 administration, leading to the improvement of post-SAH ventricular enlargement at 48 hours. The observed improvement in neurological outcomes was not necessarily consistent with the inhibitory effect related to ventricular enlargement. This discrepancy can be explained by the fact that LrFBLN5 was also protective against early brain injury after experimental SAH (unpublished data).\u003c/p\u003e \u003cp\u003eFollowing SAH, TGF-β1 levels in the CSF increased, suggesting its involvement in subarachnoid fibrosis and chronic hydrocephalus [\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, it is proposed that Smad proteins and MAPKs, which relate to this pathway, may also play a role in fibrosis [\u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. TNC, a type of MCP, is implicated in neuronal apoptosis, breakdown of the blood-brain barrier, and vasospasm following SAH [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. TNC can also promote tissue fibrosis by enhancing leptomeningeal collagen synthesis, leading to ventricular enlargement and the development of chronic hydrocephalus [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Similar to TGF-β1, TNC is associated with various inflammatory cytokines such as interleukins [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In this study, it is unclear why these proteins did not increase significantly after SAH in the WB. The possible explanations include: 1) although they increased at the meningeal level, their expression changes were not detected well in the evaluation of the entire brain; and 2) because this study was conducted 48 hours after SAH, it was too early to detect changes in their proteins\u0026rsquo; expression, which could have been captured more clearly if they had been evaluated in the chronic phase. However, this study revealed that suppression of p38 activation by LrFBLN5 may have been involved in its preventive effects against activation of PVM and microglia, as well as ventricular enlargement after SAH, although further detailed investigation is required.\u003c/p\u003e \u003cp\u003eThe present study has several limitations. First, experiments were conducted only within 48 hours of SAH. While this limitation arose from the difficulty in achieving long-term survival in SAH\u0026thinsp;+\u0026thinsp;vehicle mice, evaluations during the chronic phase are needed. Second, no experiments verified the mechanism by which LrFBLN5 affected PVM and ventricular enlargement, although the difference in the effects of LrFBLN5 and SrFBLN5 suggested the involvement of the RGD motif. Third, WB analysis was performed on the whole brains, and therefore may not have been possible to accurately detect changes in locally expressed proteins. However, this is the first study to experimentally show the relationship among FBLN5, PVM and hydrocephalus.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study first revealed that exogenous administration of FBLN5 may have a protective effect against ventricular dilatation after experimental SAH.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eANOVA\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eanalysis of variance;\u0026nbsp;\u003cstrong\u003eARRIVE\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAnimals in Research: Reporting In Vivo Experiments; \u003cstrong\u003eaVBR\u003c/strong\u003e, area of ventricle-to-brain ratio; \u003cstrong\u003eBAM\u003c/strong\u003e, border-associated macrophage; \u003cstrong\u003eCSF\u003c/strong\u003e, cerebrospinal fluid; \u003cstrong\u003eECM\u003c/strong\u003e, extracellular matrix;\u003cstrong\u003e\u0026nbsp;EGF\u003c/strong\u003e, epidermal growth factor; \u003cstrong\u003eERK\u003c/strong\u003e, extracellular signal-related kinase;\u003cstrong\u003e\u0026nbsp;FBLN5\u003c/strong\u003e, fibulin-5;\u003cstrong\u003e\u0026nbsp;Iba1\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eionized calcium-binding adaptor molecule 1; \u003cstrong\u003eICV\u003c/strong\u003e, intracerebroventricular infusion; \u003cstrong\u003eJNK\u003c/strong\u003e, c-Jun N-terminal kinase;\u003cstrong\u003e\u0026nbsp;LrFBLN5\u003c/strong\u003e, long recombinant fibulin-5; \u003cstrong\u003eMAPK\u003c/strong\u003e,\u0026nbsp;mitogen-activated protein kinase; \u003cstrong\u003eMCP\u003c/strong\u003e, matricellular protein;\u003cstrong\u003e\u0026nbsp;PBS\u003c/strong\u003e, phosphate-buffered saline; \u003cstrong\u003ePVM\u003c/strong\u003e, perivascular macrophage; \u003cstrong\u003erFBLN5\u003c/strong\u003e, recombinant fibulin-5; \u003cstrong\u003eRGD\u003c/strong\u003e, arginine-glycine-aspartic acid; \u003cstrong\u003eSAH\u003c/strong\u003e, subarachnoid hemorrhage;\u003cstrong\u003e\u0026nbsp;SrFBLN5\u003c/strong\u003e, short recombinant fibulin-5;\u003cstrong\u003e\u0026nbsp;TGF\u003c/strong\u003e,\u0026nbsp;transforming growth factor; \u003cstrong\u003eTNC\u003c/strong\u003e,\u0026nbsp;tenascin-C;\u003cstrong\u003e\u0026nbsp;VBR\u003c/strong\u003e, ventricle-to-brain ratio;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eWB\u003c/strong\u003e, Western blotting; \u003cstrong\u003ewVBR\u003c/strong\u003e, width of ventricle-to-brain ratio.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Ms. Chiduru Nakamura, Department of Neurosurgery, Mie University Graduate School of Medicine, for providing technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Sanikai Foundation (Grant Number, N/A) to Dr. Nakajima.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflicts of Interest\u003c/p\u003e\n\u003cp\u003eDr. H. Suzuki reported personal fees from Eisai, Kowa, Otsuka and Nxera Pharma, and a research fund from Japan Blood Products Organization and Nxera Pharma outside the submitted work. The other authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization and Design, Yume Suzuki; Methodology, Material Preparation, and Data Collection, Yume Suzuki, Mai Nampei; Data Analyses, Yume Suzuki; Writing \u0026ndash; Original Draft Preparation, Yume Suzuki; Writing \u0026ndash; Review \u0026amp; Editing, Mai Nampei, Fumihiro Kawakita, Hiroki Oinaka, Hideki Nakajima, and Hidenori Suzuki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from this study are available to qualified investigators upon reasonable inquiry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by the Animal Ethics Review Committee of Mie University, and were carried out according to the institution\u0026rsquo;s Guidelines for Animal Experiments (approval number 25-16-4-1).\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMacdonald RL, Schweizer TA (2017) Spontaneous subarachnoid haemorrhage. 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Pharmacol Ther 240: 108220. https://doi.org/10.1016/j.pharmthera.2022.108220\u003c/li\u003e\n\u003cli\u003eDrieu A, Du S, Storck SE, Rustenhoven J, Papadopoulos Z, Dykstra T, Zhong F, Kim K, Blackburn S, Mamuladze T, Harari O, Karch CM, Bateman RJ, Perrin R, Farlow M, Chhatwal J, Hu S, Randolph GJ, Smirnov I, Kipnis J (2022) Parenchymal border macrophages regulate the flow dynamics of the cerebrospinal fluid. Nature 611: 585\u0026ndash;593. https://doi.org/10.1038/s41586-022-05397-3 \u003c/li\u003e\n\u003cli\u003eNakamura T, Lozano PR, Ikeda Y, Iwanaga Y, Hinek A, Minamisawa S, Cheng CF, Kobuke K, Dalton N, Takada Y, Tashiro K, Ross J Jr, Honjo T, Chien KR (2002) Fibulin-5/DANCE is essential for elastogenesis in vivo. 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Stroke 25: 1400\u0026ndash;1404. https://doi.org/10.1161/01.str.25.7.1400 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hydrocephalus, Extracellular matrix protein, Perivascular macrophage, Fibulin, Subarachnoid hemorrhage","lastPublishedDoi":"10.21203/rs.3.rs-6322105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6322105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic hydrocephalus following aneurysmal subarachnoid hemorrhage (SAH) is a complication that can lead to deterioration in neurological status and cognitive impairment. Our recent clinical study reported that a high concentration of plasma fibulin-5 (FBLN5), one of matricellular proteins, was associated with the occurrence of chronic hydrocephalus after SAH. This study aimed to investigate whether and how FBLN5 was associated with hydrocephalus during acute to later phases of SAH in mice. C57BL/6 male mice underwent sham or filament perforation SAH modeling, and vehicle or two dosages (0.01 and 0.1\u0026micro;g) of short or long recombinant FBLN5 (rFBLN5) were randomly administrated by an intracerebroventricular injection. Neurobehavioral tests, measurements of the degree of ventricular enlargement, Western blotting, and immunohistochemical staining were performed to evaluate hydrocephalus 24 and 48 hours after SAH. After SAH, ventricular dilatation did not occur at 24 hours but developed at 48 hours, and both doses of long rFBLN5 with an arginine-glycine-aspartic acid domain suppressed ventricular dilatation at 48 hours after SAH. Long rFBLN5 also decreased phosphorylated p38 in the brain parenchyma and prevented post-SAH increases in perivascular macrophages as well as microglia activation in the brain parenchyma at 48 hours after SAH. Although further research is required to clarify the detailed mechanism, this study demonstrated for the first time that exogenous administration of FBLN5 may have a protective effect against ventricular dilatation after experimental SAH.\u003c/p\u003e","manuscriptTitle":"The Effect of Fibulin-5 on Hydrocephalus After Subarachnoid Hemorrhage in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 12:58:07","doi":"10.21203/rs.3.rs-6322105/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"229d9908-9f32-43c1-8a5f-21df2cf3235b","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-21T20:08:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-30 12:58:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6322105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6322105","identity":"rs-6322105","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}