The PDGFBB-PDGFRβ pathway and Laminins in pericytes are involved in the temporal change of AQP4 polarity during Epileptic Pathogenesis

The PDGFBB-PDGFRβ pathway and Laminins in pericytes are involved in the temporal change of AQP4 polarity during Epileptic Pathogenesis | 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 PDGFBB-PDGFRβ pathway and Laminins in pericytes are involved in the temporal change of AQP4 polarity during Epileptic Pathogenesis Lin Lin, Hongxia Tang, Ke Cui, Zeyi Kang, Tengwei Pan, Changqiang Feng, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6504855/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 Objective: To investigate the temporal changes in AQP4 polarity and pericyte vascularity during the pathological progression of epilepsy, with the goal of identifying potential drug targets or regulatory strategies to delay epilepsy progression and inhibit its onset. Methods: A rat model of chronic epilepsy was established via intraperitoneal injection of pilocarpine. The polarity of AQP4 and the vascular coverage of pericytes were assessed using immunofluorescence. The effects of pharmacological modulation of AQP4 polarity distribution on PTZ-induced seizures were observed. The molecular mechanisms mediating the polar distribution of AQP4 in pericytes were explored through Transwell co-culture and transcriptomics and validated at the cellular protein expression level. Immunofluorescence was employed to examine changes in the mediating molecules during the progression of epilepsy. Additionally, ELISA was used to measure the levels of PDGF-BB in serum and cerebrospinal fluid during the pathological process of epilepsy. Results: The polar distribution of AQP4 and the perivascular localization of pericytes increased rapidly after epileptic model establishment but gradually decreased, reaching their lowest levels in epileptic animals. Trifluoperazine inhibited the acute redistribution of AQP4 and reduced the latency and duration of PTZ-induced seizures, alleviated brain edema. Pericytes did not affect dystrophin-associated protein (DAP) complex components (e.g., α-syntrophin, β-dystroglycan, dystrophin, and agrin) in astrocytes compared to endothelial cells. However, astrocytes significantly enhanced pericyte-derived laminin expression. During epilepsy progression, LAMA1 and LAMA2 expression initially increased and then declined. The levels of PDGF-BB in serum and cerebrospinal fluid gradually decreased after model establishment, reaching their lowest point during epilepsy. Conclusion: The polar distribution of AQP4 plays a crucial role in the development of epilepsy. During the pathological process, AQP4 polarity is largely influenced by pericyte vascular coverage. Key regulators, such as laminins (e.g., LAMA1) and PDGF-BB, are critical for maintaining AQP4 polarity, delaying epileptic pathology, and inhibiting epileptogenesis. pericyte AQP4 polarity epileptogenesis laminins PDGFBB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Epilepsy is a neurological disorder characterized by synchronous abnormal brain discharges that result in behavioral abnormalities. Despite conventional medical therapies, approximately 30% of patients continue to experience uncontrolled seizures[ 1 ]. Common causes of epilepsy include stroke and brain trauma, with a latency period between the initial injury and the onset of epilepsy. Investigating the pathological changes occurring during this latency period and identifying therapeutic targets are crucial for advancing epilepsy management. Aquaporin 4 (AQP4) has been implicated in the pathological processes of epilepsy, with altered polar distribution observed in human epileptic tissue specimens [ 2 , 3 ]. AQP4 co-localizes with Kir4.1, and changes in AQP4 polarity influence the distribution of Kir4.1, which in turn affects extracellular potassium concentrations and seizure susceptibility [ 4 – 6 ]. AQP4 forms orthogonal arrays of particles (OAPs) around blood vessels, a process dependent on dystrophin-associated protein (DAP) complex proteins, including α-syntrophin, dystrophin, and β-dystroglycan within astrocytes, as well as agrin, α-dystroglycan, and laminin extracellularly[ 7 ]. It has been reported that AQP4 is predominantly distributed on the pericyte side of blood vessels rather than on endothelial cells, as shown by immunoelectron microscopy[ 8 , 9 ]. Perivascular pericyte distribution changes during chronic epilepsy[ 10 , 11 ], with reduced vascular coverage and decreased AQP4 polarity observed in spontaneous epileptic animals with pericyte-specific Cdk5 knockout[ 12 ]. However, the relationship between AQP4 polarity and altered perivascular pericyte distribution remains unclear, as do the mechanisms through which pericytes regulate AQP4 polarity. In this study, we dynamically analyzed the polar distribution of AQP4 and pericyte vascular coverage during epileptogenesis, as well as the factors mediating AQP4 polarity through pericytes. Our findings aim to identify potential therapeutic targets for epilepsy prevention and treatment by modulating AQP4 polarity and pericyte vascular distribution. Material and method Pilocarpine induced chronic epilepsy model The modeling method, adapted from reference[ 13 ], was slightly modified. Male Sprague-Dawley rats (300 ± 20 g) were housed in an SPF animal facility at 21–23°C with free access to food and water. After one week of acclimation, the animals received an intraperitoneal injection of 130 mg/kg lithium chloride, followed by a subcutaneous injection of 1 mg/kg scopolamine 18 hours later. After an additional 30 minutes, 30 mg/kg pilocarpine was administered intraperitoneally to induce status epilepticus (SE), which was terminated after 120 minutes with an intraperitoneal injection of 10 mg/kg diazepam. To support recovery, 2.5 mL of 5% glucose solution was administered intragastrically every 12 hours for a total of five doses. All chemicals were purchased from Sigma Aldrich, except diazepam, which was obtained from Tianjin Pharmaceuticals Group Co., Ltd. Criteria for SE: Persistent forelimb spasms lasting over 5 minutes, with interruptions no longer than 2 minutes. The animals were weighed the following day. Those exhibiting SE and a weight loss exceeding 10% were included in the subsequent study. Twenty-one days after model establishment, continuous video monitoring was used to classify animals as epileptic or non-epileptic. Epileptic animals displayed a rough, upright coat, whereas non-epileptic animals had smooth coats similar to the control group. Animals were sampled for immunofluorescence and Western blotting (WB) at the end of SE, 1, 7 and 30 days post-modeling. PTZ Seizure Model and Trifluoperazine Treatment The PTZ-induced seizure model was slightly modified from a previously described protocol[ 14 ]. C57BL/6 mice (25 ± 5 g) were selected for the study. PTZ (60 mg/kg) was administered intraperitoneally, and the trifluoperazine treatment group received an intraperitoneal injection of trifluoperazine (30 mg/kg) simultaneously with PTZ. Seizure latency and duration were observed and recorded for 30 minutes. Seizures were classified according to the Racine scale, with grade 2 seizures representing macroscopically visible epileptic symptoms [ 15 ]. Three hours after PTZ administration, the animals were anesthetized and sacrificed. The brains were harvested, weighed wet, and then dried at 110°C for 24 hours. Brain tissue water content was calculated using the formula: Water Content (%) = [(Wet Weight-Dry Weight)/Wet Weight] × 100%. Immunofluorescence The immunofluorescent assay was conducted as previously described[ 12 ]. Briefly, mice were anesthetized, perfused with PBS and 4% paraformaldehyde, and their brains were extracted. The brains were fixed in 4% paraformaldehyde for 6 hours, transferred to 30% sucrose in PBS, and sectioned at 20 µm using a cryostat (Leica CM1950). Before staining, sections were washed three times with PBS, permeabilized with 1% Triton-X100 for 15 minutes, blocked with donkey serum for 1 hour, and incubated at 4°C for 2 days with the following primary antibodies: AQP4 (MilliporeSigma, A5971), PDGFRβ (Abcam, ab32570), Glut1 (Abcam, ab40084), LAMA1 (Invitrogen, MA1-21194), or LAMA2 (Santa Cruz, Sc-59894). After washing three times with PBS, fluorescent secondary antibodies were applied and incubated in the dark for 4 hours. Sections were then washed again three times with PBS, stained with 5 µM DAPI solution for 15 minutes, washed three more times, mounted with anti-fade reagent, and imaged using confocal laser scanning microscopy (Zeiss LSM800, München, Germany). For the pericyte vascular coverage and LAMA1/LAMA2 assays, Z-stack scanning was performed. In the AQP4 polarity assay, uniform high and low stringency thresholds were applied to all images. Low stringency thresholds defined the overall AQP4 immunoreactivity region, while high stringency thresholds defined AQP4 signals localized to perivascular endfeet. The AQP4 polarity index was calculated as the ratio of fluorescence intensity at high versus low thresholds, with higher values indicating greater polarity distribution[ 16 ]. Pericyte vascular coverage was determined by dividing the PDGFRβ-positive area by the Glut1-positive area in the perivascular zone using Z-projection analysis[ 12 ]. Image analysis was performed with ImageJ software (NIH, USA). PDGF-BB Assay Animals were secured in a stereotaxic apparatus. After separating the neck skin and muscles, the medullary cistern was exposed, and cerebrospinal fluid (CSF) was carefully aspirated using a 33G syringe under a stereomicroscope (RWD Life Science Co., Ltd, model 77001) and transferred to an EP tube. Blood was collected via orbital sampling, and serum was separated by centrifugation at 1000 g for 15 minutes following a 3-hour incubation at room temperature. PDGF-BB levels were measured using an ELISA kit (R&D Systems, MBB00) according to the manufacturer's instructions. Cell Culture Transwell co-culture experiments were performed with HA1800 (Shenzhen Huatuo), HBVP (Zhongqiao Xinzhou Biotechnology Co., Ltd., ZQ0993) and hCMEC (Zhongqiao Xinzhou Biotechnology Co., Ltd., ZQ0961). Six-well transwell plates (Falcon, PET membrane 1.0 µm pore size, Cat. No. FAL-353502/353102) were used with 5◊10 5 cells in the upper and lower layers and 10% FBS in DMEM medium. The Trasnwell inserts were inverted and the lower cells were cultured on the membrane, placed upright into the culture plate 24 hours later, and the upper cells were added for culture, with a transwell insert medium volume of 3 ml. After 48 hours of transwell culture, cells on both sides of the membrane were gently scraped with a cell scraper to extract total protein, membrane protein (ThermoFisher, Mem-PerTM Plus Membrane Protein Extraction Kit), or RNA from upper and lower cells for experiments. Transcriptomics experiment Both the up and down cells of HA1800-HA1800, HA1800-HBVP, HA1800-hCMEC, HBVP-HBVP Transwell chamber (cell before “-” is the up layer, cell after “-” is the lower layer) were collected for high-throughput mRNA sequencing. Total RNA was extracted using TRIzol Reagent (Invitrogen) followed by quality check on a NanoDrop spectrophotometer (Thermo Fisher Scientific), and a 1% agarose gel. Qualified samples, which had an RNA integrity number > 7, were retained for next-generation library preparation according to the manufacturer’s protocol (an NEBNext Ultra RNA Library Prep Kit for Illumina). The resulting RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Takara, RR036A). After that, DNA sequencing of the libraries was performed on an Illumina sequencer, and data were analyzed by GENEWIZ. Western Blot The brain hippocampus and cells were extracted and then homogenized or lysised in lysis buffer containing a protease inhibitor cocktail. The supernatant was collected after centrifugation at 12,000 rpm for 10 min. The cells in the up or down layer of Transwell chamber were collected and the total or membrane protein were extracted according to the manufactory protocol. The protein concentration was determined using the rapid gold BCA protein assay kit (Thermoscientific, Rockford, United States). Equivalent amount of protein was subjected to SDS-PAGE gel (6–10%) and transferred to PVDF membranes (Millipore, Billerica, MA, United States). The blots were probed with anti-AQP4 (CST, 59678), anti-LAMA1 (Novus, NB300-144), LAMA2 antibody (Santa Cruz, Sc-59854), anti-GAPDH (abcam, ab181602), β-actin (Abway, AB0035) or anti-Vinculin (Proteintech, 66305-1-Ig) at 4°C overnight, and then incubated with HRP conjugated secondary antibodies. The proteins were visualized by an enhanced chemiluminescence detection system. The density of protein bands was quantified using ImageJ software (US National Institutes of Health) and normalized to GAPDH, β-actin or vinculin. Results 1. Changes in AQP4 polarity during the pathological process of epilepsy We used pilocarpine to create a rat model of chronic epilepsy and observed changes in AQP4 polarity during the pathological process Three quarters of all modeled animals developed seizures (including dead animals) 30 days after modeling, the success rate of which was relatively high. In order to ensure the occurrence of epilepsy, animals in the incubation period were selected to have a weight loss of more than 20% at 24 hours after modeling, rough coat color and irritable animals at 7 days after modeling. Immunofluorescence results showed that AQP4 polarity in the CA1 region of the hippocampus was significantly increased at 0 days and 1 day after modeling compared with the control group, and the mean value at 7 days after modeling was higher than that of the normal control group, but there was no significant difference (Figs. 1 A, B). AQP4 polarity in epileptic animals at 30 days after model establishment was significantly lower than that at 0 d, 1 d, and 7 d, and the mean value was slightly lower than that in the control group, but there was no significant difference (Fig. 1 A, B). 2. Inhibition of AQP4 Polar Distribution Mitigates PTZ-induced Seizures To investigate the relationship between AQP4 polarity distribution and epilepsy, we investigated the effect of trifluoperazine, a calmodulin antagonist which was reported to be able to inhibit polar distribution of AQP4[ 17 ], on PTZ-induced seizures. The results showed that trifluoperazine was able to prolong PTZ-induced seizure latency (Fig. 2 A), inhibit seizure duration (Fig. 2 B), and simultaneously inhibit brain edema resulting from seizures (Fig. 2 C). PTZ induced an increase in AQP4 polar distribution, whereas trifluoperazine was able to inhibit the increase in AQP4 polar distribution (Fig. 2 D, E). Thus, trifluoperazine suppressed PTZ-induced seizures, which was related to AQP4 polar distribution. AQP4 polarity is involved in seizures. 3. Changes in pericyte vascular coverage during epileptogenesis The results of Z-stack immunofluorescence with PDGFRβ and Glut1 co-staining showed that the vascular coverage rate of pericytes in the hippocampus increased after modeling, but gradually decreased thereafter. The vascular coverage rate of pericytes at 0 d and 1 d after model establishment was significantly higher than that of the normal control group, and at 7 d after model establishment was significantly lower than that at 0 d after model establishment. Pericyte vascular coverage in epileptic animals at 30 days after modeling was significantly lower than that at 0 d and 1 d after modeling, and the mean value was lower than that in normal controls and 7 d after modeling, but there was no significant difference (Fig. 3 A, B). Compared with the results of the second part, pericyte vascular coverage was consistent with changes in AQP4 polarity during epileptogenesis. 4. Changes of PDGF-BB during the pathological process of epilepsy It has been reported that PDGF-BB can promote the distribution of pericytes around blood vessels, and we used ELISA to detect the content of PDGF-BB in serum and cerebrospinal fluid of pilocarpine induced chronic epilepsy rats. The content of PDGF-BB in serum significantly decreased at 7 days after modeling and in epileptic animals at 30days compared with the normal group and 1 day after modeling. The content of PDGF-BB in serum in 0d, 1d, and non-epileptic animals at 30 days showed no difference to normal control (Fig. 3 C). The content of PDGF-BB in cerebrospinal fluid decreased gradually after model establishment, and only epileptic animals at 30 days after modeling were lower than normal controls (Fig. 3 D). Decreased vascularity of pericytes in pilocarpine-induced epileptic animals was associated with gradually decreased levels of PDGF-BB. 5. Pericyte culture enhanced the membrane distribution of AQP4 To investigate whether the distribution of AQP4 was associated with pericyte distribution, we co-cultured astrocyte HA1800, pericyte HBVP and endothelial cell hCMEC with HA1800 in transwells to detect the expression of AQP4 total protein and AQP4 in the cell membrane of upper HA1800 cells. WB results showed that HBVP could promote the increase of AQP4 content on the cell membrane compared with HA1800 and hCMEC (Fig. 4 A, B), but had no effect on the total amount of AQP4 (Fig. 4 C, D). Thus, pericyte distribution promotes membrane distribution of AQP4. 6. Pericyte laminins may mediate AQP4 polarity To search for factors mediating AQP4 distribution in pericytes, we performed transwell experiment with HA1800, HBVP, hCMEC and HA1800, HBVP and HBVP, and collected cells for transcriptomic sequencing analysis after cell culture (Fig. 5 ). Transcriptome results showed that pericyte co-culture resulted in decreased transcription of 470 genes and increased transcription of 179 genes in astrocytes (Fig. 5 B). Endothelial cell co-culture resulted in decreased expression of 861 genes and increased transcription of 699 genes in astrocytes (Fig. 5 C). Endothelial cells co-culture resulted in down-regulation of 230 genes and up-regulation of 164 genes in astrocytes compared to pericytes co-culture (Fig. 5 D). In parallel, astrocyte cultures increased pericyte transcription of 1,185 genes and decreased transcription of 1,733 genes (Fig. 5 E). By GO analysis, collagen-containing extracellular matrix was found to be involved in the transcriptional changes of genes in pericytes, endothelial cells and astrocytes co-culture (Fig. 5 F-I), so we performed a specific analysis of this GO genes. The result showed that: (1) AQP4 and its polarity-associated proteins, such as α-syntrophin (SNTA1), agrin (AGRN), dystrophin (DMD, dystrophin-protein71, DP-71), β-dystroglycan (DAG1), and α-dystrobrevin (DTNA) in astrocyte were not changed after co-cultured with pericyte compared to endothelial cells, while IL-33 (Cytosolic DNA-sensing pathway), ICAM1, CDH5, ITGB3, and LAMC2 were decreased and PDGFB was increased (Supplementary material Table 1-1.1). (2) Astrocyte co-culture induced increased LAMA1, LAMA2, PDGFRB, PDGFB, and LAMC1, and decreased LAMB3, LAMC2, LAMA3, LAMC3, agrin (AGRN) in pericyte compared to endothelial cells (Supplementary material Table 1-1.2). (3) Pericyte transcripted higher PDGFRB, PDGFB, LAMA2, LAMA1, LAMC1, LAMB1, LAMC2, and LAMA3 and lower LAMA5, LAMB3, and agrin (AGRN) compared to astrocyte after astrocyte co-culture (Supplementary material Table 1-1.3). Transcriptome results indicated that pericytes’ laminins might be involved in astrocyte AQP4 distribution and we performed WB validation on co-cultured cells. The results showed that pericyte LAMA1 levels were significantly higher than endothelial cells, and the levels of pericyte LAMA1 were significantly increased after co-culture with astrocytes (Fig. 6 A, B). LAMA2 also showed the same changes (Fig. 7 A, B), which is consistent with the results of transcriptomics. 7. Changes of laminins during epileptogenesis To investigate whether laminins are associated with altered AQP4 polarity during epilepsy progression, we performed temporal immunofluorescence detection of laminins in hippocampal tissues. The results revealed that hippocampal LAMA1 levels increased significantly immediately after sustained epilepsy, then gradually decreased. One day after model establishment, LAMA1 levels remained elevated compared to the control group but decreased significantly by day 7, returning to control levels. At 30 days, LAMA1 levels were significantly lower than those observed immediately after sustained epilepsy and on day 1 (Fig. 6 C-E). In contrast, LAMA2 levels did not increase immediately after epilepsy but showed significant elevation at days 1 and 7, returning to control levels by day 30 (Fig. 7 C-E). These trends paralleled changes in AQP4 polarity and pericyte vascular coverage. While LAMA1 and LAMA2 are not exclusively expressed in pericytes, their expression around pericytes increased proportionally with their overall expression levels. (Figs. 6 E, 7 E). Discussion This study investigated whether AQP4 polarity during the epileptic pathological process is influenced by the vascular distribution of pericytes, focusing on temporal changes. The findings revealed that alterations in AQP4 polarity closely align with the vascular coverage of pericytes in epilepsy. Furthermore, LAMA1 and LAMA2 laminins in pericytes were shown to influence AQP4 distribution in astrocytes. Changes in AQP4 polarity during epilepsy were also correlated with laminin expression. To our knowledge, this is the first study to identify a link between astrocyte AQP4 polarity and pericyte vascularization during epilepsy, highlighting the potential regulatory role of pericyte vascular coverage and laminins in this process. Nearly 30% of epilepsy patients are unresponsive to medications[ 1 ], highlighting the importance of early intervention in high-risk individuals before symptom onset. Understanding the pathophysiologic changes during epileptogenesis is crucial to identifying potential therapeutic targets, drugs, or modulators that can inhibit this process[ 18 ]. While neuronal damage is a key focus, the pathological roles of non-neuronal elements, including astrocytes, vascular neural units, and other cell types, also warrant significant attention[ 19 ]. The astrocytic water channel protein AQP4 plays a critical role in epileptic pathogenesis[ 2 ]. Modulating AQP4 may serve as an effective therapeutic target for epilepsy. AQP4 co-localizes with Kir4.1[ 4 ], and alterations in its polarized distribution influence potassium (K + ) ion homeostasis, a key factor in epileptogenesis. In the hippocampus, astrocytic AQP4 and Kir4.1 cooperate in K + regulation, and depletion of the perivascular AQP4 pool slows K + clearance, exacerbating seizure severity[ 5 ]. Our experiments demonstrated that disrupting AQP4 polarization during acute seizures could mitigate seizure activity. In human epileptic hippocampal tissues, AQP4 polarization is significantly reduced compared to normal tissues[ 3 , 20 ]. Notably, our results found that, in epileptic animals, AQP4 polarization initially increased during the early stages of acute injury but decreased progressively, reaching its lowest levels in chronic epilepsy. To our knowledge, this is the first study to explore the dynamics of AQP4 polarization throughout the epileptic process. These findings underscore the need to consider the stage of epilepsy when targeting AQP4 polarization for therapeutic intervention. Based on these results, the search for regulators of AQP4 polarity distribution may offer a promising strategy to delay epileptogenesis. Immunoelectron microscopy has revealed that AQP4 distribution is more pronounced around pericytes than endothelial cells, with pericytes mediating AQP4's polar distribution[ 8 , 9 ]. Consistently, the present study found that co-culture with pericytes significantly increased AQP4 expression on astrocyte membranes. In Pdgfb ret/ret transgenic mice [ 21 ], pericyte LAMC1-knockout animals[ 22 ], and pericyte Cdk5 knockout spontaneously epileptic mice[ 12 ], both AQP4 polarity distribution and pericyte vascular coverage were concurrently reduced, underscoring a potential correlation. However, the temporal dynamics of these changes during the pathological progression of epilepsy remain unexplored. In this study, pericyte vascular coverage increased in the early stages after SE injury and progressively decreased as epilepsy advanced, with the lowest expression observed in epileptic animals. Consistent with our findings, previous research reported increased pericyte vascular coverage in PDGFRβ + cells during the acute phase after SE[ 10 ]. However, decreased vascular coverage was noted in NG2-positive cells [ 10 , 11 ]. This discrepancy may be due to the distinct roles of NG2 and PDGFRβ in characterizing vascular mural cells, as NG2 primarily marks oligodendrocyte precursor cells[ 23 ], whereas PDGFRβ preferentially identifies pericyte cells[ 24 ], despite also being expressed in non-vascular brain parenchymal cells. In human brain tissue with chronic temporal lobe epilepsy, one study reported individual variability in PDGFRβ vascular distribution [ 10 ]. Another study observed an increased number of blood vessels fully encapsulated by PDGFRβ-positive cells, alongside a decrease in incompletely encapsulated vessels, indicating increased pericyte coverage in epileptic tissues[ 25 ]. However, this study focused on the cerebral white matter of the temporal gyrus, differing from our investigation of the hippocampal region, which may explain the discrepancies. In summary, our study unraveled the dynamic changed of vascular coverage of PDGFRβ-positive pericytes in the hippocampal tissue of rats with epilepsy progression. Notably, the perivascular distribution of pericytes closely paralleled changes in AQP4 polarity during epilepsy progression. This suggests that pericytes may play a critical role in regulating AQP4 polarity distribution in astrocytes. The polar distribution of AQP4 is influenced by DAP complex proteins, including β-DG, syntrophin, and extracellular matrix components[ 7 , 26 ]. Using Transwell transcriptomics, we observed no changes in the expression of AQP4, α-syntrophin, agrin, dystrophin, β-dystroglycan, or α-dystrobrevin in astrocytes co-cultured with pericytes compared to endothelial cells. In line with single-cell sequencing data[ 27 ], we found significantly higher expression of laminins, including LAMA1, LAMA2, and LAMC1, in pericytes than in endothelial cells. Co-culture with astrocytes increased LAMA1 and LAMA2 expression in pericytes, suggesting that laminins like LAMA1 in pericytes may play a key role in AQP4 distribution. This is evidenced by the fact that the interaction between laminins and dystroglycan is critical for maintaining the OAPs and polarity of AQP4[ 7 , 26 ], and knockout of LAMC1 in pericytes reduces AQP4 polarity distribution[ 22 ]. In this study, the levels of LAMA1 and LAMA2 increase in response to seizures but decrease in epileptic animals. Following KA or 3-NPA treatment, LAMA1 expression in vascular elements increases[ 28 ]. In pilocarpine-induced epilepsy, laminin expression rises on days 3 and 4 post-SE but normalizes by day 30[ 29 ], consistent with our findings. Thus, expression patterns of laminin align with AQP4 distribution and pericyte vascular coverage during epilepsy progression, and stabilizing extracellular matrix components like LAMA1 in pericytes could help delay epilepsy progression[ 30 ]. Matrix metalloproteinases (MMPs) are known to regulate neurovascular extracellular matrix stability and are involved in post-traumatic epileptogenesis, with MMP inhibitors reducing epileptogenesis[ 31 – 33 ]. Notably, pericytes release the most MMP9 upon thrombin stimulation compared to endothelial cells and astrocytes[ 34 ], highlighting the potential role of pericyte-derived MMP9 in stabilizing extracellular matrix components like laminins and AQP4 polarity. PDGF-BB/PDGFRβ signaling is crucial for vascularization by mediating pericyte recruitment to the vasculature, thereby promoting vessel integrity and function[ 35 ]. Previous studies have shown that exogenous PDGF-BB, administered 1–14 days post-KA injury, can maintain pericyte distribution and inhibit epileptogenesis[ 11 ]. However, the dynamics of PDGF-BB during epilepsy remain unclear. In this study, we are the first to examine the changes in PDGF-BB from injury to seizure, finding that its levels gradually decrease in both blood and cerebrospinal fluid as epilepsy progresses. These findings suggest that increasing PDGF-BB during the latency period after injury could potentially inhibit epileptogenesis. Conclusion The vascular distribution of pericytes plays a key role in the polarization of AQP4 during epilepsy pathology. Stabilizing extracellular matrix proteins of pericytes, such as LAMA1 and LAMA2, and administering PDGF-BB post-injury to preserve pericyte distribution around blood vessels and stabilize AQP4 polarity could be a crucial strategy for slowing epilepsy progression. Declarations Ethics statement The animal study was approved by the Committee for Animal Experiments at Taizhou Hospital of Zhejiang Province in China (No. K20190108). The study was conducted in accordance with the local legislation and institutional requirements. Funding This study was in part supported by National Natural Science Foundation of China (No. 81903584 to Gang Wu), Zhejiang Provincial Basic and Public Welfare Research Program (No. LGD20H310002 to Gang Wu), Taizhou Science and Technology Plan Project (24ywa32 to Gang Wu), Taizhou Enze Medical Center (Group) Major Project (No.2022EZZD05 to Gang Wu). Author Contribution Lin Lin: Investigation, Methodology, Visualization, Writing –original draft. Hongxia Tang: Investigation, Methodology, Visualization, Writing –original draft. Ke Cui: Resources, Writing – review & editing. Zeyi Kang: Investigation, Writing – original draft, Writing –review & editing. Tengwei Pan: Investigation, Writing – review & editing. Changqiang Feng: Resources,Writing – review & editing. XiaoHong Zhao: Investigation, Writing – original draft, Writing – review & editing. Jiewei Wang: Investigation, Writing – review & editing. Zhiyuan Chen: Investigation, Writing – review & editing. Zhengli Jiang: Resources, Writing – review & editing. Gang Wu: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review & editing. Acknowledgement We would like to thank Zhenyu Yang, at Public Scientific Research Platform, Taizhou Hospital of Zhejiang Province for the excellent technical assistance. Data Availability Sequence data that support the findings of this study have been deposited in the European Nucleotide Archive with the primary accession code PRJEB88623. References Krishnamurthy KB (2016) Epilepsy. Annals of Internal Medicine 164:ITC17 Binder DK, Nagelhus EA, Ottersen OP (2012) Aquaporin-4 and epilepsy. Glia 60:1203–1214 Eid T, Lee TS, Thomas MJ, Amiry-Moghaddam M, Bjørnsen LP, Spencer DD, Agre P, Ottersen OP, de Lanerolle NC (2005) Loss of perivascular aquaporin 4 may underlie deficient water and K + homeostasis in the human epileptogenic hippocampus. 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Brain Resrarch 1352:239–247 Kim YJ, Kim JY, Ko AR, Kang TC (2014) Over-expression of laminin correlates to recovery of vasogenic edema following status epilepticus. Neuroscience 275:146–161 Pitkänen A, Ndodeekane XE, Lukasiuk K, Wilczynski GM, Dityatev A, Walker MC, Chabrol E, Dedeurwaerdere S, Vazquez N, Powell EM (2014) Neural ECM and epilepsy. Prog Brain Res 214:229–262 Ikonomidou C (2014) Matrix metalloproteinases and epileptogenesis. Mol Cell Pediatr 1:1–6 Pijet B, Stefaniuk M, Kostrzewskaksiezyk A, Tsilibary PE, Tzinia A, Kaczmarek L (2018) Elevation of MMP-9 Levels Promotes Epileptogenesis After Traumatic Brain Injury. Mol Neurobiol 55:1–13 Jayakumar AR, Apeksha A, Norenberg MD (2016) Role of Matricellular Proteins in Disorders of the Central Nervous System. Neurochem Res 42:1–18 Machida T, Takata F, Matsumoto J, Takenoshita H, Kimura I, Yamauchi A, Dohgu S, Kataoka Y (2015) Brain pericytes are the most thrombin-sensitive matrix metalloproteinase-9-releasing cell type constituting the blood–brain barrier in vitro. Neurosci Lett 599:109–114 Tsioumpekou M, Cunha SI, Ma H, Åhgren A, Cedervall J, Olsson AK, Heldin CH, Lennartsson J (2020) Specific targeting of PDGFRβ in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression. Theranostics 10:1122–1135 Supplementary Table Supplementary Tables are not available with this version. Additional Declarations No competing interests reported. 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-6504855","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448264371,"identity":"847505c9-dcee-4644-89c5-07d29b03a9ab","order_by":0,"name":"Lin Lin","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Lin","suffix":""},{"id":448264372,"identity":"71f7b415-151d-4ecb-ba87-26907fdf3c7b","order_by":1,"name":"Hongxia Tang","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongxia","middleName":"","lastName":"Tang","suffix":""},{"id":448264373,"identity":"8b3410ed-992a-42c8-9444-01aa90bd8405","order_by":2,"name":"Ke Cui","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Cui","suffix":""},{"id":448264374,"identity":"1404fe8d-5523-45f9-b156-d5d52b6b4382","order_by":3,"name":"Zeyi Kang","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zeyi","middleName":"","lastName":"Kang","suffix":""},{"id":448264377,"identity":"f102d2c5-1954-443a-8ba8-972f4a2a6592","order_by":4,"name":"Tengwei Pan","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tengwei","middleName":"","lastName":"Pan","suffix":""},{"id":448264378,"identity":"ed590f55-8dde-4fc9-9be7-03e2ace66f52","order_by":5,"name":"Changqiang Feng","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changqiang","middleName":"","lastName":"Feng","suffix":""},{"id":448264383,"identity":"44a3b166-6a25-462d-8e9d-57978f244c87","order_by":6,"name":"Xiaohong Zhao","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohong","middleName":"","lastName":"Zhao","suffix":""},{"id":448264384,"identity":"4d2e6d0c-f043-4873-a3b1-c352f8847dac","order_by":7,"name":"Jiewei Wang","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiewei","middleName":"","lastName":"Wang","suffix":""},{"id":448264385,"identity":"bb534465-df46-43de-87e1-74164d615702","order_by":8,"name":"Zhiyuan Chen","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhiyuan","middleName":"","lastName":"Chen","suffix":""},{"id":448264388,"identity":"947bcef7-9a14-47ec-91e0-1f62919c7f5b","order_by":9,"name":"Zhengli Jiang","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhengli","middleName":"","lastName":"Jiang","suffix":""},{"id":448264391,"identity":"6a4cd61e-ab04-45b5-8a6e-1bae3e56cb3d","order_by":10,"name":"Gang Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFACxgZmOPuDgY0cCVrYgMwZBWnGRNkD18LM8+FwIkHlBsebGz8XVNxhkJ/f/OyzjQFzAgP74aMb8Go5c7BZesaZZwwGx9iMZ+cYsOUx8KSl3cCnxexGYhszb9thBgM2BmPmHAOeYgYJHjP8Wu4/BGr5d5hBvo39M7OFgURiA0EtNxiBWhoOMzAc4zFmZjAwIKzF/kxiszTPMaDDjuUUM/YYJBizEfKLZPvxh595aoAOaz6+meHHn/9y/OyHj+HVAgP1DTAWGzHKR8EoGAWjYBTgBwB09UT1nJk75QAAAABJRU5ErkJggg==","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Gang","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-04-22 13:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6504855/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6504855/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81793407,"identity":"2dabb063-8793-4e85-9749-f9bbdccfbd9b","added_by":"auto","created_at":"2025-05-02 02:27:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3854069,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Quantitative analysis of AQP4 polarity in control group (CG), 0d, 1d, 7d, 30d after SE. n=12, 3 rats per group, 4 slice per rat, ***, p \u0026lt;0.001. (B) Representative immunofluorescence images (Red: AQP4, Blue: DAPI) and gray images of AQP4 at high and low stringency to calculate the AQP4 polarity. Scale bar: 40μm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6504855/v1/b951ba3e60fe8e76fcb2113c.png"},{"id":81793409,"identity":"cf4c7713-d824-450b-aad5-bd20f375c52a","added_by":"auto","created_at":"2025-05-02 02:27:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3201722,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the AQP4 polarity distribution inhibitor trifluoperazine on PTZ-induced seizures:\u003c/p\u003e\n\u003cp\u003e(A) Seizure latency. (B) Seizure duration (*p \u0026lt; 0.05, n = 6 per group). (C) Brain edema 3 hours after PTZ injection (*p \u0026lt; 0.05, **p \u0026lt; 0.01, n = 3 per group). (D) Statistical analysis of hippocampal AQP4 polarity distribution across groups (**p \u0026lt; 0.01, n = 3 animals per group, 2 slices per animal).\u003c/p\u003e\n\u003cp\u003e(E) Representative immunofluorescence images: Red, AQP4; Green, Glut1; Blue, DAPI. Scale bar: 40μm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6504855/v1/389fd955c53f2d90ed49f999.png"},{"id":81793413,"identity":"f5015f4f-534a-407e-9190-f0d879ad315e","added_by":"auto","created_at":"2025-05-02 02:27:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8305544,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in pericyte vascular coverage and PDGF-BB concentration in chronic epilepsy rats induced by pilocarpine. (A) Representative immunofluorescence images of pericyte vascular coverage: Red, Pdgfrβ; Green, Glut1; Blue, DAPI. Scale bar: 50 μm. Yellow arrows indicate pericytes surrounding blood vessels. (B) Statistical analysis of pericyte vascular coverage in the hippocampal CA1 region. n = 12, 3 animals per group, 2 brain slices per animal, 2 hippocampal regions. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001. (C) PDGF-BB concentration in serum. n = 8 for control group(CG), 0d, 1d, 7d; n = 6 for 30d with or without epilepsy. * p \u0026lt; 0.05, ** p \u0026lt; 0.01. (D) PDGF-BB concentration in cerebrospinal fluid. n = 6 for control group, 0d, 1d, 30d with epilepsy; n = 7 for 7d; n = 5 for 30d non-epilepsy. * p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6504855/v1/df4d94f84246f3f6a04fe8f8.png"},{"id":81793552,"identity":"61672daf-fd18-4e28-9060-7bdc47457b5a","added_by":"auto","created_at":"2025-05-02 02:35:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":692161,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pericyte co-culture on astrocyte membrane AQP4 levels. HA1800, HBVP, and hCMEC were Transwell co-cultured with HA1800 cells. AQP4 levels in the membrane of upper layer of HA1800 cell: (A) Representative WB images, (B) Statistical analysis, n = 3, * p \u0026lt; 0.05. The total AQP4 levels in the upper layer of HA1800 cells: (C) Representative WB images, (D) Statistical analysis, n = 3.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6504855/v1/f7e1f5933bff0f1b21ac2f7f.png"},{"id":81793410,"identity":"5d1752f9-f29e-456b-a887-60fca205cdfd","added_by":"auto","created_at":"2025-05-02 02:27:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1655892,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic analysis of lower and upper layer cells after Transwell co-culture of HA1800, HBVP, hCMEC with HA1800, and HBVP with HBVP cells. Each group contains four specimens. (A) Principal component analysis plot. (B) Volcano plot of differentially expressed genes (DEG) comparing upper layer HA1800 in HA1800-HBVP to HA1800-HA1800 co-culture. (C) Volcano plot of DEG comparing upper layer HA1800 in HA1800-hCMEC to HA1800-HA1800 co-culture. (D) Volcano plot of DEG comparing upper layer HA1800 in HA1800-HBVP to HA1800-hCMEC co-culture. (E) Volcano plot of DEG comparing lower layer HBVP in HA1800-HBVP to HBVP-HBVP co-culture. (F-I) GO analysis of DEG in (B-E) respectively.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6504855/v1/546ea45dc24f246747abf262.png"},{"id":81793424,"identity":"671448e3-30e0-47b6-9f2d-8257b53ef434","added_by":"auto","created_at":"2025-05-02 02:27:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8562034,"visible":true,"origin":"","legend":"\u003cp\u003eWB analysis of LAMA1 in lower layer HBVP and hCMEC cells after Transwell co-culture of HA1800-HBVP, HBVP-HBVP, HA1800-hCMEC, and hCMEC-hCMEC: Representative WB image (A) and statistical analysis (B) of LAMA1 expression, n= 3, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001. Changes in hippocampal LAMA1 in chronic epilepsy rats induced by pilocarpine: representative WB image (C) and statistical analysis (D), n = 3 animals per group, * p \u0026lt; 0.05, ** p \u0026lt; 0.01. (E) Representative immunofluorescence images of LAMA1 in each group. Red, LAMA1; Green, PDGFRβ; Blue, DAPI. Yellow arrows indicate LAMA1 localized surrounding pericytes. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6504855/v1/60c2bd2adf57c194f3a266a2.png"},{"id":81793415,"identity":"19bffd9d-4a18-4aaa-855b-c163e19edb78","added_by":"auto","created_at":"2025-05-02 02:27:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7856830,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of LAMA2 expression in Transwell co-cultures and hippocampal tissue from chronic epilepsy rats: (A) Representative WB image and (B) statistical analysis of LAMA2 expression in lower-layer HBVP and hCMEC following Transwell co-culture of HA1800-HBVP, HBVP-HBVP, HA1800-hCMEC, and hCMEC-hCMEC (n = 3, *p \u0026lt; 0.05, ***p \u0026lt; 0.001). (C) Representative WB image and (D) statistical analysis of hippocampal LAMA2 expression in chronic epilepsy rats induced by pilocarpine (n = 3 animals per group, *p \u0026lt; 0.05). (E) Representative immunofluorescence images of LAMA2 expression in each group. Red: LAMA2, Green: PDGFRβ, Blue: DAPI. Yellow arrows highlight LAMA2 localization around pericytes.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6504855/v1/3c34581fb03e698071a435b5.png"},{"id":82523269,"identity":"57572029-c814-46d9-b8d5-057856e5a4cf","added_by":"auto","created_at":"2025-05-12 13:17:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30796624,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6504855/v1/3dd173a1-a4ef-449d-be9d-19a3096f0491.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The PDGFBB-PDGFRβ pathway and Laminins in pericytes are involved in the temporal change of AQP4 polarity during Epileptic Pathogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy is a neurological disorder characterized by synchronous abnormal brain discharges that result in behavioral abnormalities. Despite conventional medical therapies, approximately 30% of patients continue to experience uncontrolled seizures[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Common causes of epilepsy include stroke and brain trauma, with a latency period between the initial injury and the onset of epilepsy. Investigating the pathological changes occurring during this latency period and identifying therapeutic targets are crucial for advancing epilepsy management.\u003c/p\u003e \u003cp\u003eAquaporin 4 (AQP4) has been implicated in the pathological processes of epilepsy, with altered polar distribution observed in human epileptic tissue specimens [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. AQP4 co-localizes with Kir4.1, and changes in AQP4 polarity influence the distribution of Kir4.1, which in turn affects extracellular potassium concentrations and seizure susceptibility [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. AQP4 forms orthogonal arrays of particles (OAPs) around blood vessels, a process dependent on dystrophin-associated protein (DAP) complex proteins, including α-syntrophin, dystrophin, and β-dystroglycan within astrocytes, as well as agrin, α-dystroglycan, and laminin extracellularly[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been reported that AQP4 is predominantly distributed on the pericyte side of blood vessels rather than on endothelial cells, as shown by immunoelectron microscopy[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Perivascular pericyte distribution changes during chronic epilepsy[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], with reduced vascular coverage and decreased AQP4 polarity observed in spontaneous epileptic animals with pericyte-specific Cdk5 knockout[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the relationship between AQP4 polarity and altered perivascular pericyte distribution remains unclear, as do the mechanisms through which pericytes regulate AQP4 polarity.\u003c/p\u003e \u003cp\u003eIn this study, we dynamically analyzed the polar distribution of AQP4 and pericyte vascular coverage during epileptogenesis, as well as the factors mediating AQP4 polarity through pericytes. Our findings aim to identify potential therapeutic targets for epilepsy prevention and treatment by modulating AQP4 polarity and pericyte vascular distribution.\u003c/p\u003e"},{"header":"Material and method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePilocarpine induced chronic epilepsy model\u003c/h2\u003e \u003cp\u003eThe modeling method, adapted from reference[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], was slightly modified. Male Sprague-Dawley rats (300\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were housed in an SPF animal facility at 21\u0026ndash;23\u0026deg;C with free access to food and water. After one week of acclimation, the animals received an intraperitoneal injection of 130 mg/kg lithium chloride, followed by a subcutaneous injection of 1 mg/kg scopolamine 18 hours later. After an additional 30 minutes, 30 mg/kg pilocarpine was administered intraperitoneally to induce status epilepticus (SE), which was terminated after 120 minutes with an intraperitoneal injection of 10 mg/kg diazepam. To support recovery, 2.5 mL of 5% glucose solution was administered intragastrically every 12 hours for a total of five doses. All chemicals were purchased from Sigma Aldrich, except diazepam, which was obtained from Tianjin Pharmaceuticals Group Co., Ltd.\u003c/p\u003e \u003cp\u003eCriteria for SE: Persistent forelimb spasms lasting over 5 minutes, with interruptions no longer than 2 minutes. The animals were weighed the following day. Those exhibiting SE and a weight loss exceeding 10% were included in the subsequent study. Twenty-one days after model establishment, continuous video monitoring was used to classify animals as epileptic or non-epileptic. Epileptic animals displayed a rough, upright coat, whereas non-epileptic animals had smooth coats similar to the control group. Animals were sampled for immunofluorescence and Western blotting (WB) at the end of SE, 1, 7 and 30 days post-modeling.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePTZ Seizure Model and Trifluoperazine Treatment\u003c/h3\u003e\n\u003cp\u003eThe PTZ-induced seizure model was slightly modified from a previously described protocol[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. C57BL/6 mice (25\u0026thinsp;\u0026plusmn;\u0026thinsp;5 g) were selected for the study. PTZ (60 mg/kg) was administered intraperitoneally, and the trifluoperazine treatment group received an intraperitoneal injection of trifluoperazine (30 mg/kg) simultaneously with PTZ. Seizure latency and duration were observed and recorded for 30 minutes. Seizures were classified according to the Racine scale, with grade 2 seizures representing macroscopically visible epileptic symptoms [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Three hours after PTZ administration, the animals were anesthetized and sacrificed. The brains were harvested, weighed wet, and then dried at 110\u0026deg;C for 24 hours. Brain tissue water content was calculated using the formula: Water Content (%) = [(Wet Weight-Dry Weight)/Wet Weight] \u0026times; 100%.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eThe immunofluorescent assay was conducted as previously described[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Briefly, mice were anesthetized, perfused with PBS and 4% paraformaldehyde, and their brains were extracted. The brains were fixed in 4% paraformaldehyde for 6 hours, transferred to 30% sucrose in PBS, and sectioned at 20 \u0026micro;m using a cryostat (Leica CM1950).\u003c/p\u003e \u003cp\u003eBefore staining, sections were washed three times with PBS, permeabilized with 1% Triton-X100 for 15 minutes, blocked with donkey serum for 1 hour, and incubated at 4\u0026deg;C for 2 days with the following primary antibodies: AQP4 (MilliporeSigma, A5971), PDGFRβ (Abcam, ab32570), Glut1 (Abcam, ab40084), LAMA1 (Invitrogen, MA1-21194), or LAMA2 (Santa Cruz, Sc-59894). After washing three times with PBS, fluorescent secondary antibodies were applied and incubated in the dark for 4 hours. Sections were then washed again three times with PBS, stained with 5 \u0026micro;M DAPI solution for 15 minutes, washed three more times, mounted with anti-fade reagent, and imaged using confocal laser scanning microscopy (Zeiss LSM800, M\u0026uuml;nchen, Germany).\u003c/p\u003e \u003cp\u003eFor the pericyte vascular coverage and LAMA1/LAMA2 assays, Z-stack scanning was performed. In the AQP4 polarity assay, uniform high and low stringency thresholds were applied to all images. Low stringency thresholds defined the overall AQP4 immunoreactivity region, while high stringency thresholds defined AQP4 signals localized to perivascular endfeet. The AQP4 polarity index was calculated as the ratio of fluorescence intensity at high versus low thresholds, with higher values indicating greater polarity distribution[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Pericyte vascular coverage was determined by dividing the PDGFRβ-positive area by the Glut1-positive area in the perivascular zone using Z-projection analysis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Image analysis was performed with ImageJ software (NIH, USA).\u003c/p\u003e\n\u003ch3\u003ePDGF-BB Assay\u003c/h3\u003e\n\u003cp\u003eAnimals were secured in a stereotaxic apparatus. After separating the neck skin and muscles, the medullary cistern was exposed, and cerebrospinal fluid (CSF) was carefully aspirated using a 33G syringe under a stereomicroscope (RWD Life Science Co., Ltd, model 77001) and transferred to an EP tube. Blood was collected via orbital sampling, and serum was separated by centrifugation at 1000 g for 15 minutes following a 3-hour incubation at room temperature. PDGF-BB levels were measured using an ELISA kit (R\u0026amp;D Systems, MBB00) according to the manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eTranswell co-culture experiments were performed with HA1800 (Shenzhen Huatuo), HBVP (Zhongqiao Xinzhou Biotechnology Co., Ltd., ZQ0993) and hCMEC (Zhongqiao Xinzhou Biotechnology Co., Ltd., ZQ0961). Six-well transwell plates (Falcon, PET membrane 1.0 \u0026micro;m pore size, Cat. No. FAL-353502/353102) were used with 5\u0026loz;10\u003csup\u003e5\u003c/sup\u003e cells in the upper and lower layers and 10% FBS in DMEM medium. The Trasnwell inserts were inverted and the lower cells were cultured on the membrane, placed upright into the culture plate 24 hours later, and the upper cells were added for culture, with a transwell insert medium volume of 3 ml. After 48 hours of transwell culture, cells on both sides of the membrane were gently scraped with a cell scraper to extract total protein, membrane protein (ThermoFisher, Mem-PerTM Plus Membrane Protein Extraction Kit), or RNA from upper and lower cells for experiments.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomics experiment\u003c/h2\u003e \u003cp\u003eBoth the up and down cells of HA1800-HA1800, HA1800-HBVP, HA1800-hCMEC, HBVP-HBVP Transwell chamber (cell before \u0026ldquo;-\u0026rdquo; is the up layer, cell after \u0026ldquo;-\u0026rdquo; is the lower layer) were collected for high-throughput mRNA sequencing. Total RNA was extracted using TRIzol Reagent (Invitrogen) followed by quality check on a NanoDrop spectrophotometer (Thermo Fisher Scientific), and a 1% agarose gel. Qualified samples, which had an RNA integrity number\u0026thinsp;\u0026gt;\u0026thinsp;7, were retained for next-generation library preparation according to the manufacturer\u0026rsquo;s protocol (an NEBNext Ultra RNA Library Prep Kit for Illumina). The resulting RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Takara, RR036A). After that, DNA sequencing of the libraries was performed on an Illumina sequencer, and data were analyzed by GENEWIZ.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eThe brain hippocampus and cells were extracted and then homogenized or lysised in lysis buffer containing a protease inhibitor cocktail. The supernatant was collected after centrifugation at 12,000 rpm for 10 min. The cells in the up or down layer of Transwell chamber were collected and the total or membrane protein were extracted according to the manufactory protocol. The protein concentration was determined using the rapid gold BCA protein assay kit (Thermoscientific, Rockford, United States). Equivalent amount of protein was subjected to SDS-PAGE gel (6\u0026ndash;10%) and transferred to PVDF membranes (Millipore, Billerica, MA, United States). The blots were probed with anti-AQP4 (CST, 59678), anti-LAMA1 (Novus, NB300-144), LAMA2 antibody (Santa Cruz, Sc-59854), anti-GAPDH (abcam, ab181602), β-actin (Abway, AB0035) or anti-Vinculin (Proteintech, 66305-1-Ig) at 4\u0026deg;C overnight, and then incubated with HRP conjugated secondary antibodies. The proteins were visualized by an enhanced chemiluminescence detection system. The density of protein bands was quantified using ImageJ software (US National Institutes of Health) and normalized to GAPDH, β-actin or vinculin.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e1. Changes in AQP4 polarity during the pathological process of epilepsy\u003c/h2\u003e\n\u003cp\u003eWe used pilocarpine to create a rat model of chronic epilepsy and observed changes in AQP4 polarity during the pathological process Three quarters of all modeled animals developed seizures (including dead animals) 30 days after modeling, the success rate of which was relatively high. In order to ensure the occurrence of epilepsy, animals in the incubation period were selected to have a weight loss of more than 20% at 24 hours after modeling, rough coat color and irritable animals at 7 days after modeling. Immunofluorescence results showed that AQP4 polarity in the CA1 region of the hippocampus was significantly increased at 0 days and 1 day after modeling compared with the control group, and the mean value at 7 days after modeling was higher than that of the normal control group, but there was no significant difference (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). AQP4 polarity in epileptic animals at 30 days after model establishment was significantly lower than that at 0 d, 1 d, and 7 d, and the mean value was slightly lower than that in the control group, but there was no significant difference (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e2. Inhibition of AQP4 Polar Distribution Mitigates PTZ-induced Seizures\u003c/h2\u003e\n\u003cp\u003eTo investigate the relationship between AQP4 polarity distribution and epilepsy, we investigated the effect of trifluoperazine, a calmodulin antagonist which was reported to be able to inhibit polar distribution of AQP4[\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e], on PTZ-induced seizures. The results showed that trifluoperazine was able to prolong PTZ-induced seizure latency (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), inhibit seizure duration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), and simultaneously inhibit brain edema resulting from seizures (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). PTZ induced an increase in AQP4 polar distribution, whereas trifluoperazine was able to inhibit the increase in AQP4 polar distribution (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Thus, trifluoperazine suppressed PTZ-induced seizures, which was related to AQP4 polar distribution. AQP4 polarity is involved in seizures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3. Changes in pericyte vascular coverage during epileptogenesis\u003c/h2\u003e\n\u003cp\u003eThe results of Z-stack immunofluorescence with PDGFR\u0026beta; and Glut1 co-staining showed that the vascular coverage rate of pericytes in the hippocampus increased after modeling, but gradually decreased thereafter. The vascular coverage rate of pericytes at 0 d and 1 d after model establishment was significantly higher than that of the normal control group, and at 7 d after model establishment was significantly lower than that at 0 d after model establishment. Pericyte vascular coverage in epileptic animals at 30 days after modeling was significantly lower than that at 0 d and 1 d after modeling, and the mean value was lower than that in normal controls and 7 d after modeling, but there was no significant difference (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Compared with the results of the second part, pericyte vascular coverage was consistent with changes in AQP4 polarity during epileptogenesis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e4. Changes of PDGF-BB during the pathological process of epilepsy\u003c/h2\u003e\n\u003cp\u003eIt has been reported that PDGF-BB can promote the distribution of pericytes around blood vessels, and we used ELISA to detect the content of PDGF-BB in serum and cerebrospinal fluid of pilocarpine induced chronic epilepsy rats. The content of PDGF-BB in serum significantly decreased at 7 days after modeling and in epileptic animals at 30days compared with the normal group and 1 day after modeling. The content of PDGF-BB in serum in 0d, 1d, and non-epileptic animals at 30 days showed no difference to normal control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). The content of PDGF-BB in cerebrospinal fluid decreased gradually after model establishment, and only epileptic animals at 30 days after modeling were lower than normal controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Decreased vascularity of pericytes in pilocarpine-induced epileptic animals was associated with gradually decreased levels of PDGF-BB.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e5. Pericyte culture enhanced the membrane distribution of AQP4\u003c/h2\u003e\n\u003cp\u003eTo investigate whether the distribution of AQP4 was associated with pericyte distribution, we co-cultured astrocyte HA1800, pericyte HBVP and endothelial cell hCMEC with HA1800 in transwells to detect the expression of AQP4 total protein and AQP4 in the cell membrane of upper HA1800 cells. WB results showed that HBVP could promote the increase of AQP4 content on the cell membrane compared with HA1800 and hCMEC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), but had no effect on the total amount of AQP4 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Thus, pericyte distribution promotes membrane distribution of AQP4.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e6. Pericyte laminins may mediate AQP4 polarity\u003c/h2\u003e\n\u003cp\u003eTo search for factors mediating AQP4 distribution in pericytes, we performed transwell experiment with HA1800, HBVP, hCMEC and HA1800, HBVP and HBVP, and collected cells for transcriptomic sequencing analysis after cell culture (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Transcriptome results showed that pericyte co-culture resulted in decreased transcription of 470 genes and increased transcription of 179 genes in astrocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Endothelial cell co-culture resulted in decreased expression of 861 genes and increased transcription of 699 genes in astrocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Endothelial cells co-culture resulted in down-regulation of 230 genes and up-regulation of 164 genes in astrocytes compared to pericytes co-culture (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). In parallel, astrocyte cultures increased pericyte transcription of 1,185 genes and decreased transcription of 1,733 genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). By GO analysis, collagen-containing extracellular matrix was found to be involved in the transcriptional changes of genes in pericytes, endothelial cells and astrocytes co-culture (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF-I), so we performed a specific analysis of this GO genes.\u003c/p\u003e\n\u003cp\u003eThe result showed that: (1) AQP4 and its polarity-associated proteins, such as \u0026alpha;-syntrophin (SNTA1), agrin (AGRN), dystrophin (DMD, dystrophin-protein71, DP-71), \u0026beta;-dystroglycan (DAG1), and \u0026alpha;-dystrobrevin (DTNA) in astrocyte were not changed after co-cultured with pericyte compared to endothelial cells, while IL-33 (Cytosolic DNA-sensing pathway), ICAM1, CDH5, ITGB3, and LAMC2 were decreased and PDGFB was increased (Supplementary material Table\u0026nbsp;1-1.1). (2) Astrocyte co-culture induced increased LAMA1, LAMA2, PDGFRB, PDGFB, and LAMC1, and decreased LAMB3, LAMC2, LAMA3, LAMC3, agrin (AGRN) in pericyte compared to endothelial cells (Supplementary material Table\u0026nbsp;1-1.2). (3) Pericyte transcripted higher PDGFRB, PDGFB, LAMA2, LAMA1, LAMC1, LAMB1, LAMC2, and LAMA3 and lower LAMA5, LAMB3, and agrin (AGRN) compared to astrocyte after astrocyte co-culture (Supplementary material Table\u0026nbsp;1-1.3). Transcriptome results indicated that pericytes\u0026rsquo; laminins might be involved in astrocyte AQP4 distribution and we performed WB validation on co-cultured cells. The results showed that pericyte LAMA1 levels were significantly higher than endothelial cells, and the levels of pericyte LAMA1 were significantly increased after co-culture with astrocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). LAMA2 also showed the same changes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, B), which is consistent with the results of transcriptomics.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e7. Changes of laminins during epileptogenesis\u003c/h2\u003e\n\u003cp\u003eTo investigate whether laminins are associated with altered AQP4 polarity during epilepsy progression, we performed temporal immunofluorescence detection of laminins in hippocampal tissues. The results revealed that hippocampal LAMA1 levels increased significantly immediately after sustained epilepsy, then gradually decreased. One day after model establishment, LAMA1 levels remained elevated compared to the control group but decreased significantly by day 7, returning to control levels. At 30 days, LAMA1 levels were significantly lower than those observed immediately after sustained epilepsy and on day 1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC-E). In contrast, LAMA2 levels did not increase immediately after epilepsy but showed significant elevation at days 1 and 7, returning to control levels by day 30 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC-E). These trends paralleled changes in AQP4 polarity and pericyte vascular coverage. While LAMA1 and LAMA2 are not exclusively expressed in pericytes, their expression around pericytes increased proportionally with their overall expression levels. (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated whether AQP4 polarity during the epileptic pathological process is influenced by the vascular distribution of pericytes, focusing on temporal changes. The findings revealed that alterations in AQP4 polarity closely align with the vascular coverage of pericytes in epilepsy. Furthermore, LAMA1 and LAMA2 laminins in pericytes were shown to influence AQP4 distribution in astrocytes. Changes in AQP4 polarity during epilepsy were also correlated with laminin expression. To our knowledge, this is the first study to identify a link between astrocyte AQP4 polarity and pericyte vascularization during epilepsy, highlighting the potential regulatory role of pericyte vascular coverage and laminins in this process.\u003c/p\u003e \u003cp\u003eNearly 30% of epilepsy patients are unresponsive to medications[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], highlighting the importance of early intervention in high-risk individuals before symptom onset. Understanding the pathophysiologic changes during epileptogenesis is crucial to identifying potential therapeutic targets, drugs, or modulators that can inhibit this process[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. While neuronal damage is a key focus, the pathological roles of non-neuronal elements, including astrocytes, vascular neural units, and other cell types, also warrant significant attention[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe astrocytic water channel protein AQP4 plays a critical role in epileptic pathogenesis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Modulating AQP4 may serve as an effective therapeutic target for epilepsy. AQP4 co-localizes with Kir4.1[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and alterations in its polarized distribution influence potassium (K\u003csup\u003e+\u003c/sup\u003e) ion homeostasis, a key factor in epileptogenesis. In the hippocampus, astrocytic AQP4 and Kir4.1 cooperate in K\u003csup\u003e+\u003c/sup\u003e regulation, and depletion of the perivascular AQP4 pool slows K\u003csup\u003e+\u003c/sup\u003e clearance, exacerbating seizure severity[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Our experiments demonstrated that disrupting AQP4 polarization during acute seizures could mitigate seizure activity. In human epileptic hippocampal tissues, AQP4 polarization is significantly reduced compared to normal tissues[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Notably, our results found that, in epileptic animals, AQP4 polarization initially increased during the early stages of acute injury but decreased progressively, reaching its lowest levels in chronic epilepsy. To our knowledge, this is the first study to explore the dynamics of AQP4 polarization throughout the epileptic process. These findings underscore the need to consider the stage of epilepsy when targeting AQP4 polarization for therapeutic intervention.\u003c/p\u003e \u003cp\u003eBased on these results, the search for regulators of AQP4 polarity distribution may offer a promising strategy to delay epileptogenesis. Immunoelectron microscopy has revealed that AQP4 distribution is more pronounced around pericytes than endothelial cells, with pericytes mediating AQP4's polar distribution[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consistently, the present study found that co-culture with pericytes significantly increased AQP4 expression on astrocyte membranes. In Pdgfb\u003csup\u003eret/ret\u003c/sup\u003e transgenic mice [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], pericyte LAMC1-knockout animals[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and pericyte Cdk5 knockout spontaneously epileptic mice[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], both AQP4 polarity distribution and pericyte vascular coverage were concurrently reduced, underscoring a potential correlation. However, the temporal dynamics of these changes during the pathological progression of epilepsy remain unexplored.\u003c/p\u003e \u003cp\u003eIn this study, pericyte vascular coverage increased in the early stages after SE injury and progressively decreased as epilepsy advanced, with the lowest expression observed in epileptic animals. Consistent with our findings, previous research reported increased pericyte vascular coverage in PDGFRβ\u003csup\u003e+\u003c/sup\u003e cells during the acute phase after SE[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, decreased vascular coverage was noted in NG2-positive cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This discrepancy may be due to the distinct roles of NG2 and PDGFRβ in characterizing vascular mural cells, as NG2 primarily marks oligodendrocyte precursor cells[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], whereas PDGFRβ preferentially identifies pericyte cells[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], despite also being expressed in non-vascular brain parenchymal cells. In human brain tissue with chronic temporal lobe epilepsy, one study reported individual variability in PDGFRβ vascular distribution [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Another study observed an increased number of blood vessels fully encapsulated by PDGFRβ-positive cells, alongside a decrease in incompletely encapsulated vessels, indicating increased pericyte coverage in epileptic tissues[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, this study focused on the cerebral white matter of the temporal gyrus, differing from our investigation of the hippocampal region, which may explain the discrepancies. In summary, our study unraveled the dynamic changed of vascular coverage of PDGFRβ-positive pericytes in the hippocampal tissue of rats with epilepsy progression. Notably, the perivascular distribution of pericytes closely paralleled changes in AQP4 polarity during epilepsy progression. This suggests that pericytes may play a critical role in regulating AQP4 polarity distribution in astrocytes.\u003c/p\u003e \u003cp\u003eThe polar distribution of AQP4 is influenced by DAP complex proteins, including β-DG, syntrophin, and extracellular matrix components[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Using Transwell transcriptomics, we observed no changes in the expression of AQP4, α-syntrophin, agrin, dystrophin, β-dystroglycan, or α-dystrobrevin in astrocytes co-cultured with pericytes compared to endothelial cells. In line with single-cell sequencing data[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], we found significantly higher expression of laminins, including LAMA1, LAMA2, and LAMC1, in pericytes than in endothelial cells. Co-culture with astrocytes increased LAMA1 and LAMA2 expression in pericytes, suggesting that laminins like LAMA1 in pericytes may play a key role in AQP4 distribution. This is evidenced by the fact that the interaction between laminins and dystroglycan is critical for maintaining the OAPs and polarity of AQP4[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and knockout of LAMC1 in pericytes reduces AQP4 polarity distribution[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, the levels of LAMA1 and LAMA2 increase in response to seizures but decrease in epileptic animals. Following KA or 3-NPA treatment, LAMA1 expression in vascular elements increases[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In pilocarpine-induced epilepsy, laminin expression rises on days 3 and 4 post-SE but normalizes by day 30[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], consistent with our findings. Thus, expression patterns of laminin align with AQP4 distribution and pericyte vascular coverage during epilepsy progression, and stabilizing extracellular matrix components like LAMA1 in pericytes could help delay epilepsy progression[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Matrix metalloproteinases (MMPs) are known to regulate neurovascular extracellular matrix stability and are involved in post-traumatic epileptogenesis, with MMP inhibitors reducing epileptogenesis[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Notably, pericytes release the most MMP9 upon thrombin stimulation compared to endothelial cells and astrocytes[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], highlighting the potential role of pericyte-derived MMP9 in stabilizing extracellular matrix components like laminins and AQP4 polarity.\u003c/p\u003e \u003cp\u003ePDGF-BB/PDGFRβ signaling is crucial for vascularization by mediating pericyte recruitment to the vasculature, thereby promoting vessel integrity and function[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Previous studies have shown that exogenous PDGF-BB, administered 1\u0026ndash;14 days post-KA injury, can maintain pericyte distribution and inhibit epileptogenesis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the dynamics of PDGF-BB during epilepsy remain unclear. In this study, we are the first to examine the changes in PDGF-BB from injury to seizure, finding that its levels gradually decrease in both blood and cerebrospinal fluid as epilepsy progresses. These findings suggest that increasing PDGF-BB during the latency period after injury could potentially inhibit epileptogenesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe vascular distribution of pericytes plays a key role in the polarization of AQP4 during epilepsy pathology. Stabilizing extracellular matrix proteins of pericytes, such as LAMA1 and LAMA2, and administering PDGF-BB post-injury to preserve pericyte distribution around blood vessels and stabilize AQP4 polarity could be a crucial strategy for slowing epilepsy progression.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e The animal study was approved by the Committee for Animal Experiments at Taizhou Hospital of Zhejiang Province in China (No. K20190108). The study was conducted in accordance with the local legislation and institutional requirements.\u003c/p\u003e \u003c/div\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was in part supported by National Natural Science Foundation of China (No. 81903584 to Gang Wu), Zhejiang Provincial Basic and Public Welfare Research Program (No. LGD20H310002 to Gang Wu), Taizhou Science and Technology Plan Project (24ywa32 to Gang Wu), Taizhou Enze Medical Center (Group) Major Project (No.2022EZZD05 to Gang Wu).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLin Lin: Investigation, Methodology, Visualization, Writing \u0026ndash;original draft. Hongxia Tang: Investigation, Methodology, Visualization, Writing \u0026ndash;original draft. Ke Cui: Resources, Writing \u0026ndash; review \u0026amp; editing. Zeyi Kang: Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash;review \u0026amp; editing. Tengwei Pan: Investigation, Writing \u0026ndash; review \u0026amp; editing. Changqiang Feng: Resources,Writing \u0026ndash; review \u0026amp; editing. XiaoHong Zhao: Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Jiewei Wang: Investigation, Writing \u0026ndash; review \u0026amp; editing. Zhiyuan Chen: Investigation, Writing \u0026ndash; review \u0026amp; editing. Zhengli Jiang: Resources, Writing \u0026ndash; review \u0026amp; editing. Gang Wu: Conceptualization, Funding acquisition, Project administration, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Zhenyu Yang, at Public Scientific Research Platform, Taizhou Hospital of Zhejiang Province for the excellent technical assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSequence data that support the findings of this study have been deposited in the European Nucleotide Archive with the primary accession code PRJEB88623.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKrishnamurthy KB (2016) Epilepsy. Annals of Internal Medicine 164:ITC17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBinder DK, Nagelhus EA, Ottersen OP (2012) Aquaporin-4 and epilepsy. Glia 60:1203\u0026ndash;1214\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEid T, Lee TS, Thomas MJ, Amiry-Moghaddam M, Bj\u0026oslash;rnsen LP, Spencer DD, Agre P, Ottersen OP, de Lanerolle NC (2005) Loss of perivascular aquaporin 4 may underlie deficient water and K\u0026thinsp;+\u0026thinsp;homeostasis in the human epileptogenic hippocampus. Proc Natl Acad Sci U S A 102:1193\u0026ndash;1198\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasaki H, Wakayama Y, Hara H, Jimi T, Unaki A, Iijima S, Oniki H, Nakano K, Kishimoto K, Hirayama Y (2010) Immunocytochemical Studies of Aquaporin 4, Kir4.1, and α1-syntrophin in the Astrocyte Endfeet of Mouse Brain Capillaries. Acta Histochem Cytochem 43:99\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmiry-Moghaddam M, Williamson A, Palomba M, Eid T, Lanerolle NCD, Nagelhus EA, Adams ME, Froehner SC, Agre P, Ottersen OP (2003) Delayed K\u0026thinsp;+\u0026thinsp;Clearance Associated with Aquaporin-4 Mislocalization: Phenotypic Defects in Brains of α-Syntrophin-Null Mice. Proc Natl Acad Sci USA 100:13615\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu XX, Yang L, Shao LX, He Y, Wu G, Bao YH, Lu NN, Gong DM, Lu YP, Cui TT, Sun NH, Chen DY, Shi WX, Fukunaga K, Chen HS, Chen Z, Han F, Lu YM (2020) Endothelial Cdk5 deficit leads to the development of spontaneous epilepsy through CXCL1/CXCR2-mediated reactive astrogliosis. J Exp Med 217:e20180992\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmiry-Moghaddam M, Frydenlund DS, Ottersen OP (2004) Anchoring of aquaporin-4 in brain: molecular mechanisms and implications for the physiology and pathophysiology of water transport. Neuroscience 129:999\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGundersen GA, Vindedal GF, Skare O, Nagelhus EA (2014) Evidence that pericytes regulate aquaporin-4 polarization in mouse cortical astrocytes. Brain Struct Function 219:2181\u0026ndash;2186\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoddevik EH, Khan FH, Rahmani S, Ottersen OP, Boldt HB, Amiry-Moghaddam M (2017) Factors determining the density of AQP4 water channel molecules at the brain\u0026ndash;blood interface. Brain Struct Function 222:1753\u0026ndash;1766\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilesi S, Boussadia B, Plaud C, Catteau M, Rousset MC, De Bock F, Schaeffer M, Lerner-Natoli M, Rigau V, Marchi N (2014) Redistribution of PDGFRbeta cells and NG2DsRed pericytes at the cerebrovasculature after status epilepticus. Neurobiol Dis 71:151\u0026ndash;158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArango-Lievano M, Boussadia B, De Terdonck LDT, Gault C, Fontanaud P, Lafont C, Mollard P, Marchi N, Jeanneteau F (2018) Topographic Reorganization of Cerebrovascular Mural Cells under Seizure Conditions. Cell Rep 23:1045\u0026ndash;1059\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin L, Hu X, Hong W, Pan T, Wang Z, Wang E, Wu G (2024) A novel animal model of spontaneous epilepsy: Cdk5 knockout in pericyte-specific mice. Front Cell Neurosci 18:1474231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLian XY, Xu K, Fau - Stringer JL, Stringer JL (2008) Oral administration of fructose-1,6-diphosphate has anticonvulsant activity. Neurosci Lett 446:75\u0026ndash;77\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen Y, Qin H, Chen J, Mou L, He Y, Yan Y, Zhou H, Lv Y, Chen Z, Wang J, Zhou YD (2016) Postnatal activation of TLR4 in astrocytes promotes excitatory synaptogenesis in hippocampal neurons. J Cell Biol 215:719\u0026ndash;734\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRacine RJ (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32:281\u0026ndash;294\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Iliff JJ, Liao Y, Chen MJ, Shinseki MS, Venkataraman A, Cheung J, Wang W, Nedergaard M (2012) Cognitive deficits and delayed neuronal loss in a mouse model of multiple microinfarcts. J Neurosci 32:17948\u0026ndash;17960\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKitchen P, Salman MM, Halsey AM, Clarke-Bland C, MacDonald JA, Ishida H, Vogel HJ, Almutiri S, Logan A, Kreida S, Al-Jubair T, Winkel Missel J, Gourdon P, T\u0026ouml;rnroth-Horsefield S, Conner MT, Ahmed Z, Conner AC, Bill RM (2020) Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema. Cell 181:784\u0026ndash;799\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShariff S, Nouh HA, Inshutiyimana S, Kachouh C, Abdelwahab MM, Nazir A, Wojtara M, Uwishema O (2024) Advances in understanding the pathogenesis of epilepsy: Unraveling the molecular mechanisms: A cross-sectional study. Health Sci Rep 7:e1896\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanigro D, Walker MC (2014) What non-neuronal mechanisms should be studied to understand epileptic seizures? Adv Exp Med Biol 813:253\u0026ndash;264\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeixoto-Santos JE, Kandratavicius L, Velasco TR, Assirati JA, Carlotti CG, Scandiuzzi RC, Salmon CE, Santos AC, Leite JP (2017) Individual hippocampal subfield assessment indicates that matrix macromolecules and gliosis are key elements for the increased T2 relaxation time seen in temporal lobe epilepsy. Epilepsia 58:149\u0026ndash;159\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, He L, Norlin J, Lindblom P, Strittmatter K, Johansson BR, Betsholtz C (2010) Pericytes regulate the blood-brain barrier. Nature 468:557\u0026ndash;561\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGautam J, Zhang X, Yao Y (2016) The role of pericytic laminin in blood brain barrier integrity maintenance. Sci Rep 6:36450\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBottero M, Pessina G, Bason C, Vigo T, Uccelli A, Ferrara G (2024) Nerve-Glial antigen 2: unmasking the enigmatic cellular identity in the central nervous system. Front Immunol 15:1393842\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaranjo O, Osborne O, Torices S, Toborek M (2022) In Vivo Targeting of the Neurovascular Unit: Challenges and Advancements. Cell Mol Neurobiol 42:2131\u0026ndash;2146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Binding L, Puntambekar I, Patodia S, Lim YM, Mryzyglod A, Xiao F, Pan S, Mito R, de Tisi J, Duncan JS, Baxendale S, Koepp M, Thom M (2024) Microangiopathy in temporal lobe epilepsy with diffusion MRI alterations and cognitive decline. Acta Neuropathol 148:49\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaite A Brown Sc Fau - Blake DJ, Blake DJ The dystrophin-glycoprotein complex in brain development and disease. Trends Neurosci 35:487\u0026ndash;496\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVanlandewijck M, He L, Mae MA, Andrae J, Ando K, Del Gaudio F, Nahar K, Lebouvier T, Lavina B, Gouveia L, Sun Y, Raschperger E, Rasanen M, Zarb Y, Mochizuki N, Keller A, Lendahl U, Betsholtz C (2018) A molecular atlas of cell types and zonation in the brain vasculature. Nature 554:475\u0026ndash;480\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkar S, Schmued L (2010) Kainic acid and 3-Nitropropionic acid induced expression of laminin in vascular elements of the rat brain. Brain Resrarch 1352:239\u0026ndash;247\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim YJ, Kim JY, Ko AR, Kang TC (2014) Over-expression of laminin correlates to recovery of vasogenic edema following status epilepticus. Neuroscience 275:146\u0026ndash;161\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePitk\u0026auml;nen A, Ndodeekane XE, Lukasiuk K, Wilczynski GM, Dityatev A, Walker MC, Chabrol E, Dedeurwaerdere S, Vazquez N, Powell EM (2014) Neural ECM and epilepsy. Prog Brain Res 214:229\u0026ndash;262\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkonomidou C (2014) Matrix metalloproteinases and epileptogenesis. Mol Cell Pediatr 1:1\u0026ndash;6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePijet B, Stefaniuk M, Kostrzewskaksiezyk A, Tsilibary PE, Tzinia A, Kaczmarek L (2018) Elevation of MMP-9 Levels Promotes Epileptogenesis After Traumatic Brain Injury. Mol Neurobiol 55:1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJayakumar AR, Apeksha A, Norenberg MD (2016) Role of Matricellular Proteins in Disorders of the Central Nervous System. Neurochem Res 42:1\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachida T, Takata F, Matsumoto J, Takenoshita H, Kimura I, Yamauchi A, Dohgu S, Kataoka Y (2015) Brain pericytes are the most thrombin-sensitive matrix metalloproteinase-9-releasing cell type constituting the blood\u0026ndash;brain barrier in vitro. Neurosci Lett 599:109\u0026ndash;114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsioumpekou M, Cunha SI, Ma H, \u0026Aring;hgren A, Cedervall J, Olsson AK, Heldin CH, Lennartsson J (2020) Specific targeting of PDGFRβ in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression. Theranostics 10:1122\u0026ndash;1135\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Table","content":"\u003cp\u003eSupplementary Tables are not available with this version.\u003c/p\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":"pericyte, AQP4 polarity, epileptogenesis, laminins, PDGFBB","lastPublishedDoi":"10.21203/rs.3.rs-6504855/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6504855/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To investigate the temporal changes in AQP4 polarity and pericyte vascularity during the pathological progression of epilepsy, with the goal of identifying potential drug targets or regulatory strategies to delay epilepsy progression and inhibit its onset.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A rat model of chronic epilepsy was established via intraperitoneal injection of pilocarpine. The polarity of AQP4 and the vascular coverage of pericytes were assessed using immunofluorescence. The effects of pharmacological modulation of AQP4 polarity distribution on PTZ-induced seizures were observed. The molecular mechanisms mediating the polar distribution of AQP4 in pericytes were explored through Transwell co-culture and transcriptomics and validated at the cellular protein expression level. Immunofluorescence was employed to examine changes in the mediating molecules during the progression of epilepsy. Additionally, ELISA was used to measure the levels of PDGF-BB in serum and cerebrospinal fluid during the pathological process of epilepsy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The polar distribution of AQP4 and the perivascular localization of pericytes increased rapidly after epileptic model establishment but gradually decreased, reaching their lowest levels in epileptic animals. Trifluoperazine inhibited the acute redistribution of AQP4 and reduced the latency and duration of PTZ-induced seizures, alleviated brain edema. Pericytes did not affect dystrophin-associated protein (DAP) complex components (e.g., α-syntrophin, β-dystroglycan, dystrophin, and agrin) in astrocytes compared to endothelial cells. However, astrocytes significantly enhanced pericyte-derived laminin expression. During epilepsy progression, LAMA1 and LAMA2 expression initially increased and then declined. The levels of PDGF-BB in serum and cerebrospinal fluid gradually decreased after model establishment, reaching their lowest point during epilepsy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The polar distribution of AQP4 plays a crucial role in the development of epilepsy. During the pathological process, AQP4 polarity is largely influenced by pericyte vascular coverage. Key regulators, such as laminins (e.g., LAMA1) and PDGF-BB, are critical for maintaining AQP4 polarity, delaying epileptic pathology, and inhibiting epileptogenesis.\u003c/p\u003e","manuscriptTitle":"The PDGFBB-PDGFRβ pathway and Laminins in pericytes are involved in the temporal change of AQP4 polarity during Epileptic Pathogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 02:27:18","doi":"10.21203/rs.3.rs-6504855/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":"c92ff5cb-4802-46ec-ae8c-eb3f158f4f6f","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T13:09:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-02 02:27:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6504855","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6504855","identity":"rs-6504855","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}