Calpeptin mitigates ventriculomegaly and microvascular impairment in a novel LPS- dependent mouse model of acquired hydrocephalus

doi:10.21203/rs.3.rs-3762291/v1

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Calpeptin mitigates ventriculomegaly and microvascular impairment in a novel LPS- dependent mouse model of acquired hydrocephalus

https://doi.org/10.21203/rs.3.rs-3762291/v1

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Hydrocephalus, characterized by ventricular distension due to impaired cerebrospinal fluid (CSF) pathways, is a multifactorial condition with perinatal infection as a predominant global cause. Over three decades, alterations in periventricular cell junctions, especially cadherins, have been linked to pediatric hydrocephalus pathogenesis. We propose that the Toll-like receptor (TLR)–calpain (calcium-dependent proteinase) pathway induces cadherin cleavage, playing a central role in initiating vascular impairment associated with vascular permeability and ventriculomegaly—hallmark events in acquired hydrocephalus (AH).

In this manuscript, perinatal C57BL/6 mice and endothelial cell cultures were exposed to lipopolysaccharide (LPS) toxin, a gram-negative bacteria component, modeled post-infectious hydrocephalus. For treatment, calpain inhibitor (calpeptin) and TLR-4 receptor inhibitor were employed in cell cultures, with calpeptin used in mice. Magnetic resonance imaging (MRI) quantified ventricular volume, and immunofluorescence and DiI assessed microvascular integrity. Additionally, endothelial permeability was evaluated using a transwell system.

Results showed that in vitro LPS induced increased endothelial permeability associated with calpain-dependent cleavage of VE-cadherin. In vivo, intraventricular LPS-induced ventriculomegaly linked to microvascular impairments dorsally to the ventricles (p<0.05). In vitro, TLR and calpain inhibition exhibited similar vascular permeability and VE-cadherin cleavage after LPS exposure compared to the control. In vivo, calpain inhibition prevented or attenuated ventriculomegaly and significantly improved survival after LPS injection (p<0.05).

This research offers a comprehensive analysis from in vitro experiments on C57BL/6 mice primary brain microvascular endothelial cells to in vivo mouse models, highlighting the pivotal role of the TLR4-Calpain pathway, VE-cadherin cleavage, and vascular impairment in acquired hydrocephalus pathogenesis. The findings emphasize the potential of calpain inhibition in preventing the physiopathology associated with LPS exposure.

Hydrocephalus

LPS

Calpain

TLR4

calpeptin

microvascular permeability

Hydrocephalus is characterized by abnormal cerebrospinal fluid (CSF) accumulation within the brain's ventricular system^1,2. The acquired forms, postinfectious (PIH) and posthemorrhagic (PHH) hydrocephalus are the most common forms of hydrocephalus worldwide³, sharing common acute inflammatory pathways^4,5. While surgical cerebrospinal fluid shunting has been the primary treatment, its efficacy remains limited^6,7

CSF circulation, traditionally rooted in the bulk flow theory^8,9, faces challenges with emerging evidence suggesting alternative absorption pathways in pediatric patients^10–13. Notably, the “glymphatic system," involving astrocyte end-feet and perivascular channels, has gained recognition as part of the parenchymal CSF absorption pathway^14,15.

Neuroinflammation, a hallmark of hydrocephalus pathogenesis, is linked to changes in vascular integrity. Toll-like receptors (TLRs) recognize damage-associated and pathogen-associated molecular patterns (DAMPs and PAMPs), triggering an inflammatory cascade^4,5, activating calpains, calcium-dependent proteases, which cleave vascular endothelial cadherins (VE-cadherin) and increase vascular permeability^16–18 to recruit immune response-associated cells into the affected areas¹⁹.

Given that PAMPs and DAMPs constitute the primary etiological factors in acquired hydrocephalus (AH)^4,5 and the pivotal role of perivascular regions in CSF absorption^14,20, our hypothesis posits that intraventricular lipopolysaccharide (LPS) as an acute inflammatory PAMP may activate calpain hyperactivity via TLR4 pathway, leading to parenchymal CSF flow alterations and ventriculomegaly. This manuscript presents a novel mouse model of acquired hydrocephalus, offering a comprehensive analysis across multiple levels to explore the inflammatory pathway linked to pediatric hydrocephalus pathogenesis, explicitly focusing on the vascular system.

Experimental Animals:

Wild-type mice (C57BL/6) were used. All mice were obtained from The Jackson Laboratory and bred by the Animal Experimentation Service of the Children's Hospital Orange County in a room at 22°C with a 12:12 light/dark cycle and standard food and water available ad libitum. The design of the experiments, housing, handling, care, and processing of the animals was conducted following accepted protocols and legislation. Experimental procedures were approved by the Children's Hospital in Orange County Institutional Animal Care and Use Committee and performed in an AAALAC-accredited facility in compliance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. Mice were divided into two main experimental groups: LPS-dependent hydrocephalus optimization and calpain modulation experimental testing. For ventriculomegaly optimization, 3 groups (n = 6 per group) of 3-day-old mice were injected with 1, 2, and 3 µL of LPS (Sigma-Aldrich #L3024) at a concentration of 2.5 µg per µL of 10% glycerol and 90% PBS Ph 7.4. Mice were divided into vehicle, LPS, & LPS + calpeptin (Sigma-Aldrich #C8999) groups for the calpain modulation experiments. All of them received 2 µL of the following solutions: control (10% glycerol, 80% PBS, 10% DMSO), LPS (10 µg of LPS, 5% glycerol, 85% PBS, 10% DMSO), and LPS + calpeptin (10 µg of LPS, 5% glycerol, 85% PBS, 10% of 15µg of calpeptin dissolved in DMSO). The injections were performed in the right lateral ventricle following stereotactic coordinates (0.8 mm lateral, 1.7 mm anterior, and 1.5 mm deep from the posterior fontanelle) as previously described²¹. The solutions were administered over 30 seconds using a 25-microliter syringe (Hamilton, 80366) adapted to a UMP3 Ultra Micropump (World Precision Instruments) as previously reported^22,23. The animals were anesthetized for hypothermia. Pretreatment was performed in the LPS + calpeptin group in which 75 µg of calpeptin dissolved in 10 µL of 20% DMSO, and 80% PBS was injected subcutaneously 24 and 3 hours before intraventricular induction.

Magnetic Resonance Imaging in Mice:

Fourteen days after the induction of neuroinflammation with LPS, in vivo MRI scans were conducted on the mice at the Preclinical and Translational Neuroimaging Center at the University of California, Irvine (UCI). The mice (including dams and pups) had magnetic resonance imaging of the brain under the UC Irvine Preclinical and Translational Neuroimaging Center mouse protocol (IACUC protocol AUP 20–172; MRI Services). Imaging was performed using a 4.7T horizontal bore Agilent MRI system with a 2 cm ID quadrature RF coil. Animals were anesthetized with isoflurane, and their core temperature was maintained through regulated airflow. Respiratory parameters were monitored and adjusted as needed. T2-weighted images of each mouse were employed for ventricular volume segmentation using the ITK-SNAP program (http://www.itksnap.org), and the volume of the telencephalic ventricles was quantified according to Botfield et al. (2013)²⁴.

Mouse perfusion and vessel painting:

Mice were sacrificed via transcranial perfusion after MRI imaging. Vessel painting is based on 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) affinity to endothelial cells. The mice were perfused with PBS (150 ml), followed by 50 ml of DiI (13 µg/ml), and finally fixed with paraformaldehyde (4%, 200 ml) as previously reported²⁵. After brain extraction, cortical vessels were photographed using a fluorescence Keyence BZ-X810 microscope (Keyence).

Cell Growth:

C57BL/6 Mouse Primary Brain Microvascular Endothelial Cells (CellBiologics, C57-6023) were cultured in complete endothelial cell medium (Kit M1168). The cells were incubated under standard conditions (37°C, 5% CO2) for 7 days with media refreshed every 48 hours as previously reported²⁶.

Cell cultures were divided into four groups: vehicle, LPS, LPS + calpeptin & LPS + TLR4 inhibitor (Novus Biologicals, #NBP2-26244) with 6 wells each. All the wells received 1 µL of the different experimental solutions in 1 ml of complete vascular endothelial cell medium. The testing solutions were prepared as follows: vehicle (10% glycerol, 80% PBS, 10% DMSO), LPS (5 µg of LPS, 5% glycerol, 85% PBS, 10% DMSO), LPS + calpeptin (5 µg of LPS, 5% glycerol, 85% PBS, 10% of 7.5 µg of calpeptin dissolved in DMSO) and LPS + TLR4 inhibitor (5 µg of LPS, 1 µg 5% glycerol, 85% PBS, 10% DMSO). Cells were exposed to the LPS solutions for two hours before undergoing permeability and Western blot tests.

Western blot:

The 24-well plate was divided into 4 sections, with 6 wells per experimental condition (described in the cell growth section). Cells were incubated under standard conditions for 1 hour. After additional PBS washes, cells were lysed using RIPA Lysis and Extraction Buffer (ThermoFisher, #89900) combined with Laemmli sample buffer (98%) plus a reducing agent (2%) and heated at 70°C for 10 minutes. Western blot analysis was performed using 4 samples for each condition at 20 µL per well. The gel was transferred using a Trans-Blot Turbo Transfer Pack (BIO-RAD Cat. #1704158). Following transfer, the membrane was blocked in a solution of nonfat milk powder and PBS for 45 minutes, rinsed in PBS and incubated with primary antibodies: anti-GAPDH at 1:1000 (Abcam, ab8245) and VE-cadherin at 1:250 (Invitrogen, PA5-19612), overnight at 4°C. After further PBS rinses, the membrane was treated with fluorescent goat anti-mouse (Invitrogen #A21127) at 1:10,000 and fluorescent goat anti-rabbit (Thermo Fisher Scientific, #A11034) at 1:10,000 for 2 hours at room temperature.

Calpain activity:

VE-cadherin cleavage under calpain modulation was quantified as the ratio of the cleaved (95 KD) vs full-length portion (130 KD) of VE-cadherin as previously reported²⁷. A ratio of cleaved divided by full length indicates the cleavage index.

Immunocytochemistry:

After the experimental procedures, the cells were rinsed twice in two 5-minute PBS washes. Subsequently, cells were fixed with paraformaldehyde for 7 minutes and rinsed in PBS. Cells were stained with Anti-Pan-cadherin at 1:150 (Sigma, SAB4200731), anti-VE-cadherin at 1:25 (Invitrogen, PA5-19612) or anti-GFAP (Abcam, ab7260) at 1:400 in PBSt. Following a 24-hour incubation at room temperature, fluorescent goat anti-mouse (Invitrogen, #A21127) at 1:400 and fluorescent anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, #A11034) at 1:400 were used. Cells were stained with DAPI at 1:5000 in PBS for 5 min to label nuclei. After two 5-minute PBS washes, imaging was conducted with a fluorescence ECHO-Revolve microscope (Echo), and quantification was performed using ImageJ (NIH image software).

Permeability:

After incubation with peroxidase and the experimental solutions, media from the bottom of the wells (50 µL) was combined with SuperSignal West Pico Plus chemiluminescent (Thermofisher #34577) (50 µL) and imaged using a ChemiDoc system as previously reported²².

Generative AI and AI-assisted technologies in the writing process:

While preparing this work, the authors used Grammarly and Chat GPT to improve the language and readability of the manuscript. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the publication's content.

Quantification and Statistical Analysis:

Statistical analyses were carried out using GraphPad Prism9 Software (San Diego, CA, USA). Data are presented as the mean ± SD. Nonparametric data were subjected to Kruskal-Wallis's test for hypothesis testing, while parametric data were analyzed using ANOVA. Chi-square tests were used to quantify mouse survival. Statistical significance was considered at p < 0.05.

LPS intraventricular injection induces acquired hydrocephalus.

Mice were exposed to LPS in three groups to investigate the role LPS plays in the development of hydrocephalus; mice were injected with 2.5, 5, and 7.5 µg of LPS. Results revealed a proportional increase in ventricular volumes with increasing LPS exposure, showing a positive correlation with r = 0.9901 and p = 0.0099. Therefore, the mice injected with 7.5 µg of LPS showed larger ventricular volumes (23.43 ± 1.848) than the mice injected with 5 µg (12.79 ± 2.897) and 2.5 µg (7.141 ± 1.894) of LPS (Fig. 1). Conversely, while LPS exposure increased, the survival percentage decreased, showing a negative correlation (r = -0.9129, p = 0.0871). Thus, the mice injected with 7.5 µg of LPS showed a lower survival rate (40%) than mice injected with 5 µg (80%) and 2.5 µg (100%) of LPS (χ2 = 4.86, p = 0.089). These findings suggest that LPS is a promising toxin for reliably inducing acquired hydrocephalus.

TLR-Calpain Pathway Modulation Prevents Cytopathology Associated with Acquired Hydrocephalus In Vitro

Using C57BL/6 Mouse Primary Brain Microvascular Endothelial Cells, we investigated the role of the TLR-Calpain pathway in cadherin vascular alterations associated with acquired hydrocephalus. LPS triggered VE-cadherin-endothelial disruption, while calpeptin and TLR4 inhibitors were administered as treatments. Calpain cleavage of VE-cadherin results in a 95 KD fragment, and the ratio of cleaved VE-cadherin vs. full-length VE-cadherin is indicative of cleavage activity and was significantly higher (median, 0.35) in the LPS-exposed group compared to the control (median, 0.211 p = 0.028). Treatment with a TLR4 inhibitor and calpeptin effectively prevented this cleavage activity (median 0.179 and 0.143, respectively). After the permeability assay, the dot-blot mean gray analysis showed that LPS exposure significantly increased permeability (median, 90.14 density units) compared to the control (median, 41.98 density units p = 0.043). At the same time, treatment with calpeptin or TLR4 inhibitor restored permeability to control levels (respectively, median, 67.79 & 77.7 density units p > 0.05). These results suggest a relationship between the TLR-Calpain cleavage pathway and endothelial cell permeability.

Calpain inhibition attenuates ventriculomegaly in LPS injected mice.

To test the influence of calpain on the pathophysiology of acquired hydrocephalus after LPS induction, mice were injected with LPS (5 µg) or LPS and calpain inhibitor (calpeptin). For the control, another group was created and injected with the vehicle for LPS and calpeptin. After ~ 14 days of LPS injection, MRI was performed to quantify ventricular volume, and then the mice were vessel painted. Several mice did not survive until the experimental endpoint. Vehicle-treated mice showed a survival rate of 91.6% (11 out of 12), and LPS-treated mice showed a significant decrease in survival (10 out of 21, 47.6%). At the same time, LPS + calpeptin treated mice exhibited significantly increased survival (10 out of 12, 83.3%) (χ² = 8.941, p = 0.0114). All mice that died were within the first 4 days of LPS induction, indicating that acute toxicity and associated inflammation may play a role in the mouse mortality. In turn, the treatment with calpeptin in LPS-injected mice showed significantly attenuated ventriculomegaly (2.48 ± 1.84 mm³) when compared with the nontreated LPS-injected mice (8.92 ± 4.1 mm³, p < 0.0001). No significant differences were found when vehicle control was compared with calpeptin-treated mice (0.47 ± 0.36 mm³, p = 0.13), indicating the potential involvement of the calpain pathways in the etiology of acquired hydrocephalus and supporting its inhibition as a possible target for future treatments (Fig. 3).

LPS Induces Calpain-Dependent Vascular Alteration an in vivo Model of AH

The vascular system was stained with DiI stain before sacrifice to assess vascular status. Our results showed areas of scarce DiI staining in cortical regions dorsal to the lateral ventricles of mice exposed to intraventricular LPS. Thus, the mean gray intensity indicated significantly decreased leveling in LPS-injected mice (27.46 ± 11.75 density units, p = 0.0018). In contrast, calpeptin-treated mice did not exhibit mean density alterations (69.90 ± 18.20 density units) when compared with vehicle control injected mice (55.36 ± 29.35 density units, p = 0.1460) (Fig. 4). These results were confirmed by transversally slicing the brains to examine the frontal cortex superior to the lateral ventricles. In this region, VE-cadherin immunodensity was quantified, showing a similar patron of expression than DiI with decreased expression in LPS-injected mice (30.89 ± 5.41 density units, P = 0.0747) when compared with control (40.57 ± 8.254 density units). Interestingly, calpeptin-treated mice demonstrated increased VE-cadherin expression (42.67 ± 8.151 density units), compared with LPS-injected mice (P = 0.0528), demonstrating similar expression levels to vehicle control. Thus, LPS-dependent calpain activity seems to contribute to microvascular impairment associated with acquired hydrocephalus.

Periventricular vascular impairments, marked by blood-brain barrier disruptions, increased permeability, and vascular collapse, are key events in the pathophysiology of AH^28–31. Our investigation centered on elucidating the role of calpain-dependent cleavage of VE-cadherin within the vascular system, proposing a mechanistic link between the pathogenesis of AH and the TLR4-Calpain pathway.

LPS-containing bacteria are a common cause of acquired hydrocephalus^32–34. LPS serves as a canonical PAMP for TLR4^35,36. Our study proved that intraventricular injection of LPS at perinatal age induces AH in mice as previously reported by Stephanie et al. (2023)³⁷ but different in that they used adult rats (8 weeks old) where the infusion of LPS into the lateral ventricles induced hydrocephalus. Our findings revealed a dose-dependent increase in ventricular volumes coupled with a concomitant decrease in survival rates. The deceased animals were consistently found within 4 days after LPS injection. The mortality was probably related to acute toxicity and associated inflammation coupled with the immaturity of the mice. This finding cements LPS as a robust inducer of AH, isolating the TLR4-dependent inflammation cascade as the main etiological factor. The work reported by Stephanie et al. (2023)³⁷ defined LPS-dependent, ChP hypersecretion secondary to a cytokine storm released by macrophages that express TLRs as the main etiological factor for hydrocephalus. However, the LPS effects that may take place on the periventricular parenchyma were disregarded, such as changes in the blood-brain barrier permeability or the CSF efflux associated with perivascular transport, including intramural periarterial drainage. The parenchyma blood vessels play a fundamental role in CSF homeostasis, and considering that vascular collapse in the germinal matrix is one of the most common causes of IVH, this ultimately leads to PHH, the most common form of acquired hydrocephalus in the USA³. This manuscript delves into the effects of the TLR4-calpain pathway as critical mediator on the vascular alterations associated with acquired hydrocephalus.

The term “cell junction pathology” was coined an etiological factor in hydrocephalus since cell-to-cell junction alterations within the ventricular zone have been described as fundamental in the pathophysiology of both congenital and acquired hydrocephalus conditions by affecting ependymal cells (ECs)^30,38–49. Since multiciliate EC plays a critical role in the local movement of CSF, a lack or malfunction of these cells can be associated with inefficient CSF flow and ventriculomegaly^39,50–53. However, in the lateral ventricles of mice, the EC are not fully differentiated until 15 days after birth⁵⁴. Considering that our induction experiments were performed at P3 (3 postnatal days), effects on the EC may not be the putative cause of ventriculomegaly in our model. Thus, we decided to explore whether the cell junction pathology could be extended to the cerebral vascular system in which VE-cadherin is mostly associated with the permeability of the blood vessels⁵⁵. Our in vitro experiments aimed to define the role of the LPS-TLR4 pathway in AH-related cytopathology, especially in the context of VE-cadherin vascular alterations. The exposure of C57BL/6 mouse primary brain microvascular endothelial cells to LPS initiated VE cadherin-endothelial disruption associated with calpain cleavage and increased permeability. The introduction of TLR4 inhibitors as treatment effectively preserved the integrity of VE-cadherin and successfully restored permeability to control levels. Calpain plays a fundamental role in VE-cadherin cleavage after TLR activation, and has long been reported to play a role in the pathophysiology of hydrocephalus associated with damage due to calcium-activated proteolytic processes⁵⁶. We also inhibited calpain by introducing calpeptin in our experimental design. Our results proved that calpeptin effectively inhibits VE-cadherin cleavage and that the permeability is comparable to that of controls after LPS exposure. These findings suggest that calpain is involved in the LPS-dependent cleavage of endothelial VE-cadherin and can be proposed as a target for developing alternative treatments for acquired hydrocephalus.

Our in vivo experimental design included intraventricular LPS injections and calpain inhibitors, resulting in reduced mortality and attenuated ventricular volume compared to untreated LPS-injected mice. Another study reported using calpain inhibitors in a model of hydrocephalus⁵⁷. The authors did not find benefits by using calpain inhibitors in their experiments. However, they used a kaolin-induced rat model of hydrocephalus, which is likely to not activate the calpain pathway as in our model. The authors also acknowledged that the drug delivery may not have been adequate in their experiments. Our survival improvement found in the LPS-injected mice treated with calpeptin is consistent with the body of evidence that supports the beneficial effect of calpain inhibition in organ failure during sepsis by intervening in the modulation of cell death and the inflammatory response^58–60 in which calpain-dependent vascular endothelial injury plays a relevant role due to pro-apoptotic effects^61–63. Our results showed vascular alterations in LPS-injected mice exhibiting diminished DiI staining and VE-cadherin in dorso-ventricular regions, indicative of vascular impairment, likely associated with endothelial apoptosis. Interestingly, mice treated with calpeptin did not show such vascular alterations, suggesting that the calpain pathway is also involved in the vascular pathology associated with AH.

Our research suggests that PAMPs and DAMPs activate TLRs, elevating cytoplasmic calcium levels. This increase in cytoplasmic triggers calpain activation as a pivotal factor in modulating vascular permeability by cleaving VE-cadherin and affecting endothelial integrity by initiating caspase-dependent pro-apoptotic pathways likely resulting in alterations of the blood vessels-dependent CSF homeostasis (Fig. 5).

In summary, our research proposes a pathway that interplays the TLR-Calpain pathway, cell junction pathology, and vascular impairment within the realm of AH, providing the backbone of future avenues of complementary treatments to reduce the burden of neurological complications experienced by individuals with this complex condition.

Limitations

This study is specifically centered on investigating the impact of lipopolysaccharide (LPS) on the parenchymal vascular system. Notably, the examination did not extend to other anatomical structures, such as the choroid plexus, as it was beyond the scope of the current manuscript. Additionally, our focus was directed solely towards elucidating the role of calpain activity on VE-cadherin and did not encompass an exploration of other cellular junctions. Therefore, the data presented herein are specifically applicable to the parameters and objectives outlined in this study.

Availability of data and materials: The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.

Acknowledgements: We thank Anthony Rangel for all the logistic support to carry out this work.

Funding: This study was supported by the Levecke Family Foundation and with a CHOC, CSO small grant 2021.

Author information

Authors and Affiliations

CHOC Children’s Research Institute, 1201 W. La Veta Avenue, Orange, CA, 92868, USA

Jenna Ledbetter, Sora Sato, Seunghyun Lee, Gio Jison, Bianca Romero, Amirhossein Shahriari,

Celine Tran, Leandro Castañeyra-Ruiz

Neurosurgery Department, CHOC Children’s Hospital, 505 S Main St., Orange, CA,

92868, USA

Michael Muhonen

Contributions: JL conducted experiments. SS and SL performed data analysis, provided technical support, and contributed to literature support. GJ offered technical and analytical advice and participated in manuscript editing. AS and CT contributed to literature support and participated in manuscript editing. ET and AP conducted animal procedures. MM contributed to the study's conception, provided guidance, and offered critical literature references. LCR conceived the idea, drafted the initial and revised manuscript, created and formatted figures, and oversaw experimental procedures.

Corresponding author: Correspondence to Leandro Castañeyra-Ruiz

Ethics approval and consent to participate: The authors have no conflict of interest to declare.

Consent for publication: Not applicable.

Competing interests: The authors declare that they have no competing interests.

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Calpeptin mitigates ventriculomegaly and microvascular impairment in a novel LPS- dependent mouse model of acquired hydrocephalus

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Abstract

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Introduction

Methods

Experimental Animals:

Magnetic Resonance Imaging in Mice:

Mouse perfusion and vessel painting:

Cell Growth:

Western blot:

Calpain activity:

Immunocytochemistry:

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Generative AI and AI-assisted technologies in the writing process:

Quantification and Statistical Analysis:

Results

TLR-Calpain Pathway Modulation Prevents Cytopathology Associated with Acquired Hydrocephalus In Vitro

LPS Induces Calpain-Dependent Vascular Alteration an in vivo Model of AH

Discussion

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References

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