Early immune responses to systemic inflammation in the postnatal mouse brain initiated by migrating macrophages and leptomeningeal fibroblasts

Early immune responses to systemic inflammation in the postnatal mouse brain initiated by migrating macrophages and leptomeningeal fibroblasts | 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 Early immune responses to systemic inflammation in the postnatal mouse brain initiated by migrating macrophages and leptomeningeal fibroblasts Rei Settsu, Aki Obara, Takehiko Tarui, Sanae Hasegawa-Ishii, Atsuyoshi Shimada This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6850479/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Oct, 2025 Read the published version in Journal of Neuroinflammation → Version 1 posted 13 You are reading this latest preprint version Abstract Bacterial infection is a key trigger of inflammatory pathways that damage the preterm brain, even without direct bacterial brain invasion. However, the mechanisms underlying the involvement of intracranial tissues and cells in early immune responses to acute systemic inflammation in preterm infants remain unknown. Lipopolysaccharide (LPS) was administered with a single intraperitoneal injection into postnatal day (P) 7 mice. Four hours later, the leptomeningeal macrophages initiated an intracranial reaction by producing IL-1β, which set the stage for the arachnoid, pial, and perivascular fibroblasts to act. These brain fibroblasts produced CCL2, which increased the number of brain parenchymal macrophages/microglia and led to their hypertrophy. The macrophages/microglia were then isolated from the brain 24 hours after LPS administration. Microarray analysis showed marked transcript expression of genes including Saa3, Irg1, Lcn2, and Cxcl9. RT-qPCR assays and histological staining showed that the expression level of Saa3 was upregulated in the leptomeningeal macrophages, cerebral perivascular macrophages, and cerebellar medulla macrophages, as well as in the fibroblasts of the leptomeninges, choroid plexus stroma, and perivascular space. The leptomeningeal macrophages expressing Saa3 continued their developmental migration from the leptomeninges to the parenchyma, such as the bundles that traverse the roof of the third ventricle and the corpus callosum. The expression level of Saa3 reached their peak 4 and 12 hours after LPS administration. The blood-brain barrier (BBB) of P7 pups remained intact up to 72 hours after LPS administration. The expression level of Irg1 was remarkably increased in the leptomeningeal macrophages and reached their peak 4 and 12 hours after LPS administration. The expression level of Lcn2 was the most increased in the perivascular fibroblasts and choroid plexus epithelial cells, peaking 4 and 12 hours after LPS administration. The expression level of Cxcl9 was also increased in the leptomeningeal fibroblasts, peaking 4 and 12 hours after LPS administration. The early enhancement of Saa3 expression played a key role in priming inflammation, initiated by macrophages and fibroblasts. Early enhancement of Irg1 expression was crucial for determining the direction of the inflammatory response. The increase of Lcn2 was vital, sequestering the iron siderophore to limit bacterial growth. It was interesting to observe how the early immune responses to systemic inflammation in the postnatal mouse brain were initiated by migrating macrophages and leptomeningeal fibroblasts, leading to immediate gene upregulation to protect immature brain tissue. prematurity endotoxin leptomeninges fibroblasts macrophages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Introduction In the year 2020, an estimated 13.4 million newborns were born preterm (before 37 completed weeks of gestation) [1]. Bacterial infection triggers inflammatory pathways that damage the preterm brain even without direct bacterial invasion into the brain [2]. Bacteria-induced systemic inflammation occurring in preterm infants, including bacteremia, sepsis, chorioamnionitis, and necrotizing enterocolitis, is a risk factor for adverse neurodevelopmental outcomes [3]. The neuropathological findings of this disability in preterm infants consist of mixed injuries of the cerebral white matter and gray matter [4, 5]. However, the mechanisms underlying the involvement of intracranial tissues and cells in early immune responses to acute systemic inflammation in preterm infants remain unknown. In our previous studies, we treated two-month-old C57BL/6N mice with a single intraperitoneal administration of lipopolysaccharide (LPS). The histological analysis showed that the macrophages of the choroid plexus and leptomeninges produced interleukin (IL)-1β most rapidly in response to LPS-induced systemic inflammation 1 hour after LPS administration [6-8]. These macrophages stimulated the choroid plexus epithelial and stromal cells to produce CC-motif ligand (CCL)2, CXC-motif ligand (CXCL)1, CXCL2, and IL-6 4 hours after LPS administration. These cytokines were then transported into the brain parenchyma [9]. Of the brain parenchymal cells, astrocytes were found to be the most efficient in responding to CCL2, CXCL1, CXCL2, and IL-6 using the cytokine receptors located on the endfeet. Astrocytes then produced CCL11, CXCL10 and G-CSF 24 hours after LPS administration. The astrocyte-derived cytokines activated microglia via receptors. This collaboration led to a shift in microglial gene expression, driving them towards the M2 phenotype. Brain parenchymal cytokine concentrations returned to control levels by 72 hours after a single LPS administration. It is known that the brain developmental stage of the postnatal day (P) 7 mouse is equivalent to that of the human fetus at a gestational age of about 30 weeks [10]. Therefore, we reasoned that the brain cellular responses produced by P7 mouse pups should represent the basic responses that occur in human preterm infants [11]. The choroid plexus and leptomeningeal macrophages may be key players in the initial response to systemic inflammation in newborn mice as well [12]. However, the roles of these leptomeningeal macrophages differ significantly between neonatal and adult mice. The leptomeningeal macrophages in pre- and postnatal mice are destined to migrate into the brain parenchyma to differentiate into parenchymal microglia [13, 14]. Whether the macrophages that are to become parenchymal microglia have enough ability to respond to systemic inflammation becomes an interesting question. We are exploring the possibility that leptomeningeal macrophages play key roles in the initial response to systemic inflammation in newborn mice. This has generated new questions, such as which cells could be partners in this vital process. Determining the functionality of the blood-brain barrier (BBB) in newborn mice is an important topic. It is well-established that the BBB of postnatal developing mice is well-constructed with endothelial cells and pericytes, making their barrier function as robust as that of the BBB in adult animals [15-18]. In addition, the cytoplasmic processes of astrocytes surround brain capillaries, arterioles, and venules in adults. At capillaries, the astrocytic endfeet are located on the brain side of the basement membrane that ensheaths the endothelial cells and pericytes [19]. Communication between endothelial cells and astrocytes is crucial for both the barrier and interface functions. Astrocytes use their highly efficient cytoplasmic processes in response to brain parenchymal cytokines in adult mice [7]. However, astrocytes of P7 mice do not have well-developed sophisticated cytoplasmic processes [20]. The formation of most of the capillary bed occurs between P8 and P10, but the astrocytic endfeet incompletely cover the vasculature before P10 and enwrap the entire brain vasculature by P21 [21]. Furthermore, the perivascular space of the brain is in direct continuity with the subarachnoid space. The periarterial space is lined with perivascular cells that are continuous with the pia mater [22]. The brain perivascular cells originate from the meninges and are first seen on the parenchymal vasculature at P5 [23]. After P5, perivascular cell coverage of the cerebral vasculature expands by local cell proliferation and migration from the meninges. Perivascular cells and perivascular macrophages develop in parallel. Recent studies have advanced our understanding of the relationship between the major meningeal cells and the perivascular cells. The arachnoid cells, pial cells, and perivascular cells have all been identified as genuine fibroblasts [23-27]. We hypothesized that the leptomeningeal and choroid plexus stromal macrophages play the central role in the early responses to systemic inflammation in the immature brains of newborn mice, and that the leptomeningeal and perivascular fibroblasts are among the important partners in initiating immune responses to protect the brain. Methods The Guide for the Care and Use of Laboratory Animals, 8th edition (National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Washington DC: National Academies Press, 2011), was followed for the handling of all mice. The Institutional Animal Care and Use Committee of the Kyorin University Faculty of Health Sciences approved all of the experiments described (Protocols I17–08–03 to I17–08–07). 2-1. Preparation of histological frozen brain sections Systemic inflammation was induced in seven-day-old (P7), male, C57BL/6N mice using LPS (from E. coli O55:B5; Sigma-Aldrich-Merck, Burlington, MA, USA), a bacterial endotoxin, which was administered intraperitoneally to the experimental group at a dose of 0.75 mg/kg with a 33-G Hamilton syringe (systemic inflammation group). The control group received a single intraperitoneal injection of saline at the same dose of 3.75 mL/kg as the experimental group (saline control group). After treatment, the mouse pups were returned to their dams for continued rearing. At 4, 12, 24, 48, and 72 hours after LPS or saline administration, ketamine-xylazine anesthetic solution was administered intraperitoneally at a dose of 10 mL/kg. Blood was extracted transcardially using a phosphate-buffered saline (PBS) solution followed by perfusion with Zamboni fixative solution at a flow rate of 3 mL/min. Following removal of the scalp, eyes, and mandible, the skull containing the brain, the liver, and the spleen were immersed in Zamboni fixative at 4 °C for 2 days. Each experimental group consisted of four mice. The brains were then extracted from the skulls. Each brain was bisected along the parasagittal plane located in the midline interhemispheric fissure so that the first parasagittal brain section was safely cut from the right hemisphere. Small fragments of the liver and spleen were prepared and embedded in the same blocks as the bisected brains. Cryoprotection was achieved through immersion of the tissue samples in 10, 15, and 20% sucrose in PBS at room temperature overnight. Brain, liver, and spleen tissues were then embedded in Cryomatrix embedding medium (Thermo Fisher Scientific, Waltham, MA, USA) in Tissue Tech Cryomold No. 3 (Sakura Finetech, Tokyo, Japan) and subsequently frozen with dry ice-cold n-hexane. Frozen blocks were sectioned at a thickness of 14 μm using a LEICA CM 3050S cryostat (Leica Biosystems, Deer Park, IL, USA). Median brain sections were obtained by cutting sequentially from 300 μm to the right of the interhemispheric fissure of the cerebral hemispheres. Approximately 40 sections were prepared as median sections. Lateral brain sections were prepared from 600 μm to the left of the interhemispheric fissure and sequentially cut to obtain 50 lateral sections. The prepared sections were mounted on FRC-04-coated glass slides (Matsunami Glass Co., Ltd., Osaka, Japan), followed by air drying and vacuum drying using a V-100 vacuum pump (BUCHI Labortechnik, Flawil, Switzerland). The dried sections were then stored at -20 °C until use. Sections from mice in all experimental groups were stained with hematoxylin and eosin (H&E) to observe inflammation-related changes, such as inflammatory cell infiltration, ischemia, and necrosis. The sections were subsequently used for immunohistochemical, double immunofluorescence, and in situ hybridization (ISH) staining. To examine the histology of major organs other than the brain by H&E staining, the heart, lung, liver, spleen, and kidney were embedded in paraffin and sectioned at a thickness of 4 μm using a microtome. 2- 2 . Immunohistochemistry and immunofluorescence staining Frozen sections were soaked in Tris-buffered saline with Tween 20 (TBS-T) for 10 min and pretreated with 0.3% H 2 O 2 in methanol to block endogenous peroxidase activity and with 1% BSA in TBS-T to block non-specific binding. Sections were incubated with primary antibodies (Table 1) overnight at 4 °C or for 2 hours at room temperature, followed by incubation with reagents from the ImmPRESS HRP Antidody (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, CA, USA) for 60 min at room temperature. Reactions were visualized by incubating sections with an ImmPACT DAB Substrate Kit, Peroxidase (SK-4105; Vector Laboratories). Sections were sequentially dehydrated through 80%, 90%, 95%, and 100% ethanol, cleared with xylene, and coverslipped with HSR mounting medium (Sysmex, Kobe, Japan). Immunohistochemical photographs were taken with 4x, 10x, 20x, and 40x PlanApo λ objectives (Nikon, Tokyo, Japan) of an Eclipse Ci-L light microscope equipped with a DS-Fi3/DS-L4 digital camera control unit (Nikon), and with 4x, 10x, 20x, and 40x PlanApo λ objectives (Nikon) of a BZ-X710 microscope (Keyence, Osaka, Japan). For double immunofluorescence staining, frozen sections were soaked in TBS-T for 10 min, preincubated with 1% BSA in TBS-T, and incubated with primary antibodies overnight at 4 °C. The combinations of the primary antibodies were IL-1β and Iba1, CCL2 and type 1 collagen, or IL-1R1 and type 1 collagen. After incubation with two primary antibodies, sections were incubated with donkey anti-goat or anti-rabbit IgG secondary antibodies conjugated with Alexa Fluor 568 or 488 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for 60 min at room temperature. Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Sections were coverslipped with Fluorescence Mounting Medium (DAKO, Agilent, Santa Clara, CA, USA). Fluorescence images were captured using a BZ-X710 microscope equipped with structured illumination. 2-3. Morphometric analysis of sections immunohistochemically stained with anti-Iba1 To evaluate the changes of microglial morphology that occurred after LPS administration, frozen brain sections from mice at 4, 24, 48, and 72 hours after LPS or saline administration (n = 4 in each group) were immunohistochemically stained with anti-ionized calcium-binding adaptor molecule-1(Iba1) antibody (rabbit monoclonal [EPR16588], Abcam, Cambridge, UK). For morphometric studies, a computerized image analyzer (WinROOF 2018, Mitani Corporation, Tokyo, Japan) was used to calculate the area fraction of Iba1-positive cells (total area of Iba1-positive cell bodies with cytoplasmic processes divided by the area of interest). In addition to the analysis functions, WinROOF 2018 had a variety of combined manual and automatic editing functions, such as separating contiguous cytoplasmic process images, filling in parts of cells with density below the threshold, and removing small cell fragments and artifacts. Each field was reviewed by the operator on the instrument screen to make these corrections. Analysis of the cerebellum, cerebral cortex, and hippocampus was performed. The number of cell bodies of Iba1-positive cells was also counted in the hippocampus only. Two histological sections per individual mouse were used for each brain region. For the hippocampus, the area was delineated from the lateral parasagittal sections located between 700 and 1100 μm lateral to the interhemispheric fissure. Using a histological section, the entire dorsal hippocampus was analyzed to quantify the area fraction (Fig. 1A). Within the designated area of interest, the number of cell bodies of Iba1-positive cells was counted. In the same sections in which the hippocampus was analyzed, the parietal cortex was located dorsal to the hippocampus, and the six cortical layers were perfectly recognizable. To define the field of view for the analysis of the cerebral cortex, the first line was drawn from the point on the cortico-medullary junction corresponding to the caudal end of the lateral ventricle (point b) and perpendicular to the cortical surface (point a). The second line was drawn from point d, located on the cortical surface 500 μm caudal to point a, perpendicular to the cortical surface to the cortico-medullary junction (point c). The arachnoid and pia on the cortical surface were also included in the area of interest (Fig. 1B). In the cerebellum, median sections were used for morphometric analysis. Notably, the cerebellum did not include the deep cerebellar nuclei, and the entire cerebellar section consisted of the cerebellar cortex and medulla and the leptomeninges (Fig. 1C). 2-4. Immunoassay of cytokine concentrations in brain parenchymal tissues At 4, 24, 48, and 72 hours after LPS or saline administration, mice were perfused systemically with PBS to remove blood. The brains were removed and quickly divided on an ice-cold glass plate into the following seven regions: left and right cerebral cortices, left and right limbic systems (including olfactory bulb, olfactory tubercle, piriform cortex, entorhinal cortex, and hippocampus), left and right subcortical structures (striatum, diencephalon, midbrain, and brainstem), and cerebellum. These seven brain parts were individually snap frozen in liquid nitrogen and stored at -80 °C until they were used. Of the seven parts, the left cerebral cortex, left limbic system, and cerebellum were used to measure cytokine concentrations. Tissue Protein Extraction Reagent (T-PER, Thermo Fisher Scientific) was added to a Biomasher II tube (Nippi, Tokyo, Japan) at 20-fold tissue weight, and 1/100 volume of Halt Protease Inhibitor Cocktail (100x, Thermo Fisher Scientific) was added to T-PER. Tissue samples stored at -80 °C were added to these tubes and homogenized. After centrifugation at 13,000 rpm, 4 °C, for 5 min, only the supernatant was collected and used as the protein extraction solution. The extract was dispensed into 50-µL portions into microtubes and stored at -80 °C. Protein yield was measured by colorimetric quantification using bicinchoninic acid (BCA Protein Assay Kit, TaKaRa, Shiga, Japan) and serial dilution of bovine serum albumin (BSA). Using the Luminex 200 xPONENT system (Thermo Fisher Scientific), a simultaneous multiplex protein immunoassay system of protein extracts from the left cerebral cortex, left hippocampus, and cerebellum was prepared to determine the tissue concentrations of the following 15 cytokines: CCL2, CCL11, CXCL1, CXCL2, CXCL10, granulocyte colony stimulating factor (G-CSF), IL-1α, IL-1β, IL-4, IL-6, IL-10, IL-12, IL-17, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). 2-5. Isolation of CD11b(+) cells, RNA extraction, and microarray analysis At 24 hours after LPS or saline administration, mice were deeply anesthetized with ketamine and xylazine. Blood was washed out by transcardial perfusion with sterile Dulbecco’s phosphate-buffered saline [D-PBS(−)] to remove plasma and blood cells. Fresh whole brains, including the arachnoid, pia, and parenchyma, were quickly removed. Two brains from the same dam puppies, treated in the same experimental manner, were placed in a 50-mL conical tube containing ice-cold D-PBS(−) and processed as a single sample. Four samples were prepared in each of the systemic inflammation and control groups. To collect macrophages and microglial cells from fresh brains, CD11b(+) cells were isolated using magnetic-activated cell sorting (MACS) methods [28]. Whole brains were dissociated by enzymatic digestion of the extracellular matrix using the Adult Brain Dissociation Kit for mice (Miltenyi Biotec, Auburn, CA, USA). Mechanical dissociation steps were performed using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) according to the manufacturer’s protocol. Brain tissue dissociates were centrifuged at 400 × g for 5 min at 4 °C. The pellets were resuspended in cold D-PBS (+) (with calcium and magnesium) containing 0.5% bovine serum albumin (PB buffer). In the presence of the kit’s Debris Removal Solution, the cell suspensions were centrifuged at 3000 × g for 12 min at 4 °C to remove the debris phase. The pellets were resuspended in cold PB buffer and incubated with R-phycoerythrin (PE)-conjugated primary human/mouse CD11b monoclonal antibody (130–113–235, Miltenyi Biotec) and Fc receptor blocking reagent (130–092–575, Miltenyi Biotec), followed by incubation with MicroBeads UltraPure conjugated to anti-PE monoclonal antibody (130–105–639, Miltenyi Biotec). Suspended cells labeled with anti-PE MicroBeads were enriched by magnetic separation using an LS column (Miltenyi Biotec) that was placed in a QuadroMACS separator (Miltenyi Biotec) according to the manufacturer’s protocol. CD11b-positive-selected cells (positive fraction) were considered macrophages and microglia. During the positive selection process, CD11b-negative cells were also collected (negative fraction). The number of cells in the positive and negative fractions was determined using cell counting plates (OneCell counter; Fine Plus International, Kyoto, Japan) under an ECLIPSE Ts2 (Nikon) inverted phase-contrast microscope. The cells of the positive and negative fractions were finally suspended in 1 mL CELLBANKER 1 Plus (TaKaRa) and stored at −80 °C before RNA extraction. Frozen cells were thawed rapidly at 37 °C, centrifuged at 400 × g for 5 min at 4 °C, and washed with RNase-free PBS by centrifugation under the same conditions. Total RNA was extracted from the cell pellets using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. RNA quantification was performed using NanoVue (GE Healthcare Life Sciences, Chicago, IL, USA) and 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA). Eight RNA samples were of high quality with RNA Integrity Numbers (RINs) ranging from 7.9 to 9.1. Gene expression profiles were analyzed by Hokkaido System Science Co., Ltd. (Hokkaido, Japan) using the SurePrint G3 Mouse GE 8x60K Ver.2.0 Microarray (Agilent, G4852B). Cyanine-3 (Cy3)-labeled cRNA was prepared from 50 ng of total RNA using the Low Input Quick Amp Labeling Kit (Agilent), followed by RNeasy column purification (QIAGEN). Then, 0.6 μg of Cy3-labeled cRNA (specific activity > 6 pmol Cy3/μg cRNA) was fragmented at 60 °C for 30 min in a reaction volume of 25 μL containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent. On completion of the fragmentation, 25 μL of 2x Agilent GE Hi-RPM hybridization buffer were added. The fragmentation mixture was hybridized to SurePrint G3 Mouse GE 8x60K Ver.2.0 Microarray at 65 °C for 17 hours in an Agilent rotating hybridization oven. After hybridization, the microarrays were washed with GE Wash Buffer 1 (Agilent) for 1 min at room temperature and with GE Wash Buffer 2 (Agilent) for 1 min at 37 °C. The slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G2600D) using one color scan setting for 8x60K array slides (dye channel set to green, and green photomultiplier tube set to 100%). The scanned images were analyzed with Feature Extraction Software 12.0.3.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended processed signal intensities. The 75th percentile shift normalization was performed using Agilent GeneSpring GX 14.9, and baseline transformation was performed using the median of all samples. 2-6. Real-time reverse transcription-polymerase chain reaction (RT-qPCR) At 4, 12, 24, 48, and 72 hours after LPS or saline administration (n = 4 samples in each group), mice were deeply anesthetized with ketamine-xylazine, blood was poured out, and fresh whole brains were quickly removed. Two brains of P7 mice from the same dam, treated in the same experimental manner, were pooled in ice-cold D-PBS(−) and processed as a single sample. CD11b(+) cells were isolated by MACS, and total RNA was extracted from CD11b(+) and CD11b(−) cells. RNA quantification was performed using NanoVue (GE Healthcare Life Sciences) and using 4150 TapeStation System (Agilent). Eighty RNA samples were of high quality with RINs ranging from 8.9 to 9.8. Fifty nanograms of total RNA were used for reverse transcription to cDNA using SuperScript III Reverse Transcriptase (Invitrogen-Thermo Fisher Scientific). The real-time reverse transcription-polymerase chain reaction (RT-qPCR) was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems -Thermo Fisher Scientific), TaqMan primer/probe sets for 10 targets (Applied Biosystems), and a 7500 Fast Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocols. The targets were as follows: (1) Saa3 (encoding serum amyloid A3), Mm00441203_m1; (2) Saa1 (encoding serum amyloid A1), Mm00656927_g1; (3) Saa2 (encoding serum amyloid A2), Mm04208126_mH; (4) Irg1 (encoding immune-responsive gene 1 [IRG1], also known as aconitate decarboxylase 1), Mm01224532_m1; (5) Ccl5 (encoding chemokine CCL5), Mm01302428_m1; (6) Cxcl13 (encoding chemokine CXCL13), Mm00444534_m1; (7) Slfn4 (encoding schlafen-4), Mm01298330_m1; (8) Cxcl9 (encoding chemokine CXCL9), Mm00434946_m1; (9) Lcn2 (encoding lipocalin-2), Mm01324470_m1; (10) internal control, Hprt (encoding hypoxanthine phosphoribosyltransferase), Mm03024075_m1. Analysis of relative transcript levels was performed using the ΔΔCT method. All assays were performed in triplicate. 2-7. In situ hybridization (ISH) Frozen sections were prepared from mice at 4, 12, 24, and 48 hours after LPS or saline administration (n = 4 mice in each group) and used for ISH. ISH was performed with RNAscope 2.5 HD Assay-Brown for fixed frozen tissue (#322310; Advanced Cell Diagnostics, Newark, CA, USA) according to the manufacturer’s protocol with minor modification. The modification was as follows: Protease Plus was diluted 1:2 and incubated for 10 min, and DAB precipitation was performed using the ImmPACT DAB Substrate Kit, Peroxidase. Target probes for the RNAscope manual assay (Advanced Cell Diagnostics) were Mm-Saa3 (Cat. No. 446841), Mm-Irg1 (450241), Mm-Cxcl13 (406311), Mm-Ccl5 (469601), Mm-Cxcl9 (489341), Mm-Lcn2 (313971), Mm-Slfn4 (573011), and Mm-Col1a1 (319371). RNAscope DAB precipitation was coupled to immunohistochemistry using anti-Iba1 antibody (rabbit monoclonal [EPR16588], Abcam) or anti-type I collagen antibody (rabbit monoclonal [EPR24331-53], Abcam). The ImmPRESS-AP Horse Anti-Rabbit IgG Polymer Detection Kit, Alkaline Phosphatase (MP-5401, Vector), was used as the secondary antibody. Immunohistochemistry was visualized using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) Substrate Kit, Alkaline Phosphatase (AP) (SK-5400, Vector). Sections were covered with G-Mount (Genostaff, Tokyo, Japan) and then coverslipped with HSR (Sysmex). 2-8. Statistical analysis For the tissue cytokine immunoassay and the morphometry of Iba1-immunopositive cells, data were obtained from 8 experimental conditions (saline vs. LPS administration; 4, 24, 48, and 72 hours after administration), and the results were analyzed by two-way analysis of variance (ANOVA; main effects of treatment and time). For RT-qPCR analyses, the mean ΔCT values for each gene target were obtained from 10 experimental conditions (saline vs. LPS administration; 4, 12, 24, 48, and 72 hours after administration). The results were analyzed by two-way ANOVA. Post hoc tests were performed using Tukey’s procedure. P values less than 0.05 were considered significant in all analyses. Results 3-1. IL-1β expressed by meningeal macrophages and IL-1R1 expressed by leptomeningeal fibroblasts The mice used in the present study ranged in age from P7 to P10 and thus exhibited a postnatal developmental increase in body weight. The pattern of body weight change was affected by the administration of LPS or saline to the P7 mice. The change in body weight was evaluated by taking the body weight just before tissue collection minus the body weight just before intraperitoneal administration, as shown in Fig. 2A. The mice injected with 0.75 mg/kg LPS lost 0.21 g at 12 hours (n = 8) and 0.22 g at 24 hours (n = 40), followed by a gain of 0.72 g at 48 hours (n = 16) and 1.21 g at 72 hours (n = 19) after LPS injection. Mice with systemic inflammation exhibited remarkable sickness behavior during the period 1 to 12 hours after LPS administration, and 98% of LPS-treated mice survived after LPS administration. In preliminary experiments to determine the dose of LPS, about 15% of mice died within one day after a dose of 1.0 mg/kg of LPS was administered (data not shown). Mice with systemic inflammation showed a slight increase in the number of polymorphonuclear leukocytes in the leptomeninges 12 and 24 hours after LPS administration, but the extent of inflammatory cell infiltration was much less than seen in meningitis (Fig. 2B,C). There was no evidence of focal or global acute ischemic changes in the brain parenchyma, with no hemorrhagic or edematous lesions that could attract inflammatory leukocytes. A small number of polymorphonuclear leukocytes also infiltrated the other major organs, including the lungs, liver, and kidneys (Fig. 2D,E). In all mice 4 hours after LPS administration, the protein expression of IL-1β was evident in Iba1-positive macrophages located in the subarachnoid space and in the choroid plexus stroma (Fig. 3A-E). In the medulla of the cerebellum, there were many Iba1-positive cells, which were considered to be the macrophages that migrate and invade the nearby brain parenchyma to become microglia at 7 days of age. Twenty-eight percent of the Iba1-positive cells in the medulla of the cerebellum were immunopositive for IL-1β 4 hours after LPS administration (Fig. 3F). The microglia located in the circumventricular organs (CVOs), including the area postrema, median eminence, and subfornical organ, expressed IL-1β, probably due to plasma LPS leakage from the blood vessels lacking the BBB (Fig. 3G). In contrast, microglia located in most of the other brain parenchyma, except CVOs, did not express IL-1β, which suggested the presence of a functional BBB in P7 mice. IL-1R1, a receptor for IL-1β, was expressed by arachnoid cells and pial cells, which are the major cells of the leptomeninges and were recently identified as genuine fibroblasts [23-27]. Double immunohistochemical staining showed that leptomeningeal cells immunopositive for type I collagen exhibited IL-1R1 immunopositivity at the edge of the cytoplasm (Fig. 3H). 3-2. Immunoassay of tissue cytokine concentration in the brain parenchyma The yields of protein extracts from the left cerebral cortex, left limbic system, cerebellum, and spleen were measured before the tissue cytokine concentrations were immunoassayed. The protein concentrations were approximately 2000 µg/mL in the left cerebral cortex and left limbic system and 2300 µg/mL in the cerebellum and spleen, confirming that the protein extraction was stable and reliable. The levels of CCL2, G-CSF, CXCL10, IL-6, and CXCL1 in the brain parenchyma and spleen tissues were significantly increased in mice with LPS-induced systemic inflammation compared with saline control (Figs. 4-8). The time-dependent changes in the tissue concentrations of each cytokine were similar among the three brain regions. CCL2 levels were increased in the cerebral cortex, limbic system, and cerebellum 4 and 24 hours after LPS administration (Fig. 4). The time-dependent changes in CCL2 levels in the brain parenchyma were similar to those in the spleen, but CCL2 levels in the spleen appeared to decrease more rapidly toward the control levels later than 4 hours after LPS administration. Immunohistochemistry using anti-CCL2 antibody was effective in identifying the cells involved in CCL2 production. CCL2-immunopositive cells were lined along the blood vessels in the leptomeninges and brain parenchyma 4 hours after LPS administration (Fig. 4 E-H). Double immunofluorescence staining for CCL2 and type I collagen showed that the CCL2-immunopositive cells were perivascular fibroblasts (Fig. 4I). G-CSF levels were increased in the cerebral cortex, limbic system, and cerebellum 4 and 24 hours after LPS administration (Fig. 5A). The time-dependent changes in G-CSF levels in the brain parenchyma were similar to those in the spleen, and G-CSF levels in the spleen were higher than those in the brain 4 and 24 hours after LPS administration. CXCL10 levels were higher in the cerebral cortex, limbic system, and cerebellum of mice with systemic inflammation than in saline control mice 4 and 24 hours after LPS administration (Fig. 5B). CXCL10 levels in the spleen of mice with systemic inflammation were significantly higher than in saline control mice only 4 hours after LPS administration. IL-6 levels were increased in the cerebral cortex, limbic system, and cerebellum 4 hours after LPS administration (Fig. 5C). The time-dependent changes in IL-6 levels in the brain parenchyma were the same as those in the spleen, but IL-6 levels in the spleen were much higher than those in the brain 4 hours after LPS administration. CXCL1 levels were higher in the cerebral cortex, limbic system, and cerebellum of mice with systemic inflammation than in saline control mice 4 hours after LPS administration (Fig. 5D). The time-dependent changes in CXCL1 levels in the brain parenchyma were the same as those in the spleen, but CXCL1 levels in the spleen were much higher than those in the brain in mice with systemic inflammation 4 hours after LPS administration. CCL11 levels were higher in the cerebral cortex, limbic system, and cerebellum of mice with systemic inflammation than in saline control mice 4, 24, and 48 hours after LPS administration (Fig. 6A). However, CCL11 levels in brain parenchymal tissues prepared from mice with systemic inflammation were relatively low compared with the five cytokines described above. CCL11 levels in the spleen of mice with systemic inflammation were highly elevated 4 hours after LPS administration. The pattern of increase in CXCL2 levels varied depending on the part of the brain (Fig. 6B). There were higher CXCL2 levels in the cerebral cortex of mice with systemic inflammation than in saline control mice 4 and 24 hours after LPS administration. CXCL2 levels were significantly higher in the cerebellum 4 hours after LPS administration than in saline-injected mice. However, there was no significant difference in CXCL2 levels in the limbic system at any time point after LPS administration. In contrast, CXCL2 levels in the spleen of LPS-treated mice were significantly and markedly higher in the cerebellum 4 hours after LPS administration than in saline-injected mice. IL-10 levels were increased only in the cerebral cortex, among the brain parenchyma, of LPS-treated mice compared with saline-injected mice 24 hours after LPS administration (Fig. 6C). In contrast, IL-10 levels were increased in the spleen 4 and 24 hours after LPS administration. There were no significant changes in IL-1α or IL-1β levels in any brain parenchymal region at any time point after LPS administration (Fig. 7). Levels of IL-1α and IL-1β were higher only in the spleen of mice with systemic inflammation than in saline control mice 4 and 24 hours after LPS administration. An increase in IFN-γ levels was not evident in the brain at any time point, but only in the spleen of mice with systemic inflammation compared with saline control mice 4 hours after LPS administration (Fig. 8A). Higher IL-17 levels were not evident in the brain at any time point, but only in the spleen of mice with systemic inflammation compared with saline control mice 24 hours after LPS administration (Fig. 8B). Higher TNF-α levels were only evident in the spleen of LPS-treated mice compared with saline-injected mice 4 hours after LPS administration (Fig. 8C). TNF-α levels in the brain were below the minimum detectable concentration according to the manufacturer’s assay characteristics in any region at any time point after saline or LPS injection. IL-12 and IL-4 levels in the brain and spleen were below the minimum detectable concentrations in all experimental groups. 3-3. Histological morphometry of glial cells Immunohistochemical staining for Iba1 was performed to clarify time-dependent changes in microglial morphology induced by LPS administration. Microglial changes differed between the cerebellar and non-cerebellar regions. First, in non-cerebellar regions, such as the cerebral cortex and hippocampus, of mice with systemic inflammation, microglia became hypertrophic with thicker and shorter cytoplasmic processes 4 hours after LPS administration (Fig. 9A,B). Subsequently, a maximum degree of cellular hypertrophy accompanied by an increased number of Iba1-positive cells was evident 24 hours after LPS administration compared with saline control mice (Fig. 9C,D). In particular, perivascular macrophages became extremely hypertrophic in mice with systemic inflammation 24 hours after LPS administration; they were probably destined to enter the cortical parenchyma (Fig. 9E,F). These results were confirmed by the morphometric analysis. The Iba1-positive area fraction of the cerebral cortex and hippocampus was significantly higher in mice with systemic inflammation than in saline control mice 24 hours after LPS administration (Fig. 10A,B). In the hippocampus, the number of Iba1-positive cells was significantly higher in mice with systemic inflammation than in saline control mice 24 and 48 hours after LPS administration (Fig. 10C). In general, there was a time-dependent increase in the number of Iba1-positive cells in the brain parenchyma of both the LPS-treated and saline-injected mice, due to the postnatal development of microglia in which leptomeningeal macrophages migrate into the brain parenchyma. In contrast, cerebellar microglia or macrophages were densely distributed in the medulla and less so in the cortex in saline control mice (Fig. 9G). In the normal development of the cerebellum, there was no increase in the Iba1-positive area fraction during the developmental period between P7 and P10 in saline control mice (Fig. 10D). The dense distribution of macrophages in the medulla was specific to pup mice, and the macrophages in the medulla were likely to be distributed to the nearby cerebellar cortex to become microglia by adulthood. Systemic inflammation led to an increased number of microglia and their hypertrophy in the cerebellar cortex 24 and 48 hours after LPS administration (Fig. 9H and 10D). Immunohistochemical staining for GFAP and S-100 was also performed to observe morphological changes in astrocytes. Regarding the astrocytes in the cerebral cortex, it is well known that the protein expression level of GFAP is quite different between the astrocytes of the superficial and deep layers and those of the middle layers, and the findings apply to both pups and adults. The middle-layer astrocytes, in which GFAP expression was undetectable immunohistologically, but the S-100 level was detectable, became hypertrophic and were detectable by immunohistochemistry for GFAP and S-100 in mice with systemic inflammation (Fig. 11). These hypertrophic changes in the cortical astrocytes represented some important homeostatic changes in response to systemic inflammation. 3-4. Microarray analysis of CD11b-positive cells Total RNA was extracted from the brain-derived CD11b-positive cells isolated from mice 24 hours after administration of LPS or saline. The microarray data are available in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE289767) under accession number GSE289767. [A secure token has been created to allow review of record GSE289767 while it remains in private status: ujqxmmiqvlkjxux] A comparison of the changes in gene expression between LPS-treated and saline-injected mice identified 1122 gene probes with significantly increased transcript expression levels and 1616 gene probes with significantly decreased transcript expression levels (Fig. 12). Of the genes that showed significant upregulation, the top 20 genes whose transcript expression levels were the highest in mice with systemic inflammation compared with saline control were as listed in Table 2. Upregulation of the top 9 genes was as follows: serum amyloid A3 ( Saa3 ) was 4924-fold (corrected p = 0.000094), C-X-C motif chemokine ligand 13 ( Cxcl13 ) was 289-fold (corrected p = 0.0023), serum amyloid A1 ( Saa1 ) was 265-fold (corrected p = 0.000094), serum amyloid A2 ( Saa2 ) was 135-fold (corrected p = 0.0022), C-C motif chemokine ligand 5 ( Ccl5 ) was 79-fold (corrected p = 0.00041), immune-responsive gene 1 ( Irg1 , or cis-aconitate decarboxylase 1 ( Acod1 )) was 69-fold (corrected p = 0.0022), schlafen 4 ( Slfn4 ) was 41-fold (corrected p = 0.0027), C-X-C motif chemokine ligand 9 ( Cxcl9 ) was 31-fold (corrected p = 0.0046), and lipocalin-2 ( Lcn2 ) was 29-fold (corrected p = 0.0022). In contrast, CCL2, G-CSF, CXCL10, IL-6, and CXCL1, which showed marked increases in tissue concentrations based on the LUMINEX immunoassay, the fold changes in gene expression were 1.10-fold (CCL2), 9.61-fold (G-CSF), 2.15-fold (CXCL10), -1.82-fold (IL-6), and -1.13-fold (CXCL1). The release system of these cytokines was not via gene transcription. 3-5. RT-qPCR for validation of microarray results and time-dependent changes in gene transcript expression RT-qPCR analyses of the top 9 genes were performed using total RNA extracted from the CD11b(+) and CD11b(−) cells prepared 4, 12, 24, 48, and 72 hours after LPS or saline injection. The upregulation of transcript expressions of the nine genes in CD11b(+) cells isolated from mice with systemic inflammation and saline control mice was validated overall (Figs. 13-15). For each gene, fold changes in transcript expression due to LPS treatment compared with saline injection were evaluated. The changes in expression of CD11b(+) and CD11b(−) cells were examined in detail. The changes were analyzed as a function of time after LPS administration. Each gene is described according to the degree of fold change. The transcript expression of Saa3 in CD11b(+) cells showed the most significant increase, reaching an astonishing 10,790-fold upregulation compared with the saline control 4 hours and a marked 4,459-fold increase 12 hours after administration of LPS or saline (Fig. 13A). Saa3 expression in CD11b(+) cells then decreased gradually, but it was still significantly upregulated up to 72 hours after administration. Saa3 expression in CD11b(−) cells also showed marked increases, reaching 6735-fold and 4230-fold 4 and 12 hours after LPS or saline administration, respectively (Fig. 13B). This indicates that Saa3 expression was markedly increased in both CD11b(+) and CD11b(−) cells as early as 4 and 12 hours after LPS administration. Systemic inflammation-activated changes in Cxcl9 transcript expression were upregulated as high as 4862-fold compared with saline control in CD11b(−) cells at 12 hours and 1294-fold at 4 hours after administration of LPS or saline, and thereafter decreased gradually (Fig. 14B). This marked increase in Cxcl9 expression was primarily driven by CD11b(−), since the degree of Cxcl9 upregulation in CD11b(+) cells was much lower, reaching 99-fold at 12 hours and 101-fold at 4 hours (Fig. 14A). The transcript expression of Irg1 (or Acod1 ) in CD11b(+) cells showed the most marked increase, reaching 1301-fold upregulation compared with the saline control at 4 hours and a notable 866-fold increase at 12 hours after administration of LPS or saline. Thereafter, the expression decreased gradually (Fig. 14C). Irg1 expression in CD11b(−) cells also showed marked upregulation, reaching 1012-fold at 4 hours and 659-fold at 12 hours after LPS or saline treatment, followed by a gradual decrease (Fig. 14D). This suggests that Irg1 expression is strongly increased in both CD11b(+) and CD11b(−) cells as early as 4 and 12 hours after LPS administration. Systemic inflammation-activated changes in Lcn2 transcript expression were as high as 957-fold upregulation compared with saline control in CD11b(−) cells at 12 hours and 359-fold at 4 hours after administration of LPS or saline, and decreased gradually thereafter (Fig. 13F). This surge in Lcn2 expression was primarily driven by CD11b(−), aligning with the pattern seen in Cxcl9 expression. The degree of Lcn2 upregulation in CD11b(+) cells was much lower, reaching 56-fold at 12 hours and 72-fold at 4 hours (Fig. 13E). The transcript expressions of Saa2 and Saa1 in CD11b(+) cells were the most marked, exhibiting a 750-fold increase and 491-fold increase, respectively, compared with the saline control. This was seen 4 hours after administration of LPS or saline, and decreased gradually thereafter (Fig. 14E,G). The expressions of Saa2 and Saa1 in CD11b(−) cells also exhibited marked upregulation, reaching 205-fold and 77-fold upregulation, respectively, compared with saline control at 12 hours (Fig. 14F,H). The transcript expression of Ccl5 in CD11b(+) cells showed the most significant increase, reaching 348-fold upregulation compared with the saline control. This marked response occurred just 4 hours after the administration of LPS or saline, and then decreased gradually over time (Fig. 15A). The degree of Ccl5 upregulation in CD11b(−) cells also showed a high response, with 172-fold upregulation at 4 hours, followed by a gradual decrease (Fig. 15B). The transcript expression of Cxcl13 in CD11b(+) cells showed the most marked increase, reaching 345-fold upregulation compared with the saline control. This response occurred 12 hours after the administration of LPS or saline, and then decreased gradually over time (Fig. 15C). Cxcl13 expression in CD11b(−) cells reached 51-fold upregulation at 12 hours, followed by a steady decrease (Fig. 15D). The transcript expression of Slfn4 in CD11b(+) cells showed the most marked increase, reaching 105-fold upregulation compared with the saline control. This marked response occurred just 4 hours after the administration of LPS or saline, and then decreased gradually over time (Fig. 15E). In addition, Slfn4 expression in CD11b(−) cells reached 22-fold upregulation at 12 hours, followed by a steady decrease (Fig. 15F). 3-6. Identification of cells upregulating gene expression by ISH ISH was performed to determine which cells upregulated gene expression using the histological brain sections. Mouse brain areas were identified with the help of anatomical atlases [29-31]. Saa3 transcript levels were highly expressed in cells located in the leptomeninges and choroid plexus stroma, with frequent cells in the perivascular space 4 to 24 hours after LPS administration (Fig. 13C,D). No Saa3 expression was observed by ISH in any region of the brain or leptomeninges at any timepoint in saline control mice. The major cells with high Saa3 expression were located in the leptomeninges, perivascular space, choroid plexus stroma, and cerebellar medulla part (Fig. 16A-H). Tangential sections of the leptomeninges, covering the interhemispheric fissure, showed a high distribution density of Saa3 -expressing cells (Fig. 16J). These Saa3 -expressing cells were identified as macrophages, based on double staining with ISH targeting Saa3 , followed by immunohistochemical examination using anti-Iba1 antibody (Fig. 17A,B). The other major cells with high Saa3 expression were identified as fibroblasts by double staining with ISH targeting Saa3 , followed by immunohistochemical examination using anti-type I collagen antibody (Fig. 17C). When the midsagittal and parasagittal sections were compared, the midsagittal sections, which contained the 3rd ventricle and its choroid plexus, highlighted the presence of Saa3 -expressing cells much more frequently than the parasagittal sections (Fig. 18A,B). This was because a long rostro-caudal extension of the choroid plexus stroma and the parallel leptomeninges (the roof of the third ventricle) were well populated with macrophages and fibroblasts (Fig. 18C). Further findings, by comparing the midsagittal sections made at 4, 12, and 24 hours after LPS administration, showed that the Saa3 -expressing macrophages located in the leptomeninges forming the roof of the third ventricle enter the brain parenchyma in a time-dependent manner to become microglia (Fig. 18D-F). Cell counting showed that the number of Saa3 -positive macrophages/microglia was initially low 4 hours after LPS administration. However, at 12 hours, there was a significant surge in the number of Saa3 -positive macrophages/microglia in the ventral hippocampal commissure (Fig. 17D), and at 24 hours, a significantly increased number of Saa3 -positive macrophages/microglia was evident in the ventral hippocampal commissure, fornix, medial habenula, and corpus callosum (Fig. 18G-J). The movement of these Saa3 -positive macrophages did not represent an expansion of inflammation-related events, since the migration rate was relatively slow, no lesion such as ischemia or necrosis was found in the brain parenchyma, Saa3-positive macrophages/microglia bore fine cytoplasmic processes different from activated morphology (Fig. 17D), and the migration routes were consistent among all LPS-treated mice. This migration by macrophages was interpreted as representing an important stage of neurodevelopment, that of microglial formation. This microglial formation was considered to occur in the same fashion in saline-injected mice, although normal macrophages/microglia were not detectable due to the absence of Saa3 expression. In addition, microglia located in the CVOs, such as the area postrema, median eminence, vascular organ of the lamina terminalis, subfornical organ, and subcommissural organ, also expressed Saa3 (Fig. 16I). In contrast, the microglia located in most of the brain parenchyma except the CVOs bore multiple cytoplasmic processes and never expressed Saa3 . The transcript expression of Irg1 was marked in the macrophages in the choroid plexus, with many of these being epiplexus cells, and in the leptomeningeal macrophages (Fig. 19A,B). Macrophages surrounding the blood vessels in the medulla part of the cerebellum showed high Irg1 expression as well (Fig. 19C). Microglia in the CVOs also showed clear upregulation of Irg1 (Fig. 19D), but brain parenchymal microglia did not express Irg1 . These Irg1 -positive cells were identified as macrophages by double staining with ISH targeting Irg1 followed by immunohistochemical examination using anti-Iba1 antibodies (Fig. 19E,F). The other Irg1 -positive cells in the leptomeninges that did not express Iba1 were not identified as a cell type. Macrophages showed Irg1 upregulation most markedly at 4 hours, and Irg1 expression was decreased 12 hours after LPS administration. Irg1 expression was not detected in saline control mice at any time point, underscoring the specificity of the observations. ISH targeting Lcn2 showed high expression in the perivascular cells and in the arachnoid and pial cells (Fig. 13G,H and Fig. 20A-D). These cells were identified as fibroblasts by immunohistochemical double staining for type 1 collagen (Fig. 20E,F). The Lcn2 upregulation in fibroblasts was the most marked 4 and 12 hours after LPS administration. Considering that our LPS was originally prepared from E. coli, perivascular fibroblasts played an essential role in protecting the brain tissue from potential E. coli invasion because lipocalin-2 mediates the innate immune response to inhibit bacterial growth by sequestering the iron-laden siderophore (Flo et al., 2004). In addition, the choroid plexus epithelial cells started clear Lcn2 expression slightly later than fibroblasts, peaking at 12 hours and still being high at 24 hours after LPS administration (Fig. 20G,H). Lcn2 expression was not detected in saline control mice at any time point. The transcript expression of Cxcl9 was remarkable in the arachnoid, pial, and perivascular fibroblasts (Fig. 21A-D). Cxcl9 was expressed in the choroid plexus fibroblasts but not in the epithelium (Fig. 21E,F). These results were confirmed by double staining of ISH targeting Cxcl9 followed by immunohistochemical examination for type I collagen. The upregulation of Cxcl9 in brain fibroblasts was most remarkable at 4 and 12 hours after LPS administration but not detected in saline control mice. The transcript expressions of Ccl5 and Cxcl13 were confirmed using the sections prepared 12 hours after saline or LPS injection, and these expressions were mainly detected in the choroid plexus in mice with systemic inflammation (Fig. 22A,C). Ccl5 transcript expression was also detected in the CVO (Fig. 22B). Slfn4 transcript expression was increased by systemic inflammation in the meningeal space and choroid plexus 4 h after LPS administration (Fig. 22D). ISH conclusively showed that the early major immune reactions of the brain in response to systemic inflammation were initiated by leptomeningeal, perivascular, and choroid plexus macrophages and arachnoid, pial, and perivascular fibroblasts in the postnatal mouse brain. Discussion 4-1. Summary of main results The present study explored how intracranial tissues respond to systemic inflammation to protect the brain in preterm babies. To create an animal model, E. coli -derived LPS at 0.75 mg/kg was administered by a single intraperitoneal injection into P7 mice and induced systemic inflammation. Just 4 hours later, the leptomeningeal and perivascular macrophages initiated an intracranial reaction by producing IL-1β, which set the stage for the arachnoid, pial, and perivascular fibroblasts to act. The brain fibroblasts produced CCL2, which increased the number of brain parenchymal macrophages/microglia and led to their hypertrophy. The macrophages/microglia were then isolated from the brain 24 hours after LPS administration, and total RNA was extracted. The microarray analysis showed markedly upregulated transcript expressions of Saa3 , Irg1 , Lcn2 , and Cxcl9 . RT-qPCR assays and histological staining showed that the expression level of Saa3 was increased in leptomeningeal macrophages, cerebral perivascular macrophages, and cerebellar perivascular macrophages, as well as in the fibroblastic cells of the leptomeninges, choroid plexus stroma, and perivascular space. Interestingly, the leptomeningeal macrophages expressing Saa3 continued their developmental migration from the leptomeninges to the parenchyma, such as the ventral hippocampal commissure, fornix, medial habenula, and corpus callosum. The expression level of Saa3 reached their peak 4 and 12 hours after LPS administration. The BBB of P7 pups was so well constructed that the barrier function was not disrupted up to 72 hours after LPS administration, and thus the microglia located in all brain parenchyma regions except for the CVOs never increased expressions of inflammation-related genes. The expression level of Irg1 was remarkably increased in the choroid plexus and leptomeningeal macrophages and reached their peak 4 and 12 hours after LPS administration. The expression level of Lcn2 was the most increased in the perivascular fibroblasts and choroid plexus epithelial cells, peaking 4 and 12 hours after LPS administration. The expression level of Cxcl9 was increased in the fibroblasts in the leptomeninges, perivascular space, and choroid plexus stroma, peaking 4 and 12 hours after LPS administration. Early enhancement of the expression of Saa3 is a key step in priming inflammation, initiated by macrophages and fibroblasts [32].The early enhancement of Irg1 expression is crucial for determining the direction of the inflammatory response [33]. The increase of Lcn2 is also crucial because it limits E. coli growth by sequestering the iron-laden siderophore [34]. Whereas the biological function of Cxcl9 upregulation in the present model is still being explored, CXCL9 has been reported to recruit and induce T cells to accumulate in the brain [35]. The early immune responses to systemic inflammation in the postnatal mouse brain were initiated by migrating macrophages and leptomeningeal fibroblasts, leading to immediate gene upregulations that were essential for protecting immature brain tissue. 4-2. Intracranial early immune responses to intraperitoneal LPS Intraperitoneally administered LPS is absorbed into the systemic circulation and distributed to various tissues within 1-2 hours [36, 37]. In the present study, LPS reached the vasculature of the intracranial tissues and stimulated the macrophages in the leptomeninges and choroid plexus stroma by 4 hours after LPS administration. Such macrophages were detected by immunostaining for IL-1β. However, microglial cells in the brain parenchyma except for CVOs were not highlighted with immunohistochemistry for IL-1β because the functional BBB was well structured in the brain parenchyma of P7 mice. The presence of the BBB was more clearly highlighted by the experiments using ISH targeting Saa3 and other genes, as will be discussed in 4-4. In contrast, the IL-1R1 protein, a receptor for IL-1β, was expressed by arachnoid and pial cells, which were identified as fibroblasts by immunohistochemistry for type I collagen. Many of the macrophages located in the medulla part of the cerebellum expressed IL-1β, as well as Saa3 , 4 hours after LPS administration. The cerebellar medulla is challenging to study because of its unique features during postnatal development. For example, vascularization of the cerebellum occurs in an inside-out pattern [38], and the macrophages/microglia have a specific gene expression pattern that is different from the cortical grey matter [39]. In the present study, the immune response to systemic inflammation carried out in the cerebellar area was remarkable: IL-1β production and Saa3 expression were carried out by macrophages located not only in the leptomeninges covering the cerebellar surface, but also in the leptomeninges within the cerebellum. Of interest, the fibroblasts bearing IL-1R1 were in close proximity to meningeal macrophages producing IL-1β in widespread brain areas. These fibroblasts were identified as being composed of genuine arachnoid cells and pial cells [23-27]. 4-3. Role of brain fibroblasts in immune responses following systemic inflammation Measuring tissue levels of cytokines using multiple immunoassays showed significant increases in CCL2, G-CSF, CXCL10, IL-6, and CXCL1 levels in the brain parenchyma 4 and 24 hours after LPS administration. Of the commercially available antibodies that were tried for immunohistochemistry, anti-CCL2 antibody was clearly effective for determining which cells were involved in producing CCL2. CCL2-producing cells were arachnoid and pial fibroblasts, as well as parenchymal perivascular fibroblasts. These results indicated that the leptomeningeal fibroblasts stimulated by the leptomeningeal macrophages via the IL-1β-IL-1R1 system conveyed the intercellular communications across the cell junctions, such as the gap junction, tight junction, and intermediate junction, between meningothelial fibroblasts and to parenchymal perivascular fibroblasts [40-42]. These fibroblasts then produced and released CCL2 into the brain tissue 4 hours after LPS administration. We consider that CCL2 derived from parenchymal perivascular fibroblasts attracted more leptomeningeal macrophages into the parenchyma, which enhanced the bilateral intercellular interaction. CCL2 may have other actions, since a recent article reported that maternal stress during pregnancy is associated with increased CCL2, which plays a key role in mediating offsprings’ behavioral sequelae [43]. The other strikingly important role played by perivascular fibroblasts after stimulation by the IL-1β-IL-1R1 system was the production of lipocalin-2 in widespread areas of the brain parenchyma. As reported, lipocalin-2 mediates the innate immune response to inhibit E. coli growth by sequestering the iron-laden siderophore [34]. Considering that our LPS was originally prepared from E. coli, perivascular fibroblasts played an essential role in protecting the brain tissue from potential E. coli invasion. 4-4. Well-constructed blood-brain barrier functionality after intraperitoneal LPS SAA proteins are small (about 100 amino acids) acute phase reactants associated with high-density lipoprotein (HDL), produced in the liver and increasing 1000-fold in plasma during inflammation [44]. SAAs probably play a critical role in the propagation of the primordial acute phase response [32]. In mice, SAAs induce Nod-like receptor protein 3 (NLRP3) inflammasome activation in macrophages and stimulate IL-1β release [45, 46]. Murine SAA1, SAA2, and SAA3 selectively contribute to Th17-mediated pathogenesis in inflammatory bowel disease and experimental autoimmune encephalomyelitis through loss- and gain-of-function with STAT3-activating cytokines [47]. Consistent with such previous publications, the present data indicated that LPS-induced systemic inflammation caused extreme upregulation of Saa3 in the macrophages located in the subarachnoid space and choroid plexus stroma. Saa3 was also upregulated in the arachnoid and pial cells, which are composed of genuine fibroblasts. It was important that the brain parenchymal microglia, with the exception of microglia in the CVOs, did not upregulate Saa3 in response to systemic inflammation. Similarly, brain parenchymal microglia did not upregulate Irg1 or Ccl5 in response to systemic inflammation, but microglia in the CVOs upregulated these genes. These findings clearly indicated that the BBB of postnatally developing mice was so well constructed that the endothelial cells and pericytes ensured that the barrier function was as complete as that of adult mice [15-18]. In contrast, the immunohistochemistry for GFAP showed that astrocytes of P7-P10 mice did not have sophisticated endfeet compared with those of adults (data not shown). This suggested that the functions performed by the endothelial cells and the pericytes were much more important than the functions performed by the astrocytic endfeet to provide a simple barrier function to the blood vessels. 4-5. Brain structures that may contain possible changes in microglial development following systemic inflammation Given that the duration of Saa3 transcript expression detected by ISH was longer than that of other genes, the time-dependent changes in the location of Saa3 -positive macrophages/microglia were followed. The number of Saa3 -positive macrophages/microglia in the ventral hippocampal commissure was significantly higher at 12 hours than at 4 hours after LPS administration. By 24 hours, a further increase in the number of Saa3 -positive cells was observed in the fornix, medial habenula, and corpus callosum. These Saa3 -positive cells migrated into the brain parenchyma to complete the developmental transformation to become microglia. It is interesting that the migrating macrophages, which were essential for the transformation into parenchymal microglia, showed responsiveness to systemic inflammation, ensuring their timely participation in every crucial stage of microglial development. Several interhemispheric commissure bundles traverse the roof of the third ventricle: the ventral hippocampal commissure, dorsal hippocampal commissure, fornix, habenular commissure, and posterior commissure. The corpus callosum is located just dorsal to the dorsal hippocampal commissure in the caudal part and dorsal to the fornix in the central part. Immunohistochemical staining for type I collagen and ISH targeting Col1a1 showed that the collagen-rich leptomeninges, which cover the brain surface, were in close proximity to the ventral hippocampal commissure, fornix, medial habenula, and corpus callosum (Fig. 18). The leptomeninges contain numerous cerebrovascular branches that are destined for the brain parenchyma. The parenchymal blood vessels are accompanied by the perivascular spaces, which represent a major route of entry for macrophages to migrate into the brain parenchyma [14]. Once these macrophages enter the midline structure of the commissural fibers, they gain straightforward access to migrate to bilateral distal areas along the neuroaxonal fiber bundles. Indeed, some Saa3 -positive microglia detected in the medial habenula 24 hours after LPS administration were interpreted to originate from the habenular commissure. The habenula, a system that has existed since the evolution of the nervous system, functions as a regulatory hub, thereby enabling the forebrain to modulate the activity of cholinergic and ascending monoaminergic pathways in the midbrain [48-50]. Dysfunction of the habenula has been associated with significant mental disorders, including depression and addiction [51, 52]. The ventral hippocampal commissure facilitates interhemispheric connections between the left and right hippocampi [53, 54]. The fornix, a white matter tract, connects the hippocampus to several subcortical regions to contribute to the memory system [55]. In the present study, macrophages that showed a response to systemic inflammation entered the midline commissural bundles and gained access to migrate bilaterally to distal areas along the neuroaxonal bundles. In P7 mice, the likelihood may be high that neuroanatomical structures related to the roof of the third ventricle may contain abnormal microglia following systemic inflammation. 4-6. Possible clinical association with encephalopathy of prematurity The central feature of the encephalopathy of prematurity (EP) is periventricular leukomalacia (PVL) [5]. Focal or large necrotic lesions are formed in the periventricular area of the white matter. The EP is characterized not only by hypoxia-ischemia, but also by multiple gray and white matter lesions. Therefore, developmental factors must be considered [5, 56]. In the present experimental model involving P7 mice, migration of leptomeningeal macrophages was highlighted in the commissural bundles that traverse the roof of the third ventricle. The brain developmental stage of P7 mice is equivalent to that of the human fetus at a gestational age of 30 weeks [10]. It is postulated that the macrophages that showed a response to systemic inflammation may retain abnormalities to maintain brain histological homeostasis. These abnormalities, if present, could potentially contribute to the pathogenesis of human EP pathology and related neurological sequelae, including depression, addiction, and pain. 4-7. Limitations The weakness of the present study was that, of the commercially available antibodies used for CCL2, G-CSF, CXCL10, IL-6, and CXCL1, only anti-CCL2 antibody exhibited an effective staining pattern that matched the treatment difference and time-dependent changes in the concentration. The antibodies against the other cytokines did not produce clear findings that matched the biochemical immunoassay data. Therefore, it was not possible to identify all cells that were involved in cytokine release. Second, the total RNA extracted from the CD11b(-) cells was effective in showing the upregulation of Lcn2 and Cxcl9 genes. However, it was not possible to isolate more purified fibroblasts from the brain. Isolation of brain fibroblasts was attempted, but some further undetermined technological improvements appear to be necessary. The total RNA extracted from the brain fibroblasts would have enabled more sophisticated analysis of fibroblasts. 4-8. Conclusions In conclusion, in response to systemic inflammation, macrophages and fibroblasts in the leptomeninges and choroid plexus stroma initiated immune reactions via the IL-1β-IL-1R1 system by 4 hours after LPS administration. Perivascular fibroblasts were stimulated and produced CCL2 in the brain parenchyma, which contributed to an increased number of parenchymal macrophages/microglia and their hypertrophy. Microarray and RT-qPCR showed that extreme upregulation of Saa3 occurred in macrophages and fibroblasts in the leptomeninges, choroid plexus stroma, and perivascular space 4 and 12 hours after LPS administration. Extreme upregulation of Irg1 also occurred in macrophages in the leptomeninges and choroid plexus stroma 4 and 12 hours after LPS administration. The profound upregulation of Lcn2 in the perivascular and leptomeningeal fibroblasts reached its peak 4 and 12 hours after LPS administration. The choroid plexus epithelial cells also showed extensive upregulation of Lcn2 . This gene upregulation was a dynamic response, preparing for the inflammatory reaction and preventing bacterial growth, both of which are essential for protecting immature brain tissue. Declarations Acknowledgments This study was supported by the following: Kyorin University Research Encouragement Award (2021: AS, RS, AO, and TT; 2023: AS), Subsidy to Private Institutions of Higher Education for Current Expenditure (2021: AS and AO; 2022: AS and AO; 2023: AS, RS, and AO; 2024: AS, RS, and AO), and Grants-in-Aid for Scientific Research from JSPS (21K07280 to SHI, 24K10495 to SHI, and 24K08792 to AO). We thank the students for their technical contributions to the experiments. Author contributions AS contributed to the conception of the work, taught the other authors how to conduct the experiments, and wrote the manuscript. RS conducted most of the experiments and wrote the manuscript. AO participated in the research project and performed some of the experiments. TT contributed to the discussion from a clinical perspective. SHI helped interpret the data and assisted the other authors with some experiments. All authors reviewed and approved the manuscript. Funding This study was supported by the following: Kyorin University Research Encouragement Award (2021: AS, RS, AO, and TT; 2023: AS), Subsidy to Private Institutions of Higher Education for Current Expenditure (2021: AS and AO; 2022: AS and AO; 2023: AS, RS, and AO; 2024: AS, RS, and AO), and Grants-in-Aid for Scientific Research from JSPS (21K07280 to SHI, 24K10495 to SHI, and 24K08792 to AO). Ethics approval and consent for participation We affirm that this article contains original data that have not been submitted elsewhere for publication and that all authors have read and approved the manuscript. 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Top twenty gene probes with highest fold changes Cite Share Download PDF Status: Published Journal Publication published 11 Oct, 2025 Read the published version in Journal of Neuroinflammation → Version 1 posted Editorial decision: Revision requested 05 Aug, 2025 Reviews received at journal 04 Aug, 2025 Reviewers agreed at journal 01 Aug, 2025 Reviewers agreed at journal 29 Jul, 2025 Reviewers agreed at journal 28 Jul, 2025 Reviewers agreed at journal 28 Jul, 2025 Reviewers agreed at journal 28 Jul, 2025 Reviews received at journal 22 Jul, 2025 Reviewers agreed at journal 01 Jul, 2025 Reviewers invited by journal 18 Jun, 2025 Editor assigned by journal 11 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 08 Jun, 2025 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-6850479","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":473092937,"identity":"68a862b7-4144-4de4-a737-1f0223181121","order_by":0,"name":"Rei Settsu","email":"","orcid":"","institution":"Kyorin University","correspondingAuthor":false,"prefix":"","firstName":"Rei","middleName":"","lastName":"Settsu","suffix":""},{"id":473092938,"identity":"5e10485a-d207-4458-9b14-5983b54771e1","order_by":1,"name":"Aki Obara","email":"","orcid":"","institution":"Kyorin University","correspondingAuthor":false,"prefix":"","firstName":"Aki","middleName":"","lastName":"Obara","suffix":""},{"id":473092939,"identity":"3166b9ca-ec9e-4e19-9ae3-4eb14097bc11","order_by":2,"name":"Takehiko Tarui","email":"","orcid":"","institution":"Kyorin University","correspondingAuthor":false,"prefix":"","firstName":"Takehiko","middleName":"","lastName":"Tarui","suffix":""},{"id":473092940,"identity":"25bdf481-b2b1-4690-8869-0cb2cc6a6d6a","order_by":3,"name":"Sanae Hasegawa-Ishii","email":"","orcid":"","institution":"Kyorin University","correspondingAuthor":false,"prefix":"","firstName":"Sanae","middleName":"","lastName":"Hasegawa-Ishii","suffix":""},{"id":473092941,"identity":"d17d1ac8-b27b-4a27-86e0-114863c921a4","order_by":4,"name":"Atsuyoshi Shimada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie2RsYrCMBzGvyJcl0BvTCjXe4VK4TjBh2kJdMozlMJBXQRXN1/DsRCISx/A0SJ0cqhbnc6IFLnBVDfh8pv+hPz48uUPWCyvidu1JUAvY9yfUZPwBrDl04pPyoFLf/BWs100rTIdxVVbr5Hkrtxhsr6vUEVCLrYSPk35Mqm0QtIQrDLEKAIpdP+AighJ8ZvkELpecd/4VG79891mvaJTvINZCRWiEbYj/bBeoQMpYyW+nHklCZs3/KJEBW3C0tQlkJsGncoCuuHSORX4WHi83jPDj/UQvMfXSS8KkuXDit5QeZud40OKxWKx/BPO2VhLUPVdR1QAAAAASUVORK5CYII=","orcid":"","institution":"Kyorin University","correspondingAuthor":true,"prefix":"","firstName":"Atsuyoshi","middleName":"","lastName":"Shimada","suffix":""}],"badges":[],"createdAt":"2025-06-09 04:08:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6850479/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6850479/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12974-025-03559-4","type":"published","date":"2025-10-11T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85387577,"identity":"b6977512-e63a-4521-82e7-728a1eaef176","added_by":"auto","created_at":"2025-06-25 10:02:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19621910,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral effects of LPS administration on mice\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eFor the hippocampus, the visual area is delineated from the lateral parasagittal sections located between 700 and 1100 μm lateral to the interhemispheric fissure. Using a histological section stained immunohistochemically with anti-Iba1 antibody, Iba-immunopositive area in the entire dorsal hippocampus is designated with green color (as shown on the right). Within the area of interest, the Iba1-positive area fraction was analyzed and the number of Iba1-positive cell bodies was counted. Scale bars, 300 μm\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eTo define the field of view for the analysis of the cerebral cortex, the first line is drawn from the point on the cortico-medullary junction corresponding to the caudal end of the lateral ventricle (point a) and perpendicular to the cortical surface (point b). The second line is drawn from point c, located on the cortical surface 500 μm caudal to point b, perpendicular to the cortical surface to the cortico-medullary junction (point d). The arachnoid and pia on the cortical surface are also included in the area of interest. Scale bars, 300 μm\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eIn the cerebellum, median sections were used for morphometric analysis. The cerebellum does not include the deep cerebellar nuclei, and the entire cerebellar section consists of the cerebellar cortex and medulla and the leptomeninges. Scale bars, 300 μm\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/9b88f6b3d51d8e03a956ccd0.png"},{"id":85386866,"identity":"e9add1f7-5790-45b0-9ff3-846a35011e1f","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11812331,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral effects of LPS administration on mice\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe change in body weight was determined by taking the body weight immediately before tissue collection minusthe body weight immediately before intraperitoneal administration. Whereas there is a loss of body weight in LPS-treated mice 12 and 24 h after LPS administration, they start to regain the weight at 48 h. Mean±SEM, n = 10 (4 h), 8 (12 h), 36 (24 h), 16 (48 h), and 18 (72 h) in saline control mice, and 10 (4 h), 8 (12 h), 40 (24 h), 16 (48 h), and 19 (72 h) in LPS-treated mice\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B and C)\u003c/strong\u003eMice with systemic inflammation show a slight increase in the number of polymorphonuclear leukocytes (arrow heads) in the leptomeninges 12 h after LPS administration (C) compared with saline control mice (B), but the extent of inflammatory cell infiltration is much less than the pathology of meningitis. Scale bars, 50 μm\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D and E)\u003c/strong\u003eA small number of polymorphonuclear leukocytes (arrow heads) infiltrate the lungs of LPS-treated mice 12 h after LPS administration (E), whereas no inflammatory cells infiltrate the lungs of saline control mice (D). Scale bars, 50 μm\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/5c04ae32174cc30fc182c4e3.png"},{"id":85387572,"identity":"826dae59-ce94-4252-8662-268bc4911cf3","added_by":"auto","created_at":"2025-06-25 10:02:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17751484,"visible":true,"origin":"","legend":"\u003cp\u003eIL-1β expression in the macrophages and IL-1R1 expression in the leptomeningeal fibroblasts in mice with systemic inflammation\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining with anti-IL-1β antibody shows that IL-1β is expressed by cells in the leptomeninges (B) and choroid plexus (D) 4 h after LPS administration, whereas no IL-1β expression is detected in the corresponding areas in saline control mice (A and C). Double immunofluorescence staining with anti-IL-1β (green) and anti-Iba1 (red) antibodies shows that the macrophages in the choroid plexus stroma and epiplexus macrophages express IL-1β 4 h after LPS administration (E). Macrophages in the leptomeninges and the medulla part of the cerebellum also express IL-1β 4 h after LPS administration (F). Microglia in the area postrema that exhibit developing morphology with multiple cytoplasmic processes also express IL-1β (G). Double immunofluorescence staining with anti-IL-1β (green) and type I collagen (red) shows that the leptomeningeal fibroblasts bear IL-1R1, a receptor for IL-1β on the cell surface (H). Scale bars, 50 μm (A-H)\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/8d24c395400dd46599d7a189.png"},{"id":85386867,"identity":"d67b4ee9-4292-4594-b9e8-990ff8480edd","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13395222,"visible":true,"origin":"","legend":"\u003cp\u003eSystemic inflammation-dependent changes of CCL2 levels in brain and spleen\u003c/p\u003e\n\u003cp\u003eCCL2 levels are elevated in the cerebral cortex (A), limbic system (B), and cerebellum (C) in mice with systemic inflammation 4 and 24 h after LPS administration compared with saline control mice. CCL2 levels in the spleen show a higher rate of increase than in the brain parenchyma 4 h after LPS administration and decrease more rapidly toward control levels at 24 h (D). CCL2-expression is increased along the blood vessels in the pial space and the brain parenchyma in mice with systemic inflammation 24 h after LPS administration (F), whereas saline control mice show no CCL2 expression (E). CCL2-immunopositive cells line the leptomeninges in mice with systemic inflammation 4 h after LPS administration (H), compared with no CCL2-immuopositivity in saline control mice (G). Double immunofluorescence staining with anti-CCL2 (green) and anti-type I collagen (red) antibodies shows that the CCL2 is produced by the perivascular fibroblasts (I). Scale bars, 100 μm (E and F) and 50 μm (G, H, and I)\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/9885750e5406a192d63f40e9.png"},{"id":85386862,"identity":"f7075a87-ae7f-44e7-ade0-8039c6fad17d","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":972359,"visible":true,"origin":"","legend":"\u003cp\u003eSystemic inflammation-dependent changes of cytokine levels in brain and spleen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A and B) \u003c/strong\u003eG-CSF levels (A) and CXCL10 levels (B) are elevated in the cerebral cortex, limbic system, and cerebellum in mice with systemic inflammation 4 and 24 h after LPS administration compared with saline control mice. G-CSF levels in the spleen are elevated with a similar time course as and higher than in the brain parenchyma after LPS administration. CXCL10 levels in the spleen are higher than in the brain parenchyma 4 h after LPS administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C and D) \u003c/strong\u003eIL-6 levels (C) and CXCL1 levels (D) are elevated in the cerebral cortex, limbic system, and cerebellum in mice with systemic inflammation 4 h after LPS administration compared with saline control mice. The levels of IL-6 and CXCL1 in the spleen are similarly elevated and higher than in the brain parenchyma 4 h after LPS administration.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/468af26bba9784d0f752455d.png"},{"id":85386865,"identity":"91976088-2e1d-478d-9f9b-d84532d2e340","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2521014,"visible":true,"origin":"","legend":"\u003cp\u003eRelatively minor changes of cytokine levels in brain and spleen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eCCL11 levels are elevated in the cerebral cortex, limbic system, and cerebellum in mice with systemic inflammation 4, 24, and 48 h after LPS administration compared with saline control mice. CCL11 levels in the spleen are elevated and higher than in the brain parenchyma 4 h after LPS administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eThere is an increase in CXCL2 levels in the cerebral cortex of mice with systemic inflammation compared with saline control mice 4 and 24 h after LPS administration. CXCL2 levels are significantly elevated in the cerebellum 4 h after LPS administration compared with saline-injected mice. In contrast, CXCL2 levels in the spleen of LPS-treated mice are significantly elevated in the cerebellum 4 h after LPS administration compared with saline-injected mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eIn the brain parenchyma, IL-10 levels are increased only in the cerebral cortex of LPS-treated mice compared with saline-injected mice 24 h after LPS administration. In contrast, IL-10 levels are increased in the spleen 4 and 24 h after LPS administration.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/76643c78403f43a1a88f1adb.png"},{"id":85387571,"identity":"54b7fa63-af01-46d3-a883-2bf9d9e06986","added_by":"auto","created_at":"2025-06-25 10:02:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":493567,"visible":true,"origin":"","legend":"\u003cp\u003eNo systemic inflammation-dependent change of IL-1α and IL-1β levels in brain\u003c/p\u003e\n\u003cp\u003eThere are no significant changes in IL-1α or IL-1β levels in any brain parenchymal region at any time point after LPS administration. These results in the brain parenchyma contrast with the early increased expression of IL-1β in the leptomeningeal and choroid plexus macrophages. \u0026nbsp;Increased levels of IL-1α and IL-1β are evident only in the spleen of mice with systemic inflammation compared with saline control mice 4 and 24 h after LPS administration.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/57e9d8ff5b75f599530b78a6.png"},{"id":85386863,"identity":"8a92820f-2286-4c05-885e-b83b1c3c4f48","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1422359,"visible":true,"origin":"","legend":"\u003cp\u003eNo systemic inflammation-associated cytokine level changes in brain\u003c/p\u003e\n\u003cp\u003eThere are no significant changes in the levels of IFN-γ (A) or IL-17 (B) in any brain parenchymal region at any time point after LPS administration. TNF-α levels (C) in the brain are below the minimum detectable concentration in any region at any time point after saline or LPS injection. Increased levels of IFN-γ and TNF-α are only evident in the spleen of LPS-treated mice compared with saline-injected mice 4 h after LPS administration. An increased level of IFN-γ IL-17 is evident only in the spleen of mice with systemic inflammation 24 h after LPS administration.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/44f323ee4d01fa50cef00817.png"},{"id":85386881,"identity":"67ea0b15-547e-45c4-902b-3646d7ecefa3","added_by":"auto","created_at":"2025-06-25 09:54:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":26259247,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of systemic inflammation on microglia\u003c/p\u003e\n\u003cp\u003eIn the cerebral cortex of mice with systemic inflammation, microglia have become hypertrophic with thicker and shorter cytoplasmic processes 4 h after LPS administration (B), compared with saline control mice (A). Subsequently, a higher degree of cellular hypertrophy accompanied by an increased number of Iba1-positive cells is evident 24 h after LPS administration (D), compared with saline control mice (C). In particular, perivascular macrophages have become extremely hypertrophic in mice with systemic inflammation 24 h after LPS administration (F), compared with saline control mice (E), which are probably destined to enter the cortical parenchyma to become microglia. Microglia are densely distributed in the cerebellar cortex of the mice with systemic inflammation 24 h after LPS administration (H). Cerebellar microglia are less distributed in the cortex and macrophages or microglia are more distributed in the medulla part in saline control mice (G). Scale bars, 50 μm (A-F) and 200 μm (G, H)\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/b8f571808f377f637d103aca.png"},{"id":85386869,"identity":"79209985-1bab-4288-b000-3c3a1b8342e8","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":372791,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of systemic inflammation on microglia with increased number and hypertrophy\u003c/p\u003e\n\u003cp\u003eThe Iba1-positive area fraction of the cerebral cortex and hippocampus in mice with systemic inflammation is significantly higher than that in saline control mice 24 h after LPS administration (A and B). In the hippocampus, the number of Iba1-positive cells is significantly increased in mice with systemic inflammation compared with saline control mice 24 and 48 h after LPS administration (C). There is a time-dependent increase in the number of Iba1-positive cells in the brain parenchyma in both the LPS-treated and saline-injected mice due to the postnatal development of microglia in which leptomeningeal macrophages migrate into the brain parenchyma. In contrast, systemic inflammation has led to an increased number of microglia and their hypertrophy in the cerebellum 24 and 48 h after LPS administration (D). There is no increase in the cerebellar Iba1-positive area fraction during the developmental period between P7 and P10 in saline control mice (D).\u003c/p\u003e","description":"","filename":"Fig.10.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/30f9027e991f3611ffbcb794.png"},{"id":85387574,"identity":"8438ec6c-e0da-4da5-9b67-783df426973a","added_by":"auto","created_at":"2025-06-25 10:02:43","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":14694683,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of systemic inflammation on astrocytes\u003c/p\u003e\n\u003cp\u003eAlthough the astrocytes in the middle layer of the cerebral cortex are not detectable by immunohistochemistry with anti-GFAP antibody in saline control mice (A), these astrocytes are detectable by immunohistochemistry for S-100 (C). In mice with systemic inflammation, these astrocytes have become hypertrophic enough to be detectable by immunohistochemistry for GFAP (B) and S-100 (D). Scale bars, 50 μm\u003c/p\u003e","description":"","filename":"Fig.11.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/b9158bcd913640e89c3dcaf9.png"},{"id":85387575,"identity":"0f15a92c-a443-492d-8095-507b575ac416","added_by":"auto","created_at":"2025-06-25 10:02:43","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":491350,"visible":true,"origin":"","legend":"\u003cp\u003eMicroarray results indicating gene expression fold changes with significant p values\u003c/p\u003e\n\u003cp\u003eA comparison of the changes in gene expression between LPS-treated and saline-injected mice shows that 1122 gene probes have significantly increased transcript expression levels (red dots), and 1616 gene probes have significantly decreased transcript expression levels (blue dots). The dots indicating increased expression with extremely high fold changes are labeled with arrows from a to i. Arrow i indicates two different probes that represent a single gene.\u003c/p\u003e","description":"","filename":"Fig.12.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/667c51b3f84549b30fdebbb6.png"},{"id":85386880,"identity":"3c18c626-b6f5-46ae-8ba5-c01e97645d4f","added_by":"auto","created_at":"2025-06-25 09:54:44","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":23656345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRT-qPCR data showing the fold changes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSaa3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLcn2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transcript expressions of brain cells in response to systemic inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A and B) \u003c/strong\u003eThe transcript expression of \u003cem\u003eSaa3\u003c/em\u003e in brain CD11b(+) cells of mice with systemic inflammation shows very marked upregulation compared with saline control mice from 4 to 72 h after LPS administration (A). Furthermore, \u003cem\u003eSaa3\u003c/em\u003eexpression in brain CD11b(−) cells of mice with systemic inflammation shows a marked increase from 4 to 72 h after LPS administration (B). **p \u0026lt; 0.01, compared with saline control\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C and D) \u003c/strong\u003eThese two sections were stained with in situ hybridization targeting \u003cem\u003eSaa3\u003c/em\u003eusing midsagittal (C) and parasagittal (D) sections. The histological photographs were tiled so that the image contains the whole brain area. Scale bars, 1000 μm\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E and F) \u003c/strong\u003eSystemic inflammation-activated changes in \u003cem\u003eLcn2\u003c/em\u003e transcript expression levels in CD11b(−) cells are highly upregulated compared with saline control from 4 to 24 h after LPS administration, and thereafter decrease gradually (F). The degree of \u003cem\u003eLcn2\u003c/em\u003e upregulation is much lower in CD11b(+) cells than in CD11b(-) cells (E). **p \u0026lt; 0.01, compared with saline control\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G and H) \u003c/strong\u003eThese two sections were stained with in situ hybridization targeting \u003cem\u003eLcn2\u003c/em\u003eusing midsagittal (G) and parasagittal (H) sections. The histological photographs were tiled so that an image contains the whole brain area. Scale bars, 1000 μm\u003c/p\u003e","description":"","filename":"Fig.13.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/0e06645a23a43569f3603520.png"},{"id":85386870,"identity":"a8eaf652-64c6-4a87-a8d2-0253334caec1","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":860807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRT-qPCR data showing fold changes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCxcl9\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIrg1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSaa2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSaa1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etranscript expression levels of brain cells in response to systemic inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystemic inflammation-activated changes in \u003cem\u003eCxcl9\u003c/em\u003e transcript expression in CD11b(−) cells are extremely upregulated compared with saline control at 4 and 12 h, and thereafter they decrease gradually, but are still significantly high up to 72 h after LPS administration (B). The degree of \u003cem\u003eCxcl9\u003c/em\u003e upregulation in CD11b(+) cells of mice with systemic inflammation is much lower than in CD11b(+) cells, but still significantly elevated up to 72 h after LPS administration (A). The transcript expression of \u003cem\u003eIrg1\u003c/em\u003e in CD11b(+) cells of mice with systemic inflammation shows the greatest increase compared with saline control mice 4 h and 12 h after LPS administration. Thereafter, expression decreases gradually but remains significantly elevated up to 48 h after LPS administration (C). \u003cem\u003eIrg1\u003c/em\u003eexpression in CD11b(−) cells of mice with systemic inflammation also shows marked upregulation 4 and 12 h after LPS treatment, followed by a gradual decrease (D). The transcript expression levels of \u003cem\u003eSaa2 \u003c/em\u003eand\u003cem\u003e Saa1 \u003c/em\u003ein CD11b(+) cells of mice with systemic inflammation are significantly and markedly increased compared with saline control mice from 4 to 24 h after LPS administration (E and G). The degree of \u003cem\u003eSaa2\u003c/em\u003e and \u003cem\u003eSaa1\u003c/em\u003e upregulation in CD11b(-) cells of saline control mice is much lower than in CD11b(+) cells, but significantly elevated from 4 to 24 h after LPS administration (F and H). **p \u0026lt; 0.01 and *p \u0026lt; 0.05, compared with saline control\u003c/p\u003e","description":"","filename":"Fig.14.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/a25b8e5d28eb0751fb6525c3.png"},{"id":85387573,"identity":"aed8b69d-331e-428b-91b5-df41b66a383d","added_by":"auto","created_at":"2025-06-25 10:02:43","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":641221,"visible":true,"origin":"","legend":"\u003cp\u003eRT-qPCR data showing fold changes in \u003cem\u003eCcl5\u003c/em\u003e, \u003cem\u003eCxcl13\u003c/em\u003e, and \u003cem\u003eSlfn4\u003c/em\u003e transcript expression levels of brain cells in response to systemic inflammation\u003c/p\u003e\n\u003cp\u003eThe transcript expression of \u003cem\u003eCcl5\u003c/em\u003e in CD11b(+) cells of mice with systemic inflammation shows a significant increase compared with saline control mice from 4 to 72 h after LPS administration (a). The degree of \u003cem\u003eCcl5\u003c/em\u003e upregulation in CD11b(−) cells also shows a significant increase from 4 to 24 h after LPS administration (B). The transcript expression of \u003cem\u003eCxcl13\u003c/em\u003ein CD11b(+) cells of mice with systemic inflammation shows a significant increase compared with saline control mice from 4 to 72 h after LPS administration (C). \u003cem\u003eCxcl13\u003c/em\u003eexpression in CD11b(−) cells shows a significant increase from 4 to 24 h after LPS administration (D). The transcript expression of \u003cem\u003eSlfn4\u003c/em\u003e in CD11b(+) cells of mice with systemic inflammation shows a significant increase compared with saline control mice from 4 to 24 h after LPS administration (E). \u003cem\u003eSlfn4\u003c/em\u003e expression in CD11b(−) cells shows a significant increase 4 and 24 h after LPS administration (F). **p \u0026lt; 0.01 and *p \u0026lt; 0.05, compared with saline control\u003c/p\u003e","description":"","filename":"Fig.15.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/907ec3793573b50153f06f25.png"},{"id":85386876,"identity":"bdc1495d-2212-4e89-a6ee-6b6d94d8ac97","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":23884628,"visible":true,"origin":"","legend":"\u003cp\u003eHistological distribution of cells expressing the \u003cem\u003eSaa3\u003c/em\u003etranscript in the brain\u003c/p\u003e\n\u003cp\u003eSystemic inflammation-associated highly elevated levels of\u003cem\u003e Saa3\u003c/em\u003e transcript expression are evident in cells located in the leptomeninges (B and D), choroid plexus stroma (F), and medulla part of the cerebellum (H) 12 h after LPS administration. Microglia with developing multiple cytoplasmic processes express Saa3 in the area postrema (I). The distribution density of Saa3-expressing cells, consisting of arachnoid cells and macrophages, is indicated by tangentially sectioned leptomeninges (J). No \u003cem\u003eSaa3\u003c/em\u003e expression is observed by ISH in any brain parenchymal region except for the CVOs (A, C, E, and G). Scale bars, 100 μm (A-I) and 50 μm (J)\u003c/p\u003e","description":"","filename":"Fig.16.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/b38fb2679366cbc39e4b8d5d.png"},{"id":85386877,"identity":"2755a5dc-463d-48dc-9fbd-492ec6b7937c","added_by":"auto","created_at":"2025-06-25 09:54:44","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":11907792,"visible":true,"origin":"","legend":"\u003cp\u003eDouble staining with in situ hybridization targeting \u003cem\u003eSaa3\u003c/em\u003e and immunohistochemistry\u003c/p\u003e\n\u003cp\u003eIn situ hybridization targeting \u003cem\u003eSaa3\u003c/em\u003e followed by immunohistochemistry for Iba1 shows that macrophages in the leptomeninges (A) and choroid plexus (B), as indicated by the red arrowheads, are major \u003cem\u003eSaa3\u003c/em\u003e-expressing cells. In situ hybridization targeting \u003cem\u003eSaa3\u003c/em\u003e followed by immunohistochemistry for type 1 collagen shows that fibroblasts in the leptomeninges (C), as indicated by “f”, are also \u003cem\u003eSaa3\u003c/em\u003e-expressing cells. At 12 h after LPS administration, migrating microglia with \u003cem\u003eSaa3\u003c/em\u003e expression enter the ventral hippocampal commissure (vhc) and start to bear Iba1-positive fine cytoplasmic processes (D). Scale bars, 50 μm\u003c/p\u003e","description":"","filename":"Fig.17.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/ab46ed071f53f03952132999.png"},{"id":85386882,"identity":"cf1df14d-5e56-40f5-bb53-45c193cf4fdb","added_by":"auto","created_at":"2025-06-25 09:54:44","extension":"png","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":26719471,"visible":true,"origin":"","legend":"\u003cp\u003eIn situ hybridization targeting \u003cem\u003eSaa3\u003c/em\u003e and \u003cem\u003eCol1a1 \u003c/em\u003earound the third ventricle\u003c/p\u003e\n\u003cp\u003eThe midsagittal sections containing the 3rd ventricle and its choroid plexus highlight the frequent presence of \u003cem\u003eSaa3\u003c/em\u003e-expressing cells in mice with systemic inflammation (B), compared with saline control mice (A). The frequent presence of \u003cem\u003eSaa3\u003c/em\u003e-expressing cells is associated with a long rostro-caudal extension of the choroid plexus stroma and the parallel leptomeninges, as clearly demonstrated by ISH targeting \u003cem\u003eCol1a1\u003c/em\u003e (C). The midsagittal sections around the 3rd ventricle show that the number of \u003cem\u003eSaa3\u003c/em\u003e-expressing cells in the ventral hippocampal commissure and fornix increases in a time-dependent manner at 4 h (D), 12 h (E), and 24 h (F) in mice with systemic inflammation. Counts of \u003cem\u003eSaa3\u003c/em\u003e-positive cells in the ventral hippocampal commissure (G), fornix (H), medial habenula (I), and corpus callosum (J) at 4, 12, and 24 h after LPS administration are shown. SFO, subfornical organ; 3V ChP, 3rd ventricular choroid plexus; vhc, ventral hippocampal commissure. Scale bars, 500 μm (A-C) and 100 μm (D-F)\u003c/p\u003e","description":"","filename":"Fig.18.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/80996a1f9a1a326df33b30f5.png"},{"id":85386883,"identity":"3e791586-0c9f-4462-9302-678f2fd1eb21","added_by":"auto","created_at":"2025-06-25 09:54:44","extension":"png","order_by":19,"title":"Figure 19","display":"","copyAsset":false,"role":"figure","size":28364120,"visible":true,"origin":"","legend":"\u003cp\u003eIn situ hybridization targeting \u003cem\u003eIrg1\u003c/em\u003e and with immunohistochemistry\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIrg1\u003c/em\u003etranscript expression is prominent in the macrophages in the choroid plexus (A), many of which are epiplexus cells, in the leptomeninges covering the cerebral cortex (B), in the cerebellar medulla (C), and in the area postrema (D) in mice with systemic inflammation. These \u003cem\u003eIrg1\u003c/em\u003e-positive cells are identified as macrophages by double staining with in situ hybridization targeting \u003cem\u003eIrg1\u003c/em\u003e and immunohistochemistry with anti-Iba1 antibody, for example in the leptomeninges (E, arrows) and in the choroid plexus (F, arrows). Scale bars, 50 μm\u003c/p\u003e","description":"","filename":"Fig.19.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/a861598cc4e1328310074e16.png"},{"id":85386885,"identity":"91fa2d65-2a30-47e6-8641-e1ae8e4f9fef","added_by":"auto","created_at":"2025-06-25 09:54:45","extension":"png","order_by":20,"title":"Figure 20","display":"","copyAsset":false,"role":"figure","size":60574481,"visible":true,"origin":"","legend":"\u003cp\u003eHistological distribution of cells expressing the \u003cem\u003eLcn2\u003c/em\u003e transcript in the brain\u003c/p\u003e\n\u003cp\u003eIn situ hybridization targeting \u003cem\u003eLcn2\u003c/em\u003e shows high expression in the perivascular cells in the parenchyma of the midbrain (A) and brainstem (B and C) and in the leptomeningeal perivascular cells with connecting cerebrocortical perivascular cells (D) in mice with systemic inflammation. These cells are identified as fibroblasts by double staining with immunohistochemistry for type 1 collagen, for example in the arachnoid of the interpeduncular fossa (E) and pontine blood vessels (F). \u003cem\u003eLcn2\u003c/em\u003e upregulation in fibroblasts is the most prominent in mice with systemic inflammation 4 and 12 h after LPS administration. The choroid plexus epithelial cells are positive for \u003cem\u003eLcn2\u003c/em\u003e expression 12 h after LPS administration (G). Thus, the choroid plexus contains many cells rich in \u003cem\u003eLcn2\u003c/em\u003e expression in both epithelial cells and stromal fibroblasts, as demonstrated by double staining with immunohistochemistry for type 1 collagen (H). Scale bars, 50 μm\u003c/p\u003e","description":"","filename":"Fig.20.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/03e5d30c7ecbf7debd58d067.png"},{"id":85386871,"identity":"4f6ab164-7168-439e-b21f-a01b84afd4e0","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"png","order_by":21,"title":"Figure 21","display":"","copyAsset":false,"role":"figure","size":15598216,"visible":true,"origin":"","legend":"\u003cp\u003eHistological distribution of cells expressing the \u003cem\u003eCxcl9\u003c/em\u003e transcript in the brain\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCxcl9\u003c/em\u003etranscript expression is prominent in the fibroblasts in the leptomeninges of mice with systemic inflammation (A and B). These results are confirmed by double staining of in situ hybridization targeting \u003cem\u003eCxcl9\u003c/em\u003e and immunohistochemistry for type I collagen, for example, in the fibroblasts of the leptomeninges covering the cerebellar cortex (C) and perivascular fibroblasts in the brainstem (D). \u003cem\u003eCxcl9\u003c/em\u003e is expressed in the choroid plexus stromal fibroblasts, but not in the epithelium (E). Double staining with immunohistochemistry for type I collagen shows double staining of \u003cem\u003eCxcl9\u003c/em\u003eand type 1 collagen in the choroid plexus stromal fibroblasts (F, arrows). Scale bars, 50 μm\u003c/p\u003e","description":"","filename":"Fig.21.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/55de5807f5f56bcafd07a7f1.png"},{"id":85387576,"identity":"73489d12-d456-4428-ad8a-174c4e5d5b07","added_by":"auto","created_at":"2025-06-25 10:02:44","extension":"png","order_by":22,"title":"Figure 22","display":"","copyAsset":false,"role":"figure","size":10561285,"visible":true,"origin":"","legend":"\u003cp\u003eHistological distribution of cells expressing \u003cem\u003eCcl5, Cxcl13, and Slfn4\u003c/em\u003e transcripts in the brain\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCcl5\u003c/em\u003e(A) and \u003cem\u003eCxcl13\u003c/em\u003e (C) transcript expressions are prominent in choroid plexus macrophages in mice with systemic inflammation. \u003cem\u003eCcl5\u003c/em\u003e transcript expression is also detected in the area postrema (B). \u003cem\u003eSlfn4 \u003c/em\u003etranscript expression, mainly by macrophages in the leptomeninges covering the midbrain (D), is prominent. Scale bars, 50 μm (A, C, and D) and 100 μm (B)\u003c/p\u003e","description":"","filename":"Fig.22.png","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/f947cd55d277d70fddb5f283.png"},{"id":94600295,"identity":"8aa2d122-e085-456c-aa75-8418eee3c291","added_by":"auto","created_at":"2025-10-28 19:15:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":103549013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/beee20b1-9588-4d40-aa49-26dd29a0099a.pdf"},{"id":85386861,"identity":"8765446e-80e1-4271-8f50-c5e91bf612cd","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29442,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1. Primary antibodies for immunohistochemical staining\u003c/p\u003e","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/9aca565bdbd7a0b4f3a36148.docx"},{"id":85386873,"identity":"fc9dd3a6-3db5-4955-8807-13432f56ff3f","added_by":"auto","created_at":"2025-06-25 09:54:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14753624,"visible":true,"origin":"","legend":"\u003cp\u003eTable 2. Top twenty gene probes with highest fold changes\u003c/p\u003e","description":"","filename":"Table2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6850479/v1/a022f4aa7ea3325b39b029ed.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Early immune responses to systemic inflammation in the postnatal mouse brain initiated by migrating macrophages and leptomeningeal fibroblasts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the year 2020, an estimated 13.4 million newborns were born preterm (before 37 completed weeks of gestation) [1]. Bacterial infection triggers inflammatory pathways that damage the preterm brain even without direct bacterial invasion into the brain [2]. Bacteria-induced systemic inflammation occurring in preterm infants, including bacteremia, sepsis, chorioamnionitis, and necrotizing enterocolitis, is a risk factor for adverse neurodevelopmental outcomes [3]. The neuropathological findings of this disability in preterm infants consist of mixed injuries of the cerebral white matter and gray matter [4, 5]. However, the mechanisms underlying the involvement of intracranial tissues and cells in early immune responses to acute systemic inflammation in preterm infants remain unknown.\u003c/p\u003e\n\u003cp\u003eIn our previous studies, we treated two-month-old C57BL/6N mice with a single intraperitoneal administration of lipopolysaccharide (LPS). The histological analysis showed that the macrophages of the choroid plexus and leptomeninges produced interleukin (IL)-1\u0026beta; most rapidly in response to LPS-induced systemic inflammation 1 hour after LPS administration [6-8]. These macrophages stimulated the choroid plexus epithelial and stromal cells to produce CC-motif ligand (CCL)2, CXC-motif ligand (CXCL)1, CXCL2, and IL-6 4 hours after LPS administration. These cytokines were then transported into the brain parenchyma [9]. Of the brain parenchymal cells, astrocytes were found to be the most efficient in responding to CCL2, CXCL1, CXCL2, and IL-6 using the cytokine receptors located on the endfeet. Astrocytes then produced CCL11, CXCL10 and G-CSF 24 hours after LPS administration. The astrocyte-derived cytokines activated microglia via receptors. This collaboration led to a shift in microglial gene expression, driving them towards the M2 phenotype. Brain parenchymal cytokine concentrations returned to control levels by 72 hours after a single LPS administration.\u003c/p\u003e\n\u003cp\u003eIt is known that the brain developmental stage of the postnatal day (P) 7 mouse is equivalent to that of the human fetus at a gestational age of about 30 weeks [10]. Therefore, we reasoned that the brain cellular responses produced by P7 mouse pups should represent the basic responses that occur in human preterm infants [11]. The choroid plexus and leptomeningeal macrophages may be key players in the initial response to systemic inflammation in newborn mice as well [12]. However, the roles of these leptomeningeal macrophages differ significantly between neonatal and adult mice. The leptomeningeal macrophages in pre- and postnatal mice are destined to migrate into the brain parenchyma to differentiate into parenchymal microglia [13, 14]. Whether the macrophages that are to become parenchymal microglia have enough ability to respond to systemic inflammation becomes an interesting question. We are exploring the possibility that leptomeningeal macrophages play key roles in the initial response to systemic inflammation in newborn mice. This has generated new questions, such as which cells could be partners in this vital process.\u003c/p\u003e\n\u003cp\u003eDetermining the functionality of the blood-brain barrier (BBB) in newborn mice is an important topic. It is well-established that the BBB of postnatal developing mice is well-constructed with endothelial cells and pericytes, making their barrier function as robust as that of the BBB in adult animals [15-18]. In addition, the cytoplasmic processes of astrocytes surround brain capillaries, arterioles, and venules in adults. At capillaries, the astrocytic endfeet are located on the brain side of the basement membrane that ensheaths the endothelial cells and pericytes [19]. Communication between endothelial cells and astrocytes is crucial for both the barrier and interface functions. Astrocytes use their highly efficient cytoplasmic processes in response to brain parenchymal cytokines in adult mice [7]. However, astrocytes of P7 mice do not have well-developed sophisticated cytoplasmic processes [20]. The formation of most of the capillary bed occurs between P8 and P10, but the astrocytic endfeet incompletely cover the vasculature before P10 and enwrap the entire brain vasculature by P21 [21]. Furthermore, the perivascular space of the brain is in direct continuity with the subarachnoid space. The periarterial space is lined with perivascular cells that are continuous with the pia mater [22]. The brain perivascular cells originate from the meninges and are first seen on the parenchymal vasculature at P5 [23]. After P5, perivascular cell coverage of the cerebral vasculature expands by local cell proliferation and migration from the meninges. Perivascular cells and perivascular macrophages develop in parallel. Recent studies have advanced our understanding of the relationship between the major meningeal cells and the perivascular cells. The arachnoid cells, pial cells, and perivascular cells have all been identified as genuine fibroblasts [23-27].\u003c/p\u003e\n\u003cp\u003eWe hypothesized that the leptomeningeal and choroid plexus stromal macrophages play the central role in the early responses to systemic inflammation in the immature brains of newborn mice, and that the leptomeningeal and perivascular fibroblasts are among the important partners in initiating immune responses to protect the brain.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe Guide for the Care and Use of Laboratory Animals, 8th edition (National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Washington DC: National Academies Press, 2011), was followed for the handling of all mice. The Institutional Animal Care and Use Committee of the Kyorin University Faculty of Health Sciences approved all of the experiments described (Protocols I17–08–03 to I17–08–07).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2-1. Preparation of histological frozen brain sections\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystemic inflammation was induced in seven-day-old (P7), male, C57BL/6N mice using LPS (from \u003cem\u003eE. coli\u003c/em\u003e O55:B5; Sigma-Aldrich-Merck, Burlington, MA, USA), a bacterial endotoxin, which was administered intraperitoneally to the experimental group at a dose of 0.75 mg/kg with a 33-G Hamilton syringe (systemic inflammation group). The control group received a single intraperitoneal injection of saline at the same dose of 3.75 mL/kg as the experimental group (saline control group). After treatment, the mouse pups were returned to their dams for continued rearing.\u003c/p\u003e\n\u003cp\u003eAt 4, 12, 24, 48, and 72 hours after LPS or saline administration, ketamine-xylazine anesthetic solution was administered intraperitoneally at a dose of 10 mL/kg. Blood was extracted transcardially using a phosphate-buffered saline (PBS) solution followed by perfusion with Zamboni fixative solution at a flow rate of 3 mL/min. Following removal of the scalp, eyes, and mandible, the skull containing the brain, the liver, and the spleen were immersed in Zamboni fixative at 4 °C for 2 days. Each experimental group consisted of four mice.\u003c/p\u003e\n\u003cp\u003eThe brains were then extracted from the skulls. Each brain was bisected along the parasagittal plane located in the midline interhemispheric fissure so that the first parasagittal brain section was safely cut from the right hemisphere. Small fragments of the liver and spleen were prepared and embedded in the same blocks as the bisected brains. Cryoprotection was achieved through immersion of the tissue samples in 10, 15, and 20% sucrose in PBS at room temperature overnight. Brain, liver, and spleen tissues were then embedded in Cryomatrix embedding medium (Thermo Fisher Scientific, Waltham, MA, USA) in Tissue Tech Cryomold No. 3 (Sakura Finetech, Tokyo, Japan) and subsequently frozen with dry ice-cold n-hexane.\u003c/p\u003e\n\u003cp\u003eFrozen blocks were sectioned at a thickness of 14 μm using a LEICA CM 3050S cryostat (Leica Biosystems, Deer Park, IL, USA). Median brain sections were obtained by cutting sequentially from 300 μm to the right of the interhemispheric fissure of the cerebral hemispheres. Approximately 40 sections were prepared as median sections. Lateral brain sections were prepared from 600 μm to the left of the interhemispheric fissure and sequentially cut to obtain 50 lateral sections. The prepared sections were mounted on FRC-04-coated glass slides (Matsunami Glass Co., Ltd., Osaka, Japan), followed by air drying and vacuum drying using a V-100 vacuum pump (BUCHI Labortechnik, Flawil, Switzerland). The dried sections were then stored at -20 °C until use.\u003c/p\u003e\n\u003cp\u003eSections from mice in all experimental groups were stained with hematoxylin and eosin (H\u0026amp;E) to observe inflammation-related changes, such as inflammatory cell infiltration, ischemia, and necrosis. The sections were subsequently used for immunohistochemical, double immunofluorescence, and in situ hybridization (ISH) staining. To examine the histology of major organs other than the brain by H\u0026amp;E staining, the heart, lung, liver, spleen, and kidney were embedded in paraffin and sectioned at a thickness of 4 μm using a microtome.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2-\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e\u003cem\u003e. Immunohistochemistry and immunofluorescence staining\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFrozen sections were soaked in Tris-buffered saline with Tween 20 (TBS-T) for 10 min and pretreated with 0.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol to block endogenous peroxidase activity and with 1% BSA in TBS-T to block non-specific binding. Sections were incubated with primary antibodies (Table 1) overnight at 4 °C or for 2 hours at room temperature, followed by incubation with reagents from the ImmPRESS HRP Antidody (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, CA, USA) for 60 min at room temperature. Reactions were visualized by incubating sections with an ImmPACT DAB Substrate Kit, Peroxidase (SK-4105; Vector Laboratories). Sections were sequentially dehydrated through 80%, 90%, 95%, and 100% ethanol, cleared with xylene, and coverslipped with HSR mounting medium (Sysmex, Kobe, Japan). Immunohistochemical photographs were taken with 4x, 10x, 20x, and 40x PlanApo λ objectives (Nikon, Tokyo, Japan) of an Eclipse Ci-L light microscope equipped with a DS-Fi3/DS-L4 digital camera control unit (Nikon), and with 4x, 10x, 20x, and 40x PlanApo λ objectives (Nikon) of a BZ-X710 microscope (Keyence, Osaka, Japan).\u003c/p\u003e\n\u003cp\u003eFor double immunofluorescence staining, frozen sections were soaked in TBS-T for 10 min, preincubated with 1% BSA in TBS-T, and incubated with primary antibodies overnight at 4 °C. The combinations of the primary antibodies were IL-1β and Iba1, CCL2 and type 1 collagen, or IL-1R1 and type 1 collagen. After incubation with two primary antibodies, sections were incubated with donkey anti-goat or anti-rabbit IgG secondary antibodies conjugated with Alexa Fluor 568 or 488 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for 60 min at room temperature. Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Sections were coverslipped with Fluorescence Mounting Medium (DAKO, Agilent, Santa Clara, CA, USA). Fluorescence images were captured using a BZ-X710 microscope equipped with structured illumination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2-3. Morphometric analysis of sections immunohistochemically stained with anti-Iba1\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the changes of microglial morphology that occurred after LPS administration, frozen brain sections from mice at 4, 24, 48, and 72 hours after LPS or saline administration (n = 4 in each group) were immunohistochemically stained with anti-ionized calcium-binding adaptor molecule-1(Iba1) antibody (rabbit monoclonal [EPR16588], Abcam, Cambridge, UK). For morphometric studies, a computerized image analyzer (WinROOF 2018, Mitani Corporation, Tokyo, Japan) was used to calculate the area fraction of Iba1-positive cells (total area of Iba1-positive cell bodies with cytoplasmic processes divided by the area of interest). In addition to the analysis functions, WinROOF 2018 had a variety of combined manual and automatic editing functions, such as separating contiguous cytoplasmic process images, filling in parts of cells with density below the threshold, and removing small cell fragments and artifacts. Each field was reviewed by the operator on the instrument screen to make these corrections. Analysis of the cerebellum, cerebral cortex, and hippocampus was performed. The number of cell bodies of Iba1-positive cells was also counted in the hippocampus only. Two histological sections per individual mouse were used for each brain region.\u003c/p\u003e\n\u003cp\u003eFor the hippocampus, the area was delineated from the lateral parasagittal sections located between 700 and 1100 μm lateral to the interhemispheric fissure. Using a histological section, the entire dorsal hippocampus was analyzed to quantify the area fraction (Fig. 1A). Within the designated area of interest, the number of cell bodies of Iba1-positive cells was counted. In the same sections in which the hippocampus was analyzed, the parietal cortex was located dorsal to the hippocampus, and the six cortical layers were perfectly recognizable. To define the field of view for the analysis of the cerebral cortex, the first line was drawn from the point on the cortico-medullary junction corresponding to the caudal end of the lateral ventricle (point b) and perpendicular to the cortical surface (point a). The second line was drawn from point d, located on the cortical surface 500 μm caudal to point a, perpendicular to the cortical surface to the cortico-medullary junction (point c). The arachnoid and pia on the cortical surface were also included in the area of interest (Fig. 1B). In the cerebellum, median sections were used for morphometric analysis. Notably, the cerebellum did not include the deep cerebellar nuclei, and the entire cerebellar section consisted of the cerebellar cortex and medulla and the leptomeninges (Fig. 1C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2-4. Immunoassay of cytokine concentrations in brain parenchymal tissues\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 4, 24, 48, and 72 hours after LPS or saline administration, mice were perfused systemically with PBS to remove blood. The brains were removed and quickly divided on an ice-cold glass plate into the following seven regions: left and right cerebral cortices, left and right limbic systems (including olfactory bulb, olfactory tubercle, piriform cortex, entorhinal cortex, and hippocampus), left and right subcortical structures (striatum, diencephalon, midbrain, and brainstem), and cerebellum. These seven brain parts were individually snap frozen in liquid nitrogen and stored at -80 °C until they were used.\u003c/p\u003e\n\u003cp\u003eOf the seven parts, the left cerebral cortex, left limbic system, and cerebellum were used to measure cytokine concentrations. Tissue Protein Extraction Reagent (T-PER, Thermo Fisher Scientific) was added to a Biomasher II tube (Nippi, Tokyo, Japan) at 20-fold tissue weight, and 1/100 volume of Halt Protease Inhibitor Cocktail (100x, Thermo Fisher Scientific) was added to T-PER. Tissue samples stored at -80 °C were added to these tubes and homogenized. After centrifugation at 13,000 rpm, 4 °C, for 5 min, only the supernatant was collected and used as the protein extraction solution. The extract was dispensed into 50-µL portions into microtubes and stored at -80 °C. Protein yield was measured by colorimetric quantification using bicinchoninic acid (BCA Protein Assay Kit, TaKaRa, Shiga, Japan) and serial dilution of bovine serum albumin (BSA). Using the Luminex 200 xPONENT system (Thermo Fisher Scientific), a simultaneous multiplex protein immunoassay system of protein extracts from the left cerebral cortex, left hippocampus, and cerebellum was prepared to determine the tissue concentrations of the following 15 cytokines: CCL2, CCL11, CXCL1, CXCL2, CXCL10, granulocyte colony stimulating factor (G-CSF), IL-1α, IL-1β, IL-4, IL-6, IL-10, IL-12, IL-17, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2-5. Isolation of CD11b(+) cells, RNA extraction, and microarray analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 24 hours after LPS or saline administration, mice were deeply anesthetized with ketamine and xylazine. Blood was washed out by transcardial perfusion with sterile Dulbecco’s phosphate-buffered saline [D-PBS(−)] to remove plasma and blood cells. Fresh whole brains, including the arachnoid, pia, and parenchyma, were quickly removed. Two brains from the same dam puppies, treated in the same experimental manner, were placed in a 50-mL conical tube containing ice-cold D-PBS(−) and processed as a single sample. Four samples were prepared in each of the systemic inflammation and control groups.\u003c/p\u003e\n\u003cp\u003eTo collect macrophages and microglial cells from fresh brains, CD11b(+) cells were isolated using magnetic-activated cell sorting (MACS) methods [28]. Whole brains were dissociated by enzymatic digestion of the extracellular matrix using the Adult Brain Dissociation Kit for mice (Miltenyi Biotec, Auburn, CA, USA). Mechanical dissociation steps were performed using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) according to the manufacturer’s protocol. Brain tissue dissociates were centrifuged at 400 × g for 5 min at 4 °C. The pellets were resuspended in cold D-PBS (+) (with calcium and magnesium) containing 0.5% bovine serum albumin (PB buffer). In the presence of the kit’s Debris Removal Solution, the cell suspensions were centrifuged at 3000 × g for 12 min at 4 °C to remove the debris phase. The pellets were resuspended in cold PB buffer and incubated with R-phycoerythrin (PE)-conjugated primary human/mouse CD11b monoclonal antibody (130–113–235, Miltenyi Biotec) and Fc receptor blocking reagent (130–092–575, Miltenyi Biotec), followed by incubation with MicroBeads UltraPure conjugated to anti-PE monoclonal antibody (130–105–639, Miltenyi Biotec). Suspended cells labeled with anti-PE MicroBeads were enriched by magnetic separation using an LS column (Miltenyi Biotec) that was placed in a QuadroMACS separator (Miltenyi Biotec) according to the manufacturer’s protocol. CD11b-positive-selected cells (positive fraction) were considered macrophages and microglia. During the positive selection process, CD11b-negative cells were also collected (negative fraction). The number of cells in the positive and negative fractions was determined using cell counting plates (OneCell counter; Fine Plus International, Kyoto, Japan) under an ECLIPSE Ts2 (Nikon) inverted phase-contrast microscope. The cells of the positive and negative fractions were finally suspended in 1 mL CELLBANKER 1 Plus (TaKaRa) and stored at −80 °C before RNA extraction.\u003c/p\u003e\n\u003cp\u003eFrozen cells were thawed rapidly at 37 °C, centrifuged at 400 × g for 5 min at 4 °C, and washed with RNase-free PBS by centrifugation under the same conditions. Total RNA was extracted from the cell pellets using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. RNA quantification was performed using NanoVue (GE Healthcare Life Sciences, Chicago, IL, USA) and 2100 \u003cu\u003eBioAnalyzer\u003c/u\u003e(Agilent, Santa Clara, CA, USA). Eight RNA samples were of high quality with RNA Integrity Numbers (RINs) ranging from 7.9 to 9.1.\u003c/p\u003e\n\u003cp\u003eGene expression profiles were analyzed by Hokkaido System Science Co., Ltd. (Hokkaido, Japan) using the SurePrint G3 Mouse GE 8x60K Ver.2.0 Microarray (Agilent, G4852B). Cyanine-3 (Cy3)-labeled cRNA was prepared from 50 ng of total RNA using the Low Input Quick Amp Labeling Kit (Agilent), followed by RNeasy column purification (QIAGEN). Then, 0.6 μg of Cy3-labeled cRNA (specific activity \u0026gt; 6 pmol Cy3/μg cRNA) was fragmented at 60 °C for 30 min in a reaction volume of 25 μL containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent. On completion of the fragmentation, 25 μL of 2x Agilent GE Hi-RPM hybridization buffer were added. The fragmentation mixture was hybridized to SurePrint G3 Mouse GE 8x60K Ver.2.0 Microarray at 65 °C for 17 hours in an Agilent rotating hybridization oven. After hybridization, the microarrays were washed with GE Wash Buffer 1 (Agilent) for 1 min at room temperature and with GE Wash Buffer 2 (Agilent) for 1 min at 37 °C. The slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G2600D) using one color scan setting for 8x60K array slides (dye channel set to green, and green photomultiplier tube set to 100%). The scanned images were analyzed with Feature Extraction Software 12.0.3.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended processed signal intensities. The 75th percentile shift normalization was performed using Agilent GeneSpring GX 14.9, and baseline transformation was performed using the median of all samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2-6. Real-time reverse transcription-polymerase chain reaction (RT-qPCR)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 4, 12, 24, 48, and 72 hours after LPS or saline administration (n = 4 samples in each group), mice were deeply anesthetized with ketamine-xylazine, blood was poured out, and fresh whole brains were quickly removed. Two brains of P7 mice from the same dam, treated in the same experimental manner, were pooled in ice-cold D-PBS(−) and processed as a single sample. CD11b(+) cells were isolated by MACS, and total RNA was extracted from CD11b(+) and CD11b(−) cells. RNA quantification was performed using NanoVue (GE Healthcare Life Sciences) and using 4150 TapeStation System (Agilent). Eighty RNA samples were of high quality with RINs ranging from 8.9 to 9.8.\u003c/p\u003e\n\u003cp\u003eFifty nanograms of total RNA were used for reverse transcription to cDNA using SuperScript III Reverse Transcriptase (Invitrogen-Thermo Fisher Scientific). The real-time reverse transcription-polymerase chain reaction (RT-qPCR) was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems -Thermo Fisher Scientific), TaqMan primer/probe sets for 10 targets (Applied Biosystems), and a 7500 Fast Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocols. The targets were as follows: (1) \u003cem\u003eSaa3\u003c/em\u003e (encoding serum amyloid A3), Mm00441203_m1; (2) \u003cem\u003eSaa1\u003c/em\u003e (encoding serum amyloid A1), Mm00656927_g1; (3) \u003cem\u003eSaa2\u003c/em\u003e (encoding serum amyloid A2), Mm04208126_mH; (4) \u003cem\u003eIrg1\u003c/em\u003e (encoding immune-responsive gene 1 [IRG1], also known as aconitate decarboxylase 1), Mm01224532_m1; (5) Ccl5 (encoding chemokine CCL5), Mm01302428_m1; (6) Cxcl13 (encoding chemokine CXCL13), Mm00444534_m1; (7) Slfn4 (encoding schlafen-4), Mm01298330_m1; (8) Cxcl9 (encoding chemokine CXCL9), Mm00434946_m1; (9) Lcn2 (encoding lipocalin-2), Mm01324470_m1; (10) internal control, \u003cem\u003eHprt\u003c/em\u003e (encoding hypoxanthine phosphoribosyltransferase), Mm03024075_m1. Analysis of relative transcript levels was performed using the ΔΔCT method. All assays were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2-7. In situ hybridization (ISH)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen sections were prepared from mice at 4, 12, 24, and 48 hours after LPS or saline administration (n = 4 mice in each group) and used for ISH. ISH was performed with RNAscope 2.5 HD Assay-Brown for fixed frozen tissue (#322310; Advanced Cell Diagnostics, Newark, CA, USA) according to the manufacturer’s protocol with minor modification. The modification was as follows: Protease Plus was diluted 1:2 and incubated for 10 min, and DAB precipitation was performed using the ImmPACT DAB Substrate Kit, Peroxidase. Target probes for the RNAscope manual assay (Advanced Cell Diagnostics) were Mm-Saa3 (Cat. No. 446841), Mm-Irg1 (450241), Mm-Cxcl13 (406311), Mm-Ccl5 (469601), Mm-Cxcl9 (489341), Mm-Lcn2 (313971), Mm-Slfn4 (573011), and Mm-Col1a1 (319371).\u003c/p\u003e\n\u003cp\u003eRNAscope DAB precipitation was coupled to immunohistochemistry using anti-Iba1 antibody (rabbit monoclonal [EPR16588], Abcam) or anti-type I collagen antibody (rabbit monoclonal [EPR24331-53], Abcam). The ImmPRESS-AP Horse Anti-Rabbit IgG Polymer Detection Kit, Alkaline Phosphatase (MP-5401, Vector), was used as the secondary antibody. Immunohistochemistry was visualized using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) Substrate Kit, Alkaline Phosphatase (AP) (SK-5400, Vector). Sections were covered with G-Mount (Genostaff, Tokyo, Japan) and then coverslipped with HSR (Sysmex).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2-8. Statistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the tissue cytokine immunoassay and the morphometry of Iba1-immunopositive cells, data were obtained from 8 experimental conditions (saline vs. LPS administration; 4, 24, 48, and 72 hours after administration), and the results were analyzed by two-way analysis of variance (ANOVA; main effects of treatment and time). For RT-qPCR analyses, the mean ΔCT values for each gene target were obtained from 10 experimental conditions (saline vs. LPS administration; 4, 12, 24, 48, and 72 hours after administration). The results were analyzed by two-way ANOVA. Post hoc tests were performed using Tukey’s procedure. P values less than 0.05 were considered significant in all analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3-1. IL-1β expressed by meningeal macrophages and IL-1R1 expressed by leptomeningeal fibroblasts\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice used in the present study ranged in age from P7 to P10 and thus exhibited a postnatal developmental increase in body weight. The pattern of body weight change was affected by the administration of LPS or saline to the P7 mice. The change in body weight was evaluated by taking the body weight just before tissue collection\u0026nbsp;minus the body weight just before intraperitoneal administration, as shown in Fig. 2A. The mice injected with 0.75 mg/kg LPS lost 0.21 g at 12 hours (n = 8) and 0.22 g at 24 hours (n = 40), followed by a gain of 0.72 g at 48 hours (n = 16) and 1.21 g at 72 hours (n = 19) after LPS injection. Mice with systemic inflammation exhibited remarkable sickness behavior during the period 1 to 12 hours after LPS administration, and 98% of LPS-treated mice survived after LPS administration. In preliminary experiments to determine the dose of LPS, about 15% of mice died within one day after a dose of 1.0 mg/kg of LPS was administered (data not shown).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMice with systemic inflammation showed a slight increase in the number of polymorphonuclear leukocytes in the leptomeninges 12 and 24 hours after LPS administration, but the extent of inflammatory cell infiltration was much less than seen in meningitis (Fig. 2B,C). There was no evidence of focal or global acute ischemic changes in the brain parenchyma, with no hemorrhagic or edematous lesions that could attract inflammatory leukocytes. A small number of polymorphonuclear leukocytes also infiltrated the other major organs, including the lungs, liver, and kidneys (Fig. 2D,E).\u003c/p\u003e\n\u003cp\u003eIn all mice 4 hours after LPS administration, the protein expression of IL-1β was evident in Iba1-positive macrophages located in the subarachnoid space and in the choroid plexus stroma (Fig. 3A-E). In the medulla of the cerebellum, there were many Iba1-positive cells, which were considered to be the macrophages that migrate and invade the nearby brain parenchyma to become microglia at 7 days of age. Twenty-eight percent of the Iba1-positive cells in the medulla of the cerebellum were immunopositive for IL-1β 4 hours after LPS administration (Fig. 3F). The microglia located in the circumventricular organs (CVOs), including the area postrema, median eminence, and subfornical organ, expressed IL-1β, probably due to plasma LPS leakage from the blood vessels lacking the BBB (Fig. 3G). In contrast, microglia located in most of the other brain parenchyma, except CVOs, did not express IL-1β, which suggested the presence of a functional BBB in P7 mice.\u003c/p\u003e\n\u003cp\u003eIL-1R1, a receptor for IL-1β, was expressed by arachnoid cells and pial cells, which are the major cells of the leptomeninges and were recently identified as genuine fibroblasts [23-27]. Double immunohistochemical staining showed that leptomeningeal cells immunopositive for type I collagen exhibited IL-1R1 immunopositivity at the edge of the cytoplasm (Fig. 3H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3-2. Immunoassay of tissue cytokine concentration in the brain parenchyma\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe yields of protein extracts from the left cerebral cortex, left limbic system, cerebellum, and spleen were measured before the tissue cytokine concentrations were immunoassayed. The protein concentrations were approximately 2000 µg/mL in the left cerebral cortex and left limbic system and 2300 µg/mL in the cerebellum and spleen, confirming that the protein extraction was stable and reliable.\u003c/p\u003e\n\u003cp\u003eThe levels of CCL2, G-CSF, CXCL10, IL-6, and CXCL1 in the brain parenchyma and spleen tissues were significantly increased in mice with LPS-induced systemic inflammation compared with saline control (Figs. 4-8). The time-dependent changes in the tissue concentrations of each cytokine were similar among the three brain regions.\u003c/p\u003e\n\u003cp\u003eCCL2 levels were increased in the cerebral cortex, limbic system, and cerebellum 4 and 24 hours after LPS administration (Fig. 4). The time-dependent changes in CCL2 levels in the brain parenchyma were similar to those in the spleen, but CCL2 levels in the spleen appeared to decrease more rapidly toward the control levels later than 4 hours after LPS administration. Immunohistochemistry using anti-CCL2 antibody was effective in identifying the cells involved in CCL2 production. CCL2-immunopositive cells were lined along the blood vessels in the leptomeninges and brain parenchyma 4 hours after LPS administration (Fig. 4 E-H). Double immunofluorescence staining for CCL2 and type I collagen showed that the CCL2-immunopositive cells were perivascular fibroblasts (Fig. 4I).\u003c/p\u003e\n\u003cp\u003eG-CSF levels were increased in the cerebral cortex, limbic system, and cerebellum 4 and 24 hours after LPS administration (Fig. 5A). The time-dependent changes in G-CSF levels in the brain parenchyma were similar to those in the spleen, and G-CSF levels in the spleen were higher than those in the brain 4 and 24 hours after LPS administration. CXCL10 levels were higher in the cerebral cortex, limbic system, and cerebellum of mice with systemic inflammation than in saline control mice 4 and 24 hours after LPS administration (Fig. 5B). CXCL10 levels in the spleen of mice with systemic inflammation were significantly higher than in saline control mice only 4 hours after LPS administration. IL-6 levels were increased in the cerebral cortex, limbic system, and cerebellum 4 hours after LPS administration (Fig. 5C). The time-dependent changes in IL-6 levels in the brain parenchyma were the same as those in the spleen, but IL-6 levels in the spleen were much higher than those in the brain 4 hours after LPS administration. CXCL1 levels were higher in the cerebral cortex, limbic system, and cerebellum of mice with systemic inflammation than in saline control mice 4 hours after LPS administration (Fig. 5D). The time-dependent changes in CXCL1 levels in the brain parenchyma were the same as those in the spleen, but CXCL1 levels in the spleen were much higher than those in the brain in mice with systemic inflammation 4 hours after LPS administration.\u003c/p\u003e\n\u003cp\u003eCCL11 levels were higher in the cerebral cortex, limbic system, and cerebellum of mice with systemic inflammation than in saline control mice 4, 24, and 48 hours after LPS administration (Fig. 6A). However, CCL11 levels in brain parenchymal tissues prepared from mice with systemic inflammation were relatively low compared with the five cytokines described above. CCL11 levels in the spleen of mice with systemic inflammation were highly elevated 4 hours after LPS administration. The pattern of increase in CXCL2 levels varied depending on the part of the brain (Fig. 6B). There were higher CXCL2 levels in the cerebral cortex of mice with systemic inflammation than in saline control mice 4 and 24 hours after LPS administration. CXCL2 levels were significantly higher in the cerebellum 4 hours after LPS administration than in saline-injected mice. However, there was no significant difference in CXCL2 levels in the limbic system at any time point after LPS administration. In contrast, CXCL2 levels in the spleen of LPS-treated mice were significantly and markedly higher in the cerebellum 4 hours after LPS administration than in saline-injected mice. IL-10 levels were increased only in the cerebral cortex, among the brain parenchyma, of LPS-treated mice compared with saline-injected mice 24 hours after LPS administration (Fig. 6C). In contrast, IL-10 levels were increased in the spleen 4 and 24 hours after LPS administration.\u003c/p\u003e\n\u003cp\u003eThere were no significant changes in\u0026nbsp;IL-1α or IL-1β levels in any brain parenchymal region at any time point after LPS administration (Fig. 7). Levels of IL-1α and IL-1β were higher only in the spleen of mice with systemic inflammation than in saline control mice 4 and 24 hours after LPS administration. An increase in\u0026nbsp;IFN-γ levels was not evident in the brain at any time point, but only in the spleen of mice with systemic inflammation compared with saline control mice 4 hours after LPS administration\u0026nbsp;(Fig. 8A). Higher IL-17\u0026nbsp;levels were not evident in the brain at any time point, but only in the spleen of mice with systemic inflammation compared with saline control mice 24 hours after LPS administration\u0026nbsp;(Fig. 8B). Higher TNF-α\u0026nbsp;levels were only evident in the spleen of LPS-treated mice compared with saline-injected mice 4 hours after LPS administration (Fig. 8C). TNF-α levels in the brain were below the minimum detectable concentration according to the manufacturer’s assay characteristics in any region at any time point after saline or LPS injection.\u0026nbsp;IL-12 and IL-4 levels in the brain and spleen were below the minimum detectable concentrations in all experimental groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3-3. Histological morphometry of glial cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining for Iba1 was performed to clarify time-dependent changes in microglial morphology induced by LPS administration. Microglial changes differed between the cerebellar and non-cerebellar regions. First, in non-cerebellar regions, such as the cerebral cortex and hippocampus, of mice with systemic inflammation, microglia became hypertrophic with thicker and shorter cytoplasmic processes 4 hours after LPS administration (Fig. 9A,B). Subsequently, a maximum degree of cellular hypertrophy accompanied by an increased number of Iba1-positive cells was evident 24 hours after LPS administration compared with saline control mice (Fig. 9C,D). In particular, perivascular macrophages became extremely hypertrophic in mice with systemic inflammation 24 hours after LPS administration; they were probably destined to enter the cortical parenchyma (Fig. 9E,F).\u003c/p\u003e\n\u003cp\u003eThese results were confirmed by the morphometric analysis. The Iba1-positive area fraction of the cerebral cortex and hippocampus was significantly higher in mice with systemic inflammation than in saline control mice 24 hours after LPS administration (Fig. 10A,B). In the hippocampus, the number of Iba1-positive cells was significantly higher in mice with systemic inflammation than in saline control mice 24 and 48 hours after LPS administration (Fig. 10C). In general, there was a time-dependent increase in the number of Iba1-positive cells in the brain parenchyma of both the LPS-treated and saline-injected mice, due to the postnatal development of microglia in which leptomeningeal macrophages migrate into the brain parenchyma.\u003c/p\u003e\n\u003cp\u003eIn contrast, cerebellar microglia or macrophages were densely distributed in the medulla and less so in the cortex in saline control mice (Fig. 9G). In the normal development of the cerebellum, there was no increase in the Iba1-positive area fraction during the developmental period between P7 and P10 in saline control mice (Fig. 10D). The dense distribution of macrophages in the medulla was specific to pup mice, and the macrophages in the medulla were likely to be distributed to the nearby cerebellar cortex to become microglia by adulthood. Systemic inflammation led to an increased number of microglia and their hypertrophy in the cerebellar cortex 24 and 48 hours after LPS administration (Fig. 9H and 10D).\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining for GFAP and S-100 was also performed to observe morphological changes in astrocytes. Regarding the astrocytes in the cerebral cortex, it is well known that the protein expression level of GFAP is quite different between the astrocytes of the superficial and deep layers and those of the middle layers, and the findings apply to both pups and adults. The middle-layer astrocytes, in which GFAP expression was undetectable immunohistologically, but the S-100 level was detectable, became hypertrophic and were detectable by immunohistochemistry for GFAP and S-100 in mice with systemic inflammation (Fig. 11). These hypertrophic changes in the cortical astrocytes represented some important homeostatic changes in response to systemic inflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3-4. Microarray analysis of CD11b-positive cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the brain-derived CD11b-positive cells isolated from mice 24 hours after administration of LPS or saline. The microarray data are available in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE289767) under accession number GSE289767. \u003cstrong\u003e[A secure token has been created to allow review of record GSE289767 while it remains in private status: ujqxmmiqvlkjxux]\u0026nbsp;\u003c/strong\u003eA comparison of the changes in gene expression between LPS-treated and saline-injected mice identified 1122 gene probes with significantly increased transcript expression levels and 1616 gene probes with significantly decreased transcript expression levels (Fig. 12). Of the genes that showed significant upregulation, the top 20 genes whose transcript expression levels were the highest in mice with systemic inflammation compared with saline control were as listed in Table 2. Upregulation of the top 9 genes was as follows: serum amyloid A3 (\u003cem\u003eSaa3\u003c/em\u003e) was 4924-fold (corrected p = 0.000094), C-X-C motif chemokine ligand 13 (\u003cem\u003eCxcl13\u003c/em\u003e) was 289-fold (corrected p = 0.0023), serum amyloid A1 (\u003cem\u003eSaa1\u003c/em\u003e) was 265-fold (corrected p = 0.000094), serum amyloid A2 (\u003cem\u003eSaa2\u003c/em\u003e) was 135-fold (corrected p = 0.0022), C-C motif chemokine ligand 5 (\u003cem\u003eCcl5\u003c/em\u003e) was 79-fold (corrected p = 0.00041), immune-responsive gene 1 (\u003cem\u003eIrg1\u003c/em\u003e, or cis-aconitate decarboxylase 1 (\u003cem\u003eAcod1\u003c/em\u003e)) was 69-fold (corrected p = 0.0022), schlafen 4 (\u003cem\u003eSlfn4\u003c/em\u003e) was 41-fold (corrected p = 0.0027), C-X-C motif chemokine ligand 9 (\u003cem\u003eCxcl9\u003c/em\u003e) was 31-fold (corrected p = 0.0046), and lipocalin-2 (\u003cem\u003eLcn2\u003c/em\u003e) was 29-fold (corrected p = 0.0022). In contrast, CCL2, G-CSF, CXCL10, IL-6, and CXCL1, which showed marked increases in tissue concentrations based on the LUMINEX immunoassay, the fold changes in gene expression were 1.10-fold (CCL2), 9.61-fold (G-CSF), 2.15-fold (CXCL10), -1.82-fold (IL-6), and -1.13-fold (CXCL1). The release system of these cytokines was not via gene transcription.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3-5. RT-qPCR for validation of microarray results and time-dependent changes in gene transcript expression\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRT-qPCR analyses of the top 9 genes were performed using total RNA extracted from the CD11b(+) and CD11b(−) cells prepared 4, 12, 24, 48, and 72 hours after LPS or saline injection. The upregulation of transcript expressions of the nine genes in CD11b(+) cells isolated from mice with systemic inflammation and saline control mice was validated overall (Figs. 13-15). For each gene, fold changes in transcript expression due to LPS treatment compared with saline injection were evaluated. The changes in expression of CD11b(+) and CD11b(−) cells were examined in detail. The changes were analyzed as a function of time after LPS administration. Each gene is described according to the degree of fold change.\u003c/p\u003e\n\u003cp\u003eThe transcript expression of \u003cem\u003eSaa3\u003c/em\u003e in CD11b(+) cells showed the most significant increase, reaching an astonishing 10,790-fold upregulation compared with the saline control 4 hours and a marked 4,459-fold increase 12 hours after administration of LPS or saline (Fig. 13A). \u003cem\u003eSaa3\u003c/em\u003e expression in CD11b(+) cells then decreased gradually, but it was still significantly upregulated up to 72 hours after administration. \u003cem\u003eSaa3\u003c/em\u003e expression in CD11b(−) cells also showed marked increases, reaching 6735-fold and 4230-fold 4 and 12 hours after LPS or saline administration, respectively (Fig. 13B). This indicates that \u003cem\u003eSaa3\u003c/em\u003e expression was markedly increased in both CD11b(+) and CD11b(−) cells as early as 4 and 12 hours after LPS administration.\u003c/p\u003e\n\u003cp\u003eSystemic inflammation-activated changes in \u003cem\u003eCxcl9\u003c/em\u003e transcript expression were upregulated as high as 4862-fold compared with saline control in CD11b(−) cells at 12 hours and 1294-fold at 4 hours after administration of LPS or saline, and thereafter decreased gradually (Fig. 14B). This marked increase in \u003cem\u003eCxcl9\u003c/em\u003e expression was primarily driven by CD11b(−), since the degree of \u003cem\u003eCxcl9\u003c/em\u003e upregulation in CD11b(+) cells was much lower, reaching 99-fold at 12 hours and 101-fold at 4 hours (Fig. 14A).\u003c/p\u003e\n\u003cp\u003eThe transcript expression of \u003cem\u003eIrg1\u003c/em\u003e (or \u003cem\u003eAcod1\u003c/em\u003e) in CD11b(+) cells showed the most marked increase, reaching 1301-fold upregulation compared with the saline control at 4 hours and a notable 866-fold increase at 12 hours after administration of LPS or saline. Thereafter, the expression decreased gradually (Fig. 14C). \u003cem\u003eIrg1\u003c/em\u003e expression in CD11b(−) cells also showed marked upregulation, reaching 1012-fold at 4 hours and 659-fold at 12 hours after LPS or saline treatment, followed by a gradual decrease (Fig. 14D). This suggests that \u003cem\u003eIrg1\u003c/em\u003e expression is strongly increased in both CD11b(+) and CD11b(−) cells as early as 4 and 12 hours after LPS administration.\u003c/p\u003e\n\u003cp\u003eSystemic inflammation-activated changes in \u003cem\u003eLcn2\u003c/em\u003e transcript expression were as high as 957-fold upregulation compared with saline control in CD11b(−) cells at 12 hours and 359-fold at 4 hours after administration of LPS or saline, and decreased gradually thereafter (Fig. 13F). This surge in \u003cem\u003eLcn2\u003c/em\u003e expression was primarily driven by CD11b(−), aligning with the pattern seen in \u003cem\u003eCxcl9\u003c/em\u003e expression. The degree of \u003cem\u003eLcn2\u003c/em\u003e upregulation in CD11b(+) cells was much lower, reaching 56-fold at 12 hours and 72-fold at 4 hours (Fig. 13E).\u003c/p\u003e\n\u003cp\u003eThe transcript expressions of \u003cem\u003eSaa2\u003c/em\u003e and \u003cem\u003eSaa1\u003c/em\u003e in CD11b(+) cells were the most marked, exhibiting a 750-fold increase and 491-fold increase, respectively, compared with the saline control. This was seen 4 hours after administration of LPS or saline, and decreased gradually thereafter (Fig. 14E,G). The expressions of \u003cem\u003eSaa2\u003c/em\u003e and \u003cem\u003eSaa1\u003c/em\u003e in CD11b(−) cells also exhibited marked upregulation, reaching 205-fold and 77-fold upregulation, respectively, compared with saline control at 12 hours (Fig. 14F,H).\u003c/p\u003e\n\u003cp\u003eThe transcript expression of \u003cem\u003eCcl5\u003c/em\u003e in CD11b(+) cells showed the most significant increase, reaching 348-fold upregulation compared with the saline control. This marked response occurred just 4 hours after the administration of LPS or saline, and then decreased gradually over time (Fig. 15A). The degree of \u003cem\u003eCcl5\u003c/em\u003e upregulation in CD11b(−) cells also showed a high response, with 172-fold upregulation at 4 hours, followed by a gradual decrease (Fig. 15B).\u003c/p\u003e\n\u003cp\u003eThe transcript expression of \u003cem\u003eCxcl13\u003c/em\u003e in CD11b(+) cells showed the most marked increase, reaching 345-fold upregulation compared with the saline control. This response occurred 12 hours after the administration of LPS or saline, and then decreased gradually over time (Fig. 15C). \u003cem\u003eCxcl13\u003c/em\u003e expression in CD11b(−) cells reached 51-fold upregulation at 12 hours, followed by a steady decrease (Fig. 15D).\u003c/p\u003e\n\u003cp\u003eThe transcript expression of \u003cem\u003eSlfn4\u003c/em\u003e in CD11b(+) cells showed the most marked increase, reaching 105-fold upregulation compared with the saline control. This marked response occurred just 4 hours after the administration of LPS or saline, and then decreased gradually over time (Fig. 15E). In addition, \u003cem\u003eSlfn4\u003c/em\u003e expression in CD11b(−) cells reached 22-fold upregulation at 12 hours, followed by a steady decrease (Fig. 15F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3-6. Identification of cells upregulating gene expression by ISH\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eISH was performed to determine which cells upregulated gene expression using the histological brain sections. Mouse brain areas were identified with the help of anatomical atlases [29-31]. \u003cem\u003eSaa3\u003c/em\u003e transcript levels were highly expressed in cells located in the leptomeninges and choroid plexus stroma, with frequent cells in the perivascular space 4 to 24 hours after LPS administration (Fig. 13C,D). No \u003cem\u003eSaa3\u003c/em\u003e expression was observed by ISH in any region of the brain or leptomeninges at any timepoint in saline control mice. The major cells with high \u003cem\u003eSaa3\u003c/em\u003e expression were located in the leptomeninges, perivascular space, choroid plexus stroma, and cerebellar medulla part (Fig. 16A-H). Tangential sections of the leptomeninges, covering the interhemispheric fissure, showed a high distribution density of \u003cem\u003eSaa3\u003c/em\u003e-expressing cells (Fig. 16J). These \u003cem\u003eSaa3\u003c/em\u003e-expressing cells were identified as macrophages, based on double staining with ISH targeting \u003cem\u003eSaa3\u003c/em\u003e, followed by immunohistochemical examination using anti-Iba1 antibody (Fig. 17A,B). The other major cells with high \u003cem\u003eSaa3\u003c/em\u003e expression were identified as fibroblasts by double staining with ISH targeting \u003cem\u003eSaa3\u003c/em\u003e, followed by immunohistochemical examination using anti-type I collagen antibody (Fig. 17C).\u003c/p\u003e\n\u003cp\u003eWhen the midsagittal and parasagittal sections were compared, the midsagittal sections, which contained the 3rd ventricle and its choroid plexus, highlighted the presence of \u003cem\u003eSaa3\u003c/em\u003e-expressing cells much more frequently than the parasagittal sections (Fig. 18A,B). This was because a long rostro-caudal extension of the choroid plexus stroma and the parallel leptomeninges (the roof of the third ventricle) were well populated with macrophages and fibroblasts (Fig. 18C). Further findings, by comparing the midsagittal sections made at 4, 12, and 24 hours after LPS administration, showed that the \u003cem\u003eSaa3\u003c/em\u003e-expressing macrophages located in the leptomeninges forming the roof of the third ventricle enter the brain parenchyma in a time-dependent manner to become microglia (Fig. 18D-F). Cell counting showed that the number of\u003cem\u003e\u0026nbsp;Saa3\u003c/em\u003e-positive macrophages/microglia was initially low 4 hours after LPS administration. However, at 12 hours, there was a significant surge in the number of \u003cem\u003eSaa3\u003c/em\u003e-positive macrophages/microglia in the ventral hippocampal commissure (Fig. 17D), and at 24 hours, a significantly increased number of \u003cem\u003eSaa3\u003c/em\u003e-positive macrophages/microglia was evident in the ventral hippocampal commissure, fornix, medial habenula, and corpus callosum (Fig. 18G-J). The movement of these \u003cem\u003eSaa3\u003c/em\u003e-positive macrophages did not represent an expansion of inflammation-related events, since the migration rate was relatively slow, no lesion such as ischemia or necrosis was found in the brain parenchyma, Saa3-positive macrophages/microglia bore fine cytoplasmic processes different from activated morphology (Fig. 17D), and the migration routes were consistent among all LPS-treated mice. This migration by macrophages was interpreted as representing an important stage of neurodevelopment, that of microglial formation. This microglial formation was considered to occur in the same fashion in saline-injected mice, although normal macrophages/microglia were not detectable due to the absence of \u003cem\u003eSaa3\u003c/em\u003e expression. In addition, microglia located in the CVOs, such as the area postrema, median eminence, vascular organ of the lamina terminalis, subfornical organ, and subcommissural organ, also expressed \u003cem\u003eSaa3\u003c/em\u003e (Fig. 16I). In contrast, the microglia located in most of the brain parenchyma except the CVOs bore multiple cytoplasmic processes and never expressed \u003cem\u003eSaa3\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe transcript expression of \u003cem\u003eIrg1\u003c/em\u003e was marked in the macrophages in the choroid plexus, with many of these being epiplexus cells, and in the leptomeningeal macrophages (Fig. 19A,B). Macrophages surrounding the blood vessels in the medulla part of the cerebellum showed high \u003cem\u003eIrg1\u003c/em\u003e expression as well (Fig. 19C). Microglia in the CVOs also showed clear upregulation of \u003cem\u003eIrg1\u003c/em\u003e (Fig. 19D), but brain parenchymal microglia did not express \u003cem\u003eIrg1\u003c/em\u003e. These \u003cem\u003eIrg1\u003c/em\u003e-positive cells were identified as macrophages by double staining with ISH targeting \u003cem\u003eIrg1\u003c/em\u003e followed by immunohistochemical examination using anti-Iba1 antibodies (Fig. 19E,F). The other \u003cem\u003eIrg1\u003c/em\u003e-positive cells in the leptomeninges that did not express Iba1 were not identified as a cell type. Macrophages showed \u003cem\u003eIrg1\u003c/em\u003e upregulation most markedly at 4 hours, and \u003cem\u003eIrg1\u003c/em\u003e expression was decreased 12 hours after LPS administration. \u003cem\u003eIrg1\u003c/em\u003e expression was not detected in saline control mice at any time point, underscoring the specificity of the observations.\u003c/p\u003e\n\u003cp\u003eISH targeting \u003cem\u003eLcn2\u003c/em\u003e showed high expression in the perivascular cells and in the arachnoid and pial cells (Fig. 13G,H and Fig. 20A-D). These cells were identified as fibroblasts by immunohistochemical double staining for type 1 collagen (Fig. 20E,F). The \u003cem\u003eLcn2\u003c/em\u003e upregulation in fibroblasts was the most marked 4 and 12 hours after LPS administration. Considering that our LPS was originally prepared from E. coli, perivascular fibroblasts played an essential role in protecting the brain tissue from potential E. coli invasion because lipocalin-2 mediates the innate immune response to inhibit bacterial growth by sequestering the iron-laden siderophore (Flo et al., 2004). In addition, the choroid plexus epithelial cells started clear \u003cem\u003eLcn2\u003c/em\u003e expression slightly later than fibroblasts, peaking at 12 hours and still being high at 24 hours after LPS administration (Fig. 20G,H). \u003cem\u003eLcn2\u003c/em\u003e expression was not detected in saline control mice at any time point.\u003c/p\u003e\n\u003cp\u003eThe transcript expression of \u003cem\u003eCxcl9\u003c/em\u003e was remarkable in the arachnoid, pial, and perivascular fibroblasts (Fig. 21A-D). \u0026nbsp; \u003cem\u003eCxcl9\u003c/em\u003e was expressed in the choroid plexus fibroblasts but not in the epithelium (Fig. 21E,F). \u0026nbsp;These results were confirmed by double staining of ISH targeting \u003cem\u003eCxcl9\u003c/em\u003e followed by immunohistochemical examination for type I collagen. \u0026nbsp;The upregulation of \u003cem\u003eCxcl9\u003c/em\u003e in brain fibroblasts was most remarkable at 4 and 12 hours after LPS administration but not detected in saline control mice.\u003c/p\u003e\n\u003cp\u003eThe transcript expressions of \u003cem\u003eCcl5\u003c/em\u003e and \u003cem\u003eCxcl13\u003c/em\u003e were confirmed using the sections prepared 12 hours after saline or LPS injection, and these expressions were mainly detected in the choroid plexus in mice with systemic inflammation (Fig. 22A,C). \u0026nbsp;\u003cem\u003eCcl5\u003c/em\u003e transcript expression was also detected in the CVO (Fig. 22B). \u0026nbsp;\u003cem\u003eSlfn4\u003c/em\u003e transcript expression was increased by systemic inflammation in the meningeal space and choroid plexus 4 h after LPS administration (Fig. 22D).\u003c/p\u003e\n\u003cp\u003eISH conclusively showed that the early major immune reactions of the brain in response to systemic inflammation were initiated by leptomeningeal, perivascular, and choroid plexus macrophages and arachnoid, pial, and perivascular fibroblasts in the postnatal mouse brain.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003e4-1. Summary of main results\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe present study explored how intracranial tissues respond to systemic inflammation to protect the brain in preterm babies. To create an animal model, \u003cem\u003eE. coli\u003c/em\u003e-derived LPS at 0.75 mg/kg was administered by a single intraperitoneal injection into P7 mice and induced systemic inflammation. Just 4 hours later, the leptomeningeal and perivascular macrophages initiated an intracranial reaction by producing IL-1\u0026beta;, which set the stage for the arachnoid, pial, and perivascular fibroblasts to act. The brain fibroblasts produced CCL2, which increased the number of brain parenchymal macrophages/microglia and led to their hypertrophy. The macrophages/microglia were then isolated from the brain 24 hours after LPS administration, and total RNA was extracted. The microarray analysis showed markedly upregulated transcript expressions of \u003cem\u003eSaa3\u003c/em\u003e, \u003cem\u003eIrg1\u003c/em\u003e, \u003cem\u003eLcn2\u003c/em\u003e, and \u003cem\u003eCxcl9\u003c/em\u003e. RT-qPCR assays and histological staining showed that the expression level of \u003cem\u003eSaa3\u003c/em\u003e was increased in leptomeningeal macrophages, cerebral perivascular macrophages, and cerebellar perivascular macrophages, as well as in the fibroblastic cells of the leptomeninges, choroid plexus stroma, and perivascular space. Interestingly, the leptomeningeal macrophages expressing \u003cem\u003eSaa3\u003c/em\u003e continued their developmental migration from the leptomeninges to the parenchyma, such as the ventral hippocampal commissure, fornix, medial habenula, and corpus callosum. The expression level of \u003cem\u003eSaa3\u0026nbsp;\u003c/em\u003ereached their peak 4 and 12 hours after LPS administration. The BBB of P7 pups was so well constructed that the barrier function was not disrupted up to 72 hours after LPS administration, and thus the microglia located in all brain parenchyma regions except for the CVOs never increased expressions of inflammation-related genes. The expression level of \u003cem\u003eIrg1\u003c/em\u003e was remarkably increased in the choroid plexus and leptomeningeal macrophages and reached their peak 4 and 12 hours after LPS administration. The expression level of \u003cem\u003eLcn2\u003c/em\u003e was the most increased in the perivascular fibroblasts and choroid plexus epithelial cells, peaking 4 and 12 hours after LPS administration. The expression level of \u003cem\u003eCxcl9\u003c/em\u003e was increased in the fibroblasts in the leptomeninges, perivascular space, and choroid plexus stroma, peaking 4 and 12 hours after LPS administration. Early enhancement of the expression of \u003cem\u003eSaa3\u003c/em\u003e is a key step in priming inflammation, initiated by macrophages and fibroblasts [32].The early enhancement of \u003cem\u003eIrg1\u003c/em\u003e expression is crucial for determining the direction of the inflammatory response [33]. The increase of \u003cem\u003eLcn2\u003c/em\u003e is also crucial because it limits \u003cem\u003eE. coli\u003c/em\u003e growth by sequestering the iron-laden siderophore [34]. Whereas the biological function of \u003cem\u003eCxcl9\u003c/em\u003e upregulation in the present model is still being explored, CXCL9 has been reported to recruit and induce T cells to accumulate in the brain [35]. The early immune responses to systemic inflammation in the postnatal mouse brain were initiated by migrating macrophages and leptomeningeal fibroblasts, leading to immediate gene upregulations that were essential for protecting immature brain tissue.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e4-2. Intracranial early immune responses to intraperitoneal LPS\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIntraperitoneally administered LPS is absorbed into the systemic circulation and distributed to various tissues within 1-2 hours [36, 37]. In the present study, LPS reached the vasculature of the intracranial tissues and stimulated the macrophages in the leptomeninges and choroid plexus stroma by 4 hours after LPS administration. Such macrophages were detected by immunostaining for IL-1\u0026beta;. However, microglial cells in the brain parenchyma except for CVOs were not highlighted with immunohistochemistry for IL-1\u0026beta; because the functional BBB was well structured in the brain parenchyma of P7 mice. The presence of the BBB was more clearly highlighted by the experiments using ISH targeting \u003cem\u003eSaa3\u003c/em\u003e and other genes, as will be discussed in 4-4. In contrast, the IL-1R1 protein, a receptor for IL-1\u0026beta;, was expressed by arachnoid and pial cells, which were identified as fibroblasts by immunohistochemistry for type I collagen. Many of the macrophages located in the medulla part of the cerebellum expressed IL-1\u0026beta;, as well as \u003cem\u003eSaa3\u003c/em\u003e, 4 hours after LPS administration. The cerebellar medulla is challenging to study because of its unique features during postnatal development. For example, vascularization of the cerebellum occurs in an inside-out pattern [38], and the macrophages/microglia have a specific gene expression pattern that is different from the cortical grey matter [39]. In the present study, the immune response to systemic inflammation carried out in the cerebellar area was remarkable: IL-1\u0026beta; production and \u003cem\u003eSaa3\u003c/em\u003e expression were carried out by macrophages located not only in the leptomeninges covering the cerebellar surface, but also in the leptomeninges within the cerebellum. Of interest, the fibroblasts bearing IL-1R1 were in close proximity to meningeal macrophages producing IL-1\u0026beta; in widespread brain areas. These fibroblasts were identified as being composed of genuine arachnoid cells and pial cells [23-27].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e4-3. Role of brain fibroblasts in immune responses following systemic inflammation\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMeasuring tissue levels of cytokines using multiple immunoassays showed significant increases in CCL2, G-CSF, CXCL10, IL-6, and CXCL1 levels in the brain parenchyma 4 and 24 hours after LPS administration. Of the commercially available antibodies that were tried for immunohistochemistry, anti-CCL2 antibody was clearly effective for determining which cells were involved in producing CCL2. CCL2-producing cells were arachnoid and pial fibroblasts, as well as parenchymal perivascular fibroblasts. These results indicated that the leptomeningeal fibroblasts stimulated by the leptomeningeal macrophages via the IL-1\u0026beta;-IL-1R1 system conveyed the intercellular communications across the cell junctions, such as the gap junction, tight junction, and intermediate junction, between meningothelial fibroblasts and to parenchymal perivascular fibroblasts [40-42]. \u0026nbsp;These fibroblasts then produced and released CCL2 into the brain tissue 4 hours after LPS administration. We consider that CCL2 derived from parenchymal perivascular fibroblasts attracted more leptomeningeal macrophages into the parenchyma, which enhanced the bilateral intercellular interaction. CCL2 may have other actions, since a recent article reported that maternal stress during pregnancy is associated with increased CCL2, which plays a key role in mediating offsprings\u0026rsquo; behavioral sequelae [43]. The other strikingly important role played by perivascular fibroblasts after stimulation by the IL-1\u0026beta;-IL-1R1 system was the production of lipocalin-2 in widespread areas of the brain parenchyma. As reported, lipocalin-2 mediates the innate immune response to inhibit E. coli growth by sequestering the iron-laden siderophore [34]. Considering that our LPS was originally prepared from E. coli, perivascular fibroblasts played an essential role in protecting the brain tissue from potential E. coli invasion.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e4-4. Well-constructed blood-brain barrier functionality after\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003cem\u003eintraperitoneal LPS\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSAA proteins are small (about 100 amino acids) acute phase reactants associated with high-density lipoprotein (HDL), produced in the liver and increasing 1000-fold in plasma during inflammation [44]. SAAs probably play a critical role in the propagation of the primordial acute phase response [32]. In mice, SAAs induce Nod-like receptor protein 3 (NLRP3) inflammasome activation in macrophages and stimulate IL-1\u0026beta; release [45, 46]. Murine SAA1, SAA2, and SAA3 selectively contribute to Th17-mediated pathogenesis in inflammatory bowel disease and experimental autoimmune encephalomyelitis through loss- and gain-of-function with STAT3-activating cytokines [47]. Consistent with such previous publications, the present data indicated that LPS-induced systemic inflammation caused extreme upregulation of \u003cem\u003eSaa3\u003c/em\u003e in the macrophages located in the subarachnoid space and choroid plexus stroma. \u003cem\u003eSaa3\u003c/em\u003e was also upregulated in the arachnoid and pial cells, which are composed of genuine fibroblasts. It was important that the brain parenchymal microglia, with the exception of microglia in the CVOs, did not upregulate \u003cem\u003eSaa3\u003c/em\u003e in response to systemic inflammation. Similarly, brain parenchymal microglia did not upregulate \u003cem\u003eIrg1\u003c/em\u003e or \u003cem\u003eCcl5\u003c/em\u003e in response to systemic inflammation, but microglia in the CVOs upregulated these genes. These findings clearly indicated that the BBB of postnatally developing mice was so well constructed that the endothelial cells and pericytes ensured that the barrier function was as complete as that of adult mice [15-18]. In contrast, the immunohistochemistry for GFAP showed that astrocytes of P7-P10 mice did not have sophisticated endfeet compared with those of adults (data not shown). This suggested that the functions performed by the endothelial cells and the pericytes were much more important than the functions performed by the astrocytic endfeet to provide a simple barrier function to the blood vessels.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e4-5. Brain structures that may contain possible changes in microglial development following systemic inflammation\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGiven that the duration of \u003cem\u003eSaa3\u003c/em\u003e transcript expression detected by ISH was longer than that of other genes, the time-dependent changes in the location of \u003cem\u003eSaa3\u003c/em\u003e-positive macrophages/microglia were followed. The number of \u003cem\u003eSaa3\u003c/em\u003e-positive macrophages/microglia in the ventral hippocampal commissure was significantly higher at 12 hours than at 4 hours after LPS administration. By 24 hours, a further increase in the number of \u003cem\u003eSaa3\u003c/em\u003e-positive cells was observed in the fornix, medial habenula, and corpus callosum. These \u003cem\u003eSaa3\u003c/em\u003e-positive cells migrated into the brain parenchyma to complete the developmental transformation to become microglia. It is interesting that the migrating macrophages, which were essential for the transformation into parenchymal microglia, showed responsiveness to systemic inflammation, ensuring their timely participation in every crucial stage of microglial development. Several interhemispheric commissure bundles traverse the roof of the third ventricle: the ventral hippocampal commissure, dorsal hippocampal commissure, fornix, habenular commissure, and posterior commissure. The corpus callosum is located just dorsal to the dorsal hippocampal commissure in the caudal part and dorsal to the fornix in the central part. Immunohistochemical staining for type I collagen and ISH targeting \u003cem\u003eCol1a1\u003c/em\u003e showed that the collagen-rich leptomeninges, which cover the brain surface, were in close proximity to the ventral hippocampal commissure, fornix, medial habenula, and corpus callosum (Fig. 18). The leptomeninges contain numerous cerebrovascular branches that are destined for the brain parenchyma. The parenchymal blood vessels are accompanied by the perivascular spaces, which represent a major route of entry for macrophages to migrate into the brain parenchyma [14]. Once these macrophages enter the midline structure of the commissural fibers, they gain straightforward access to migrate to bilateral distal areas along the neuroaxonal fiber bundles. Indeed, some \u003cem\u003eSaa3\u003c/em\u003e-positive microglia detected in the medial habenula 24 hours after LPS administration were interpreted to originate from the habenular commissure. The habenula, a system that has existed since the evolution of the nervous system, functions as a regulatory hub, thereby enabling the forebrain to modulate the activity of cholinergic and ascending monoaminergic pathways in the midbrain [48-50]. Dysfunction of the habenula has been associated with significant mental disorders, including depression and addiction [51, 52]. The ventral hippocampal commissure facilitates interhemispheric connections between the left and right hippocampi [53, 54]. The fornix, a white matter tract, connects the hippocampus to several subcortical regions to contribute to the memory system [55]. In the present study, macrophages that showed a response to systemic inflammation entered the midline commissural bundles and gained access to migrate bilaterally to distal areas along the neuroaxonal bundles. In P7 mice, the likelihood may be high that neuroanatomical structures related to the roof of the third ventricle may contain abnormal microglia following systemic inflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4-6. Possible clinical association with encephalopathy of prematurity\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe central feature of the encephalopathy of prematurity (EP) is periventricular leukomalacia (PVL) [5]. Focal or large necrotic lesions are formed in the periventricular area of the white matter. The EP is characterized not only by hypoxia-ischemia, but also by multiple gray and white matter lesions. Therefore, developmental factors must be considered [5, 56]. In the present experimental model involving P7 mice, migration of leptomeningeal macrophages was highlighted in the commissural bundles that traverse the roof of the third ventricle. The brain developmental stage of P7 mice is equivalent to that of the human fetus at a gestational age of 30 weeks [10]. It is postulated that the macrophages that showed a response to systemic inflammation may retain abnormalities to maintain brain histological homeostasis. These abnormalities, if present, could potentially contribute to the pathogenesis of human EP pathology and related neurological sequelae, including depression, addiction, and pain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4-7. Limitations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe weakness of the present study was that, of the commercially available antibodies used for CCL2, G-CSF, CXCL10, IL-6, and CXCL1, only anti-CCL2 antibody exhibited an effective staining pattern that matched the treatment difference and time-dependent changes in the concentration. The antibodies against the other cytokines did not produce clear findings that matched the biochemical immunoassay data. Therefore, it was not possible to identify all cells that were involved in cytokine release. Second, the total RNA extracted from the CD11b(-) cells was effective in showing the upregulation of \u003cem\u003eLcn2\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Cxcl9\u003c/em\u003e genes. However, it was not possible to isolate more purified fibroblasts from the brain. Isolation of brain fibroblasts was attempted, but some further undetermined technological improvements appear to be necessary. The total RNA extracted from the brain fibroblasts would have enabled more sophisticated analysis of fibroblasts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4-8. Conclusions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn conclusion, in response to systemic inflammation, macrophages and fibroblasts in the leptomeninges and choroid plexus stroma initiated immune reactions via the IL-1\u0026beta;-IL-1R1 system by 4 hours after LPS administration. Perivascular fibroblasts were stimulated and produced CCL2 in the brain parenchyma, which contributed to an increased number of parenchymal macrophages/microglia and their hypertrophy. Microarray and RT-qPCR showed that extreme upregulation of \u003cem\u003eSaa3\u003c/em\u003e occurred in macrophages and fibroblasts in the leptomeninges, choroid plexus stroma, and perivascular space 4 and 12 hours after LPS administration. Extreme upregulation of \u003cem\u003eIrg1\u003c/em\u003e also occurred in macrophages in the leptomeninges and choroid plexus stroma 4 and 12 hours after LPS administration. The profound upregulation of \u003cem\u003eLcn2\u003c/em\u003e in the perivascular and leptomeningeal fibroblasts reached its peak 4 and 12 hours after LPS administration. The choroid plexus epithelial cells also showed extensive upregulation of \u003cem\u003eLcn2\u003c/em\u003e. This gene upregulation was a dynamic response, preparing for the inflammatory reaction and preventing bacterial growth, both of which are essential for protecting immature brain tissue.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThis study was supported by the following: Kyorin University Research Encouragement Award (2021: AS, RS, AO, and TT; 2023: AS), Subsidy to Private Institutions of Higher Education for Current Expenditure (2021: AS and AO; 2022: AS and AO; 2023: AS, RS, and AO; 2024: AS, RS, and AO), and Grants-in-Aid for Scientific Research from JSPS (21K07280 to SHI, 24K10495 to SHI, and 24K08792 to AO). We thank the students for their technical contributions to the experiments. \u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eAS contributed to the conception of the work, taught the other authors how to conduct the experiments, and wrote the manuscript. RS conducted most of the experiments and wrote the manuscript. AO participated in the research project and performed some of the experiments. TT contributed to the discussion from a clinical perspective. SHI helped interpret the data and assisted the other authors with some experiments. All authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the following: Kyorin University Research Encouragement Award (2021: AS, RS, AO, and TT; 2023: AS), Subsidy to Private Institutions of Higher Education for Current Expenditure (2021: AS and AO; 2022: AS and AO; 2023: AS, RS, and AO; 2024: AS, RS, and AO), and Grants-in-Aid for Scientific Research from JSPS (21K07280 to SHI, 24K10495 to SHI, and 24K08792 to AO).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics approval and consent for participation\u003c/p\u003e\n\u003cp\u003eWe affirm that this article contains original data that have not been submitted elsewhere for publication and that all authors have read and approved the manuscript. The Guide for the Care and Use of Laboratory Animals, 8th edition (National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Washington DC: National Academies Press, 2011), was followed for the handling of all mice. The Institutional Animal Care and Use Committee of the Kyorin University Faculty of Health Sciences approved all of the experiments described (Protocols I17\u0026ndash;08\u0026ndash;03, I17-08-04, I17-08-05, I17-08-06, and I17\u0026ndash;08\u0026ndash;07).\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors have no financial conflicts of interest to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOhuma EO, Moller AB, Bradley E, Chakwera S, Hussain-Alkhateeb L, Lewin A, Okwaraji YB, Mahanani WR, Johansson EW, Lavin T, et al: \u003cstrong\u003eNational, regional, and global estimates of preterm birth in 2020, with trends from 2010: a systematic analysis.\u003c/strong\u003e \u003cem\u003eLancet 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section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"prematurity, endotoxin, leptomeninges, fibroblasts, macrophages","lastPublishedDoi":"10.21203/rs.3.rs-6850479/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6850479/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Bacterial infection is a key trigger of inflammatory pathways that damage the preterm brain, even without direct bacterial brain invasion. However, the mechanisms underlying the involvement of intracranial tissues and cells in early immune responses to acute systemic inflammation in preterm infants remain unknown. Lipopolysaccharide (LPS) was administered with a single intraperitoneal injection into postnatal day (P) 7 mice. Four hours later, the leptomeningeal macrophages initiated an intracranial reaction by producing IL-1β, which set the stage for the arachnoid, pial, and perivascular fibroblasts to act. These brain fibroblasts produced CCL2, which increased the number of brain parenchymal macrophages/microglia and led to their hypertrophy. The macrophages/microglia were then isolated from the brain 24 hours after LPS administration. Microarray analysis showed marked transcript expression of genes including Saa3, Irg1, Lcn2, and Cxcl9. RT-qPCR assays and histological staining showed that the expression level of Saa3 was upregulated in the leptomeningeal macrophages, cerebral perivascular macrophages, and cerebellar medulla macrophages, as well as in the fibroblasts of the leptomeninges, choroid plexus stroma, and perivascular space. The leptomeningeal macrophages expressing Saa3 continued their developmental migration from the leptomeninges to the parenchyma, such as the bundles that traverse the roof of the third ventricle and the corpus callosum. The expression level of Saa3 reached their peak 4 and 12 hours after LPS administration. The blood-brain barrier (BBB) of P7 pups remained intact up to 72 hours after LPS administration. The expression level of Irg1 was remarkably increased in the leptomeningeal macrophages and reached their peak 4 and 12 hours after LPS administration. The expression level of Lcn2 was the most increased in the perivascular fibroblasts and choroid plexus epithelial cells, peaking 4 and 12 hours after LPS administration. The expression level of Cxcl9 was also increased in the leptomeningeal fibroblasts, peaking 4 and 12 hours after LPS administration. The early enhancement of Saa3 expression played a key role in priming inflammation, initiated by macrophages and fibroblasts. Early enhancement of Irg1 expression was crucial for determining the direction of the inflammatory response. The increase of Lcn2 was vital, sequestering the iron siderophore to limit bacterial growth. It was interesting to observe how the early immune responses to systemic inflammation in the postnatal mouse brain were initiated by migrating macrophages and leptomeningeal fibroblasts, leading to immediate gene upregulation to protect immature brain tissue.","manuscriptTitle":"Early immune responses to systemic inflammation in the postnatal mouse brain initiated by migrating macrophages and leptomeningeal fibroblasts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 09:54:38","doi":"10.21203/rs.3.rs-6850479/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-05T15:19:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-05T00:25:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163111269976257000660976261362847579461","date":"2025-08-01T16:09:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333060040735717429798506397998677900264","date":"2025-07-29T15:36:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212271272442834894985247124344743850051","date":"2025-07-29T00:08:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50732579801096532346477444065583146697","date":"2025-07-28T20:00:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158431082552678773475127155115016433768","date":"2025-07-28T14:13:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T15:32:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220751758220938716361320914782292932514","date":"2025-07-01T07:59:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-18T12:20:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-11T04:07:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T12:48:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2025-06-09T03:56:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ec77f815-bf62-4a68-a842-91a4595331f0","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T16:07:54+00:00","versionOfRecord":{"articleIdentity":"rs-6850479","link":"https://doi.org/10.1186/s12974-025-03559-4","journal":{"identity":"journal-of-neuroinflammation","isVorOnly":false,"title":"Journal of Neuroinflammation"},"publishedOn":"2025-10-11 15:57:23","publishedOnDateReadable":"October 11th, 2025"},"versionCreatedAt":"2025-06-25 09:54:38","video":"","vorDoi":"10.1186/s12974-025-03559-4","vorDoiUrl":"https://doi.org/10.1186/s12974-025-03559-4","workflowStages":[]},"version":"v1","identity":"rs-6850479","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6850479","identity":"rs-6850479","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}