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Multiparametric MRI assessment of acute neuroinflammation in a murine model of peripheral LPS stimulation

BMC Medical Imaging volume 25, Article number: 418 (2025) Cite this article

Abstract

Background

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Neuroinflammation is implicated in the pathogenesis of several central nervous system disorders. Excessive and prolonged neuroinflammation impacts disease progression and potential interventions. A widely used model of peripherally-induced neuroinflammation involves the intraperitoneal administration of the endotoxin lipopolysaccharide (LPS) to mice. In this exploratory study, magnetic resonance imaging (MRI) was used to non-invasively investigate in the brain acute neuroinflammation induced by peripheral LPS stimulation and in combination with the microglia depleting effects of the colony stimulating factor 1 receptor kinase inhibitor BLZ945.

Methods

Multiparametric MRI, including T2-weighted signal, magnetization transfer ratio MTR, and apparent diffusion coefficient ADC, was applied to analyze the model. LPS was administered intraperitoneally once daily from day 1 to day 4 at a dose of 0.5 mg/kg. Post-mortem analyses comprised cytokine/chemokine measurements in the blood and brain, immunohistochemistry of microglia and astrocytes, and brain autoradiography using the TSPO radiotracer [3H]PK11195. BLZ945 (200 mg/kg p.o. daily) was given from day − 7 to day 4.

Results

On day 4 of LPS administration, T2-weighted signal and MTR increased, while ADC decreased in several brain regions. Neuroinflammation was confirmed by immunohistochemistry, which revealed an increase of microglia and astrocytes in the cortex. Additionally, autoradiography showed increased uptake of [3H]PK11195 in the cortex and thalamus/midbrain of animals receiving LPS. Treatment with BLZ945 resulted in a reduction of the LPS-elicited response, as revealed in vivo by MRI and confirmed by post-mortem histology and autoradiography analysis.

Conclusion

The present findings highlight the potential of non-contrast-enhanced MRI to assess acute neuroinflammation-related changes in several mouse brain areas upon peripheral LPS stimulation. The assessment of multiple parameters provided sufficient sensitivity to detect pharmacological modulation of the neuroinflammatory response elicited by the endotoxin.

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Introduction

Neuroinflammation is implicated in the pathogenesis of several central nervous system (CNS) disorders, from ischemic stroke and traumatic brain injury to Alzheimer’s disease, schizophrenia, and major depression [1,2,3]. Excessive and prolonged neuroinflammation, particularly its damaging effects on cellular and/or brain function, impact disease progression and possible interventions. Neuroinflammation has been shown to cause and accelerate long-term neurodegenerative disease, and to play a central role in the very early development of chronic conditions including dementia.

One of the most widely used models of peripherally-induced neuroinflammation comprises the intraperitoneal (i.p.) administration to mice of the endotoxin lipopolysaccharide (LPS), a bacterial macromolecular cell surface antigen known to be a potent trigger of inflammation [4]. LPS binds to the TLR4 (Toll-like Receptor 4) complex in different cell types as monocytes, dendritic cells, macrophages and B cells, thereby promoting the secretion of pro-inflammatory cytokines (e.g. IFN-γ, TNF-α or IL-1β) [5]. Moreover, LPS leads to astrocyte and microglia activation, as well as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokine expression in the brain [6]. Increase in the numbers of F4/80-, CD11-, CD45- or Iba1-positive cells as well as morphological changes related to microglia activation have been reported for the mouse brain following a single administration or multiple i.p. injections of LPS [4, 7, 8].

Imaging provides the opportunity to characterize neuroinflammatory processes in situ. Although microscopy techniques are available providing information at high spatial and temporal resolution in animals [9], nuclear imaging techniques (positron emission tomography PET; single photon emission computed tomography SPECT) and magnetic resonance imaging (MRI) are particularly important because of their translational and back-translational potential and the ability to examine patients. Translational imaging approaches of relevance include those mapping (i) the activation of CNS immunocompetent cells (e.g. PET imaging of glial activation with translocator protein (TSPO) tracers or beyond [10,11,12]), (ii) a compromised blood-brain barrier (e.g. identification of multiple sclerosis lesions with gadolinium-enhanced MRI [13, 14]), (iii) the infiltration of circulating immune cells (e.g. tracking monocyte infiltration into brain parenchyma with MRI in conjunction with the administration of iron oxide nanoparticles [15]), and (iv) pathological consequences of neuroinflammation (e.g. imaging apoptosis with [99mTc]Annexin V [16] or microhemorrhages with T2* relaxometry [17]). A review of imaging of neuroinflammation can be found in Albrecht et al. [18].

In the present work we exploited the use of non-contrast MRI techniques (T2-weighted imaging, magnetization transfer ratio (MTR), diffusion-weighted imaging) to non-invasively characterize a sub-chronic murine model of LPS-induced neuroinflammation. In vivo imaging analyses were accompanied by cytokine level assessments in plasma and brain, immunohistochemistry analyses of the brain and TSPO expression by autoradiography. Furthermore, since the colony-stimulating factor-1 (CSF-1) pathway is a key regulator of microglia differentiation and survival [19, 20], the CSF-1 receptor inhibitor, BLZ945, was tested in the same model. It has been shown that BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS [21,22,23]. This makes BLZ945 an ideal tool to modulate neruroinflammation in the LPS model and investigate its effect on MRI parameters.

Materials and methods

Statement on animal welfare

In vivo experimental procedures were performed in accordance with the Swiss laws of animal experimentation (https://www.blv.admin.ch/blv/en/home/tiere/tierversuche.html). In Switzerland, each individual animal experiment and laboratory animal facility must be approved. Protocols and experiments were approved externally by the Cantonal Veterinary Office of the City of Basel, Switzerland. The study was performed under the license number BS-2644, and prior to approval by the Cantonal Veterinary Office of the City of Basel the experimental protocol was submitted to an ethical committee by the authorities. The ethical committee is named officially “Kantonale Tierversuchskommission”. Authors complied with the ARRIVE 2.0 guidelines for reporting of animal experimentation as published by Percie du Sert et al. [24]. All assessments reported here were performed blind.

Animals

Male C57BL/6JCrl mice (Charles River, Sulzfeld, Germany; n = 99 in total for the different studies, see below) of 9–10 weeks of age were used throughout the study. Animals were housed under standard conditions (12-h light/dark cycle, room temperatures of 23–25 °C), with standard chow and water provided ad libitum. Upon arrival, animals were allowed at least two weeks of acclimatization before beginning any experiment.

This work consists of a combination of separate, independent studies, as summarized in Table 1.

Table 1 Summary of the number of mice used in the different studies described in this work
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LPS challenge

LPS from Salmonella abortus equi S-form (Enzo Life Sciences, Lausen, Switzerland; article ALX 581 009 L002) was administered i.p. once daily for four consecutive days (from day 1 to day 4), at a dose of 0.5 mg/kg. Animal health status and body weight were checked daily during the LPS challenge period. The protocol of LPS dosing was chosen based on the work of Wendeln et al. [4].

Compound administration

In a series of experiments aiming to verify the effects of microglia depletion on LPS challenge, mice received with a curved reusable feeding needle barrel (20G) either vehicle or BLZ945 (Novartis Pharma, Basel, Switzerland) p.o. daily at a dose of 200 mg/kg (vehicle: 0.5% methylcellulose + 0.1% Tween80), 10 ml/kg, from day − 7 to day 4. In other words, vehicle or BLZ945 was administered daily in the period covering 7 days prior to LPS and throughout the endotoxin challenging period. The dose of BLZ945 was chosen based on the effects of the CSF-1 receptor inhibitor in the cuprizone model [22].

Magnetic resonance imaging (MRI)

Measurements were performed with a Biospec 70/30 spectrometer (Bruker Medical Systems, Ettlingen, Germany) operating at 7 T. The operational software of the scanner was Paravision (version 6.01, Bruker Medical Systems). Images were acquired from anesthetized, spontaneously breathing animals using a mouse brain circularly polarized coil (Bruker, Model 1P T20063 V3; internal diameter 23 mm) for radiofrequency excitation and detection. Neither cardiac nor respiratory triggering was applied. Following a short period of introduction in a box, mice were maintained in anesthesia with 1.5% isoflurane (Abbott, Cham, Switzerland) in oxygen, administered via a nose cone. During MRI signal acquisitions, animals were placed in prone position in a cradle made of Plexiglas, the body temperature was kept at 37 ± 1 °C using a heating pad, and the respiration was monitored. For fixation of the head, the front teeth were hooked on a bar. Once the gas mask was slid into position over the nose the teeth bar provided a firm fixation for holding the head, which was kept stable without the use of ear bars.

A T2-weighted, two-dimensional multislice RARE (Rapid Acquisition with Relaxation Enhancement) sequence was used for determining the anatomical orientation and for evaluating signal intensities. This was followed by a two-dimensional multislice gradient-recalled FLASH (Fast Low-Angle Shot) acquisition for assessment of MTR. As both sequences had the same anatomical parameters, the choice of the regions-of-interest for evaluations, based on the Paxinos and Franklin mouse brain atlas [25], was performed manually on the RARE images and then transferred to the FLASH images (Fig. 1). MRI images were analyzed using the Paravision software. The parameters of the acquisitions were the following: (a) RARE sequence: effective echo time 80 milliseconds (ms), repetition time 3280 ms, RARE factor 16, 12 averages, field of view 20 × 18 mm2, matrix size 213 × 192, pixel size 0.094 × 0.094 mm2, slice thickness 0.5 mm, 15 adjacent slices. Hermite pulses of duration/bandwidth 1 ms/5400 Hz and 0.64 ms/5344 Hz were used for radiofrequency excitation and refocusing, respectively. Fat suppression was achieved by a gauss512 pulse of 2.61 ms/1051 Hz duration/bandwidth followed by a 2-ms-long gradient spoiler. The total acquisition time was of 7 min 52.3 s; (b) FLASH sequence: echo time 2.8 ms, repetition time 252.8 ms, 4 averages, anatomical parameters as for the RARE acquisition. A hermite pulse of 0.9 ms/6000 Hz duration/bandwidth and flipangle 30° was used for radiofrequency excitation. MTR contrast was introduced by a gauss pulse of 15 ms/182.7 Hz duration/bandwidth applied with radiofrequency peak amplitude of 7.5 µT and an irradiation offset of 2500 Hz. The acquisition was then repeated with the same parameters but without the introduction of the MTR contrast. MTR was then computed using the formula MTR = (S0-SMTR)/S0 where S0 and SMTR represent respectively the signal intensities in the FLASH acquisitions without and with the introduction of the MTR contrast. The total acquisition time for both data sets was 6 min 31.6 s.

Fig. 1
figure 1

Definition of regions-of-interest on T2-weighted images. Left-to-right: Representative caudal to rostral images displaying the manually drawn regions-of-interest in which analyses were performed. Mean values of the readouts were obtained between left and right if applicable and in case the anatomical region extended over more than one slice

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Furthermore, diffusion-weighted imaging was performed in the compound treatment experiment only using an echo-planar imaging (EPI) sequence with a spin-echo diffusion sensitizing module and the following parameters: effective spectral bandwidth 250 kHz, echo time 25 ms, repetition time 250 ms, 12 averages resulting in a total scan time of 84 s, one axial slice of thickness 0.5 mm, field-of-view 20 × 20 mm2, matrix 128 × 128, pixel size 0.156 × 0.156 mm2. The diffusion-sensitizing gradient was chosen along the slice gradient direction to achieve b-values of 0, 100, 200, 400, 600, 800 and 1000 s/mm2. The apparent diffusion coefficient (ADC) was determined by fitting the mean signal intensities in a defined region-of-interest for the images acquired with the aforementioned b-values to the formula.

$$\:\text{S}_\text{b}\:=\:\text{S}_{\text{b}=0}\:\text{*}\:\text{e}^{(-\text{b}_{\text{*}}\text{A}\text{D}\text{C})}$$
(1)

Post-mortem analyses

Prior to cull mice were perfused trans-cardiacally by phosphate-buffered saline under isoflurane anesthesia and blood was withdrawn by heart-puncture. Animals were euthanized by a high dose of isoflurane. Brains were removed from the skull and the forebrain (excluding olfactory bulb, brainstem and cerebellum) was used for biochemical assessments and histology. All brains subjected for histology were fixed in 4% paraformaldehyde for 48 h at 4 °C.

Cytokine/chemokine measurements

Half brains were homogenized 1:10 (w: v) in tris buffered saline with Precellys24 Tubes (Precellys Lysing Kit, hard tissue homogenizing CK28-R, catalogue no. 03961-1-007; Bertin Technologies, Montigny-le-Bretonneux, France) in radioimmunoprecipitation assay buffer (R0278; Thermo Fisher Scientific, Basel, Switzerland) with protease inhibitor cocktail tablets (Roche cOmplete™ mini protease inhibitor cocktail, catalogue no. 04693124001; Merck, Buchs, Switzerland), aliquoted and stored at -80 °C. A radioimmunoprecipitation assay buffer (catalogue no. 20–188; Merck Millipore, Buchs, Switzerland) was added ten times to a brain homogenate aliquot, mixed and incubated on ice for 10 min, then centrifuged for 5 min at 10,000 rcf (relative centrifugal force) at 4 °C. Supernatants from brain and plasma [1:2 and 1:10, respectively, in diluent 41 (Meso Scale Diagnostics, Rockville, MD, USA)] were used for electrochemiluminescence analyses (V-PLEX Proinflammatory Panel 1 Mouse Kit; Meso Scale Discovery, ACROBiosystems, Newark, DE, USA) according to the manufacturer`s protocol.

Histology of brains

After fixation, brains were processed for paraffin embedding by dehydration through an increasing ethanol series. Paraffin sections of 3 μm thickness (sagittal brain sections) mounted on SuperFrost + slides (Thermo Fisher Scientific, Reinach, Switzerland) were automatically immunostained using the Discovery XT technology (Ventana, Roche Diagnostics, Rotkreuz, Switzerland). Sections were de-paraffinized, rehydrated, subjected to antigen retrieval by heating with a CC1 cell conditioning buffer (Reference 05279801001, Roche Diagnostics) for 28–68 min (time depending on the antibody), then incubated for 1–3 h (time depending on the primary antibody) at room temperature, with the primary antibody diluted in antibody diluent (Ventana), incubated with the respective biotinylated secondary antibody diluted in antibody diluent, reacted with a DABMab kit (Ventana) and counterstained with Hematoxylin II and Bluing reagent (Ventana). Slides were washed with soap in hot tap water and rinsed under cold running tap water to remove the soap, then dehydrated and embedded with Pertex.

Antibodies

Rabbit anti-Iba1 (Wako 019-19741, 50 µg/100 µl; Wako, Osaka, Japan) 1:500 and rabbit anti-glial fibrillary acidic protein (anti-GFAP, Dako Z0334; Agilent Technologies, Basel, Switzerland) 1:5000 constituted the primary antibodies.

Goat anti-rabbit IgG biotinylated (catalogue no. 111-065-144; Jackson ImmunoResearch, Cambridgeshire, UK) 1:1000 and goat anti-rabbit IgG biotinylated (Vector BA-1000; Vector Laboratories, Peterborough, UK) 1:200 or 1:1000 served as secondary detection antibodies.

Analysis of histological images

For the quantitative evaluation of microglia numbers and morphology, a proprietary image analysis platform (ASTORIA, Automated Stored Image Analysis, Novartis Pharma AG, Basel, Switzerland) was developed based on MS Visual Studio 2010 (Microsoft, Seattle, WA, USA) and many functions from Matrox MIL V9 libraries (Matrox Inc., Dorval, Quebec, Canada) as described elsewhere [22, 26]. This image analysis algorithm was also used to quantify the stained areas of astrocytes (GFAP) in sagittal brain sections following the description provided in [22, 26].

Autoradiography assay

Brain sections were prepared from frozen optimal cutting temperature (OCT) blocks. Sagittal sections (10 μm thick) were produced at a cutting temperature of -16 °C and collected by thaw-mounting onto coated poly-lysine microscope glass slides. These were stored at -80° C until use. Autoradiography was conducted as previously described by Biegon et al. [27] and Tyler et al. [28]. Slides were incubated in 50 mM Tris-HCl, pH 7.4 at room temperature for 15 min while gently shaking. Pre-incubation buffer was replaced with assay buffer (1 nM [3H]PK11195, [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide] (specific activity 78.71 Ci/mmol; Perkin-Elmer, Waltham, MA, USA)) for total binding or assay buffer (1 nM [3H]PK11195 + 20 µM PK-11195 (#C0424-10 mg; Sigma, Buchs, Switzerland)) for non-specific binding (adjacent sections, no non-specific binding could be observed (supplementary Fig. 1)). Slides were further incubated for 60 min at room temperature while gently shaking and then washed 2x with cold (4 °C) 50mM Tris-HCl, pH 7.4 buffer for 6 min each time. Ice-cold distilled water for approximately 10 s to remove salts and discard supernatant was added and slides were transferred to the slide holder and dried under a stream of cold air for about 15 min.

Autoradiograms were generated by exposing the labeled tissues to a BAS-IP Tritium Phosphor Screen (cat. no. 28956482; Fujifilm, Niederscherli, Switzerland) alongside Tritium Standards [3H] (ART0123B; Hartmann Analytic, Braunschweig, Germany) at room temperature for 18 h.

Exposed phosphor screens were scanned using a Typhoon Phosphor Imager (Cytiva Life Sciences, Grens, Switzerland) and autoradiograms were analyzed and quantified using the ImageQuant™ software (version 8.2.0, Cytiva Life Sciences). The cortex was selected as region of interest and the average intensity corresponding to the specific binding was measured. All brain sections in the assay were performed in technically duplicates and randomized.

Statistics

Data are presented as means ± sd throughout the manuscript. MRI data were analyzed using ANOVA with random effects (SYSTAT 13, SYSTAT Software, Inc., San Jose, CA, USA) to take the longitudinal character of the data into account. In vitro data were analyzed by mixed-effects analyses. Sidak`s multiple comparison tests or t-tests were performed to compare two groups as specified in the figure legends. Histology data were analyzed by either Holm-Sidak’s one-way ANOVA tests with multiple test group effect per time point, ANOVA with random effects or t-tests to compare two groups as specified in the figure legends. Statistical significance was assumed for p < 0.05.

Results

Acute neuroinflammatory processes elicited by LPS translated into changes in MRI parameters in various brain regions

At day 4 of LPS administration, T2-weighted signal in the cortex, hippocampus, thalamus and striatum was significantly increased with respect to baseline, with a return to baseline levels 4 days after last LPS dosing (Fig. 2A). Increases in MTR post-endotoxin in these brain areas followed the similar acute profiles as shown by the T2-weighted acquisitions (Fig. 2B). Additional evaluations are compiled in the supplementary Figs. 2 and 3.

Fig. 2
figure 2

Acute LPS-induced effects in the mouse brain as revealed by MRI. (A) T2-weighted signal and (B) magnetization transfer ratio (MTR) normalized to the mean value at D0 in each group, before first saline or LPS administration. Values displayed as means ± sd for n = 8 and n = 10 mice in the saline and LPS group, respectively. The levels of significance *,#0.01 < p < 0.05, **,##0.001 < p < 0.01, ***p < 0.001 correspond to ANOVA with random effects comparisons. Saline or LPS (0.5 mg/kg) was dosed i.p. from day 1 (D1) to day 4 (D4). At D2 and D4, MRI was performed approximately 3 h after saline or LPS administration

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Visual inspection of immunohistochemistry sections of the cortex revealed increased Iba1 and GFAP staining at day 4 of LPS (Fig. 3A, B). Quantification of Iba1 staining showed increased microglia content in both cortex and hippocampus at day 4 of endotoxin, with baseline levels attained at day 8, i.e. 4 days post last LPS administration (Fig. 3A). In the hippocampus slightly stronger microglia response compared to cortex could be observed. On the other hand, astrogliosis revealed through quantification of astrocyte area from GFAP staining occurred predominantly in the cortex (Fig. 3B). Astrogliosis further increased between days 4 and 8, despite cessation of endotoxin injection. Size and form factor of cortical microglia were significantly increased already at day 3, indicating an early cell activation induced by LPS (Fig. 3C). Microglia activation in several brain regions returned to baseline levels at day 8 (supplementary Fig. 4).

Fig. 3
figure 3

Immunohistochemistry of microglia and astrocytes for the LPS model. (A) Top: Representative Iba1-stained sections of the brain cortex of saline- or LPS-challenged mice. Bottom: Microglia content in the cortex and hippocampus derived from Iba1 staining. (B) Top: Representative GFAP-stained sections of the brain cortex of saline- or LPS-challenged mice. Bottom: Astrocyte area in the cortex and hippocampus derived from GFAP staining. (C) Histological parameters providing information on microglia activation quantified from Iba1 staining, related to the mean values in the cortex of saline-challenged animals at day 4. Data displayed as means ± sd for n = 4 mice for each group, time point and brain area. The levels of significance *0.01 < p < 0.05, **0.001 < p < 0.01, ***p < 0.001 correspond to one-way ANOVA comparisons. Saline or LPS (0.5 mg/kg) was dosed i.p. from day 0 to day 4

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Cytokine levels in plasma peaked on the first day of LPS administration and then remained slightly elevated above background levels (Fig. 4A). In the brain, the highest cytokine levels were found at day 2 of endotoxin injection (Fig. 4B). The exception was IL-10, which displayed approximately the same elevated levels in both plasma and brain throughout the LPS dosing period.

Fig. 4
figure 4

Cytokine profiles upon LPS stimulation. Assessments in (A) plasma and (B) brain for four mice per condition and time point. The levels of significance *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001 and ****p < 0.0001 correspond to one-way ANOVA comparisons. Saline or LPS (0.5 mg/kg) was dosed i.p. from day 1 to day 4

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The LPS-induced neuroinflammation was attenuated by BLZ945 as evidenced using MRI and ex vivo analyses

To verify the effects of microglia depletion on LPS challenge, mice received either vehicle or BLZ945 p.o. daily at a dose of 200 mg/kg, from day − 7 to day 4, while the endotoxin was given from day 1 to 4 to all animals. The dose of BLZ945 was chosen based on the effects of the CSF-1 receptor inhibitor in the cuprizone model [22].

Compared to vehicle, BLZ945 treatment led at day 4 of LPS challenge to a significant reduction of T2-weighted signal in the cortex and striatum. Moreover, in the thalamus there was a trend towards signal reduction while in the hippocampus no compound effect was detected (Fig. 5A). At day 4 of endotoxin administration, MTR values in the striatum and thalamus were significantly reduced in mice treated with BLZ945 compared to vehicle-treated controls (Fig. 5B). In the cortex, a trend towards lower MTR was observed in the BLZ945 group, although this did not reach statistical significance. No differences in MTR were detected between treatment groups in the hippocampus (Fig. 5B). Finally, a significant ADC reduction was detected in all regions analyzed in the brains of LPS animals receiving vehicle (Fig. 5C). This ADC reduction was less pronounced in the cortex, striatum and thalamus of LPS mice treated with BLZ945.

Fig. 5
figure 5

BLZ945 effects in the LPS model as analyzed in vivo by MRI. (A) T2-weighted signal, (B) magnetization transfer ratio (MTR) and (C) apparent diffusion coefficient (ADC) normalized to the respective mean value at D0 in each group, before the first LPS challenge. Data displayed as means ± sd for n = 5 mice in each group. The levels of significance *,# 0.01 < p < 0.05, **0.001 < p < 0.01, ***,### p < 0.001 refer to ANOVA with random effects comparisons. LPS (0.5 mg/kg) was dosed i.p. from day 1 (D1) to day 4 (D4). Vehicle/BLZ945 (200 mg/kg) was administered p.o. daily from day − 7 to day 4. At D4, MRI was performed at approximately 3 h after LPS dosing

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Immunohistochemistry demonstrated not only a significant reduction of microglia numbers in the cortex of BLZ945-treated animals at day 4 of endotoxin administration (Fig. 6A), but throughout the brain (supplementary Fig. 5). This observation is only partly consistent with the reduced T2-weighted signal in selected brain areas (Fig. 5A). On the other hand, the CSF-1 receptor kinase inhibitor reduced the astrocyte numbers mainly in the thalamus of LPS-challenged mice but not significantly in other brain areas as assessed through GFAP staining (Fig. 6B, supplementary Fig. 5). Autoradiography revealed a significant increase of [3H]PK11195 uptake in the cortex and thalamus/midbrain of LPS-challenged, vehicle-treated mice, in comparison to control, saline-challenged animals (Fig. 6C). For LPS mice receiving BLZ945, the [3H]PK11195 uptake was similar to that of control animals (Fig. 6C).

Fig. 6
figure 6

BLZ945 effects in the LPS model as analyzed post-mortem by immunohistochemistry and autoradiography. (A) (left) Representative Iba1-stained sections of the brain cortex. (right) Microglia numbers derived from Iba1 staining relative to that on cortex of control mice receiving saline. (B) GFAP-stained sections of the brain cortex from the same mice and astrocyte numbers derived from GFAP staining relative to that on the corresponding brain area of control mice receiving saline. (C) Autoradiography of [3H]PK11195 uptake in the cortex and thalamus/midbrain. Values displayed as means ± sd for n = 4 mice for each group and technique. Animals were euthanized on day 4 for the analyses, approximately 3 h after the fourth i.p. administration of saline or LPS. Significance levels refer to ANOVA comparisons between groups: * 0.01 < p < 0.05, **0.001 < p < 0.01, ****p < 0.0001

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BLZ945 treatment led to either a significant reduction or a trend towards reduction of pro-inflammatory brain cytokines in LPS-challenged animals (Fig. 7B). Exception was IL-10, which displayed the same levels in vehicle- and BLZ945-treated, LPS-challenged mice despite being significantly reduced in the plasma of animals receiving the CSF-1 receptor inhibitor (Fig. 7A). The other cytokines analyzed in plasma had the same profile as in the brain (Fig. 7A).

Fig. 7
figure 7

Cytokine profiles at day 4 of LPS stimulation for either vehicle- or BLZ945-treated mice. Assessments in (A) plasma and (B) brain for five mice per condition. The levels of significance *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001 and ****p < 0.0001 correspond to one-way ANOVA comparisons. LPS (0.5 mg/kg) was dosed i.p. from day 1 to day 4. Vehicle/BLZ945 (200 mg/kg) was administered p.o. daily from day − 7 to day 4

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Discussion

Model characterization

It is known that peripheral inflammation may cause immune responses in the brain and that the CNS becomes more susceptible to damage after peripheral infections or other insults [29]. Here, we showed that repetitive daily i.p. injections of LPS at a dose of 0.5 mg/kg during four days to otherwise healthy mice led to changes in various brain areas that could be detected non-invasively by MRI and histology (Figs. 2 and 3). Despite not being specific parameters, the increases in T2-weighted signal and MTR were consistent with neuroinflammation and the associated elevation of protein levels elicited by the endotoxin [30]. The responses were transient, with the MRI parameters returning to baseline values already four days after interruption of LPS injection. Histology confirmed the induction of neuroinflammation by the endotoxin through the detection of elevated numbers of activated microglia in the cortex and other brain areas. At day 8 microglia numbers and activation had returned to baseline levels (Fig. 3). Astrogliosis as revealed by GFAP staining, however, persisted after cessation of LPS dosing. Moreover, increased uptake of [3H]PK11195 in the cortex and thalamus/midbrain of LPS-challenged mice as assessed by autoradiography confirmed the presence of neuroinflammation in the model (Fig. 6C). The prototypical TSPO radiotracer, (R)-[11C]PK11195, has been successfully applied to visualize neuroinflammation in several human pathologies [10,11,12].

Blood levels of the pro-inflammatory cytokines IL-6, TNF-α, KC/GRO and IL-1β peaked at day 1 (Fig. 4A), indicating peripheral immune tolerance, a critical mechanism orchestrated by multiple cellular and molecular mechanisms, with regulatory T cells playing a central role, by which the immune system prevents harmful responses to self and innocuous antigens outside the central lymphoid organs [31]. These cytokines were significantly increased in the brain at day 2 (Fig. 4B), suggesting a brain-specific training effect induced by the first LPS stimulus [32]. A prominent morphological change in microglia reflecting activation occurred at day 3 (Fig. 3C), while activated (GFAP+) astrocytes increased a day later (Fig. 3B). Importantly, IL-6, TNF-α, KC/GRO and IL-1β release in the brain was practically abolished at day 4 while IL-10 remained elevated, suggesting immune tolerance. Our data thus reproduce earlier work by Wendeln et al. [4] using the same dose of LPS and injection protocol as adopted here. The elevated IL-10 levels at day 4 might be consistent with the astrogliosis detected in the cortex at the same time point and even four days after the last LPS administration, since under inflammatory conditions, microglia and astrocytes are the main producers of this cytokine in the CNS [33, 34].

Multiparametric MRI for pathology detection

The transient increase of T2-weighted signal upon LPS stimulation detected here in several brain areas (Fig. 2A) is consistent with neuroinflammation elicited by the endotoxin, possibly reflecting significant activation and proliferation of microglia and astrocytes across multiple brain regions including the hippocampus, cortex, striatum, hypothalamus, and cerebellum as evidenced here (Fig. 3) and by others [35, 36]. That so many brain regions responded to repeated peripheral endotoxin stimulation is consistent with the fact that peripheral LPS upregulates cytokine (e.g. IL-1β, IL-6, and TNF-α) mRNA and protein in multiple brain areas, showing a robust central immune response following peripheral challenge [37]. T2-weighted MRI has been demonstrated both in animal models [22, 38,39,40] and in the clinics [41, 42] to be a sensitive but rather non-specific indicator of tissue injury.

A reduction in ADC as found here at day 4 of LPS challenge (Fig. 5C) indicates restricted diffusion of water molecules in tissue, which has been suggested to reflect either intracellular edema [43] or hypercellularity due to glial activation and inflammatory cell recruitment [44]. Blamire et al. [45] reported reduced ADC already 6 h post-injection of recombinant IL-1β into the rat striatum, with IL-1β having a direct effect on the resident cell populations. In a photothrombotic cerebral infarction model in rats, low ADC and high T2 values indicated an early pathology stage with necrosis and beginning glial activation [46]. Lodygensky et al. [47] described an ADC reduction 24 h after a 1-mg/kg injection of LPS in the corpus callosum of rat pups. Immunohistochemistry showed activated microglia and astrogliosis predominantly in the corpus callosum but also in other brain regions. A decrease of ADC was also found in patients 48 to 72 h after hippocampal ischemia, limbic encephalitis, status epilepticus or transient global amnesia [48]. Apart from stroke, restricted diffusion within the cortex has been described in infectious, metabolic, hemodynamic, and genetic diseases (see Koksel et al. [49] for a review).

MTR assesses the transfer of magnetic resonance signal from protons in water that are associated with macromolecules to protons in free water. Reductions in MTR have been linked with structural changes in tissue such as demyelination [22, 26, 40] or generalized inflammatory processes [50]. We detected an increase in MTR at day 4 of LPS dosing (Figs. 2B and 5B), which is in agreement with recently reported MTR increases in several brain areas in mice submitted to a high fat diet [51]. Of importance, an augmented presence of astrocytes and microglia occurs during high fat diet-induced cerebral inflammation [52, 53]. Quantitative magnetization transfer (qMT) [54] has also been used to investigate low-level neuroinflammation in humans. Early (3–4 h) after administration of typhoid vaccine containing endotoxin from Salmonella typhi, which can be used as an experimental stimulus to induce a low level of inflammation [55], significantly increased MT exchange rate was found in the insula of healthy volunteers [56]. Plank et al. [57] described an increased MT exchange rate in the cerebellum, where cytokine increases following an acute inflammatory stimulus had been found in animal studies [58], upon typhoid vaccine dosing to healthy individuals, with higher IL-6 blood concentrations post-vaccine compared to post-placebo. Also, an increased MT exchange rate was observed in the striatum of hepatitis C patients 4 h following IFN-α administration [59]. The infiltration of cytokines released by astroglia and neurons has been suggested as a possible mechanism influencing MT exchange [56]. Following this reasoning, the increased MTR and cytokines upon LPS dosing we observed here might suggest that the endotoxin dosing regimen induced a mild neuroinflammation. This would be consistent with the fact that four days after cessation of LPS injection, the MRI parameters as well as microglia activation returned to baseline levels.

Effects of CSF-1 receptor kinase inhibition

Increased mRNA and protein levels of CSF-1 receptor and CSF-1 in the striatum and substantia nigra upon sub-chronic LPS injection [60] provided a basis for testing BLZ945 in the model. Furthermore, we wanted to analyze the impact of microglia depletion in a neuroinflammatory situation on the MRI parameters. Compared to vehicle, treatment with BLZ945 led to significant T2-weighted signal reductions in the cortex and striatum of LPS mice, suggesting anti-neuroinflammatory effects of the compound (Fig. 5A). This has been confirmed by autoradiography, which demonstrated clear reduction of [3H]PK11195 uptake in the cortex and thalamus/midbrain of LPS-challenged, BLZ945-treated animals (Fig. 6C), in agreement with microglia depletion revealed by histology (Fig. 6A). Interestingly, the CSF-1 receptor kinase inhibitor had no impact on the astrocyte numbers. These results are in general accordance with the overall cytokine decrease in the brains of LPS mice receiving BLZ945, except for IL-10, which is primarily produced by astrocytes and microglia [61, 62]. Similarly to T2-weighted signal, MTR and ADC also detected significant effects of BLZ945 in the striatum, whereas in the cortex these readouts only showed a trend towards normalization (Fig. 5B, C). While MTR demonstrated a clear effect of BLZ945 in the thalamus, T2-weighted signal and ADC displayed solely a trend.

The non-specific nature of the MRI parameters may be an important reason for them to differentially respond to pharmacological interventions. On the other hand, regional differences in glial cell populations could also significantly contribute to the regional heterogeneity of the responses to BLZ945 brought to light by MRI. Single cell RNA-seq studies in humans and mice have shown high heterogeneity of microglia, as well as related complexity in microglial development [63]. Single-cell analyses revealed specific time- and region-dependent microglia subtypes, and demyelinating as well as neurodegenerative conditions evoked context-dependent microglia subtypes with distinct molecular hallmarks and heterogeneous cellular kinetics. Also, specialized astrocyte subtypes were found between and within regions of the mouse brain [64]. More detailed studies additionally involving advanced tools such as single-cell analyses are required to better understand the regional complexity of the pharmacological results obtained here.

CSF-1 receptor kinase inhibition has been shown to lead to significant reduction in microglial proliferation, neuroinflammation, and disease pathology in Alzheimer’s disease [65], amyotrophic lateral sclerosis [66], and prion disease models [67]. In a model of Parkinson’s disease, the CSF-1 receptor kinase inhibitor, GW2580, significantly attenuated MPTP-induced CSF-1 receptor activation and Iba1-positive cell proliferation in the substantia nigra [60]. GW2580 treatment also decreased mRNA levels of pro-inflammatory factors, and significantly attenuated the MPTP-induced loss of dopamine neurons and motor behavioral deficits. These effects occurred in the absence of overt microglial depletion, suggesting that targeting CSF-1 receptor signaling might be a viable neuroprotective strategy in Parkinson’s disease that disrupts pro-inflammatory signaling, but maintains the beneficial effects of microglia. Along these lines, partial microglia depletion with the CSF-1 receptor kinase inhibitor, PLX5622, before the induction of sepsis was sufficient to attenuate long-term neurocognitive dysfunction. PLX5622 acted by reducing microglia-induced synaptic attachment/engulfment and preventing chronic microgliosis [68].

Limitations of the study

As mentioned above, the main limitation of MRI readouts is their non-specific nature, with multiple pathomechanisms impacting their characteristics. More in-depth experimental paradigms including systematic histological analyses are needed to determine the influence of these factors individually on T2, ADC, and MTR-related signal changes. In particular, exploring qMT [54] and its parametric images reflecting intrinsic MR properties of the tissue may enable increasing the specificity of MT acquisitions. Recently, diffusion tensor imaging (DTI) or neurite orientation dispersion and density imaging (NODDI) have been applied to detect changes in the brains of rats upon systemic or local LPS administration [69, 70]. The diffusivity changes correlated with histologically measured changes in microglial and astrocyte morphology/activation, confirming the sensitivity of diffusion MRI to neuroinflammatory processes. T2 mapping might have been a better alternative for assessing tissue properties than quantifying T2-weighted signal. Practical challenges of obtaining such maps in a reasonable acquisition time could be addressed by using for instance Bloch-simulation-based reconstruction [71]. Also, the application of contrast-enhanced MRI involving the administration of contrast agents may provide additional information when studying neuroinflammation models, by allowing e.g. to monitor the disruption of the blood-brain barrier [72] and of the blood-cerebrospinal fluid barrier [73, 74] as well as the infiltration of immune cells [75] (see [76] for a review).

The contributions of the different cell populations in the brain to immune challenges is complex and not well-understood. Indeed, Brandi et al. [77] recently demonstrated that microglia and astrocytes heterogeneity lead to region-specific inflammatory response in presence of a systemic LPS treatment. The cellular heterogeneity of the CNS poses a significant obstacle to understand the molecular basis of overlapping and divergent responses to pathogens, as well as the underlying mechanisms of the brain’s response to innate immune challenges. Single cell, single nuclei RNA sequencing and spatial transcriptomics approaches may advance our understanding of cell type specific innate immune responses. Despite being beyond the scope of the present study, future work comprising a careful comparison of in vivo imaging results with these advanced analyses may help to better understand the regional differences of therapy effects on neuroinflammation.

Perspectives and conclusion

Imaging microglia and astrocytes in vivo in the context of neuroinflammation is the domain of PET through the use of tracers that bind to TSPO [78, 79]. Specifically, PET has been used to demonstrate acute widespread increases in brain TSPO binding after inflammatory LPS challenge in humans [80] and mice [81]. However, PET involves exposure to radioactivity, is invasive requiring blood sampling from an arterial catheter, and TSPO is not exclusively expressed in glia. Alternative targets such as monoamine oxidase B, matrix metalloproteinases, CSF-1 or the fractalkine receptor are under consideration with the aim to achieve higher specificity for microglia or astrocytes and their activation status [79]. Furthermore, precise quantification of the TSPO PET signal in the brain may be challenging due to potential redistribution of TSPO radiotracers across compartments upon inflammatory stimulation [82, 83].

In conclusion, the present results as well as earlier publications [69, 70] demonstrate that non-contrast-enhanced MRI has the ability to quantify acute changes in several brain areas of small rodents upon peripheral or local LPS stimulation. Assessment of multiple parameters also provided enough sensitivity to detect pharmacological modulation of the neuroinflammatory response elicited by the endotoxin. The absence of ionizing radiation, the good spatial resolution and the fact that no contrast agent is required are attributes of MRI that are particularly attractive in the context of pharmacological studies. The technique may serve as an interesting tool for assessing in vivo in small rodents the efficacy of compounds targeting neuroinflammation before considering involved PET analyses. Further combined PET/MRI examinations in the same animal as published recently by Zhu et al. [84] may shed additional light on the complementary characteristics of these imaging techniques. Finally, MRI provides translational potential, as equivalent investigations detecting with MRI acute neuroinflammatory responses demonstrated MTR increases in different brain areas of healthy human volunteers following peripheral toxin administration [56, 57, 59].

Data availability

The datasets generated and/or analyzed during the current study are not publicly available due to internal regulations from Novartis on data availablity. Data may however become available from the corresponding author upon reasonable request and pending approval by Novartis.

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Funding

This work was funded by Novartis Pharma AG, Basel, Switzerland. The funding source played no role in designing the study, analyzing the data, writing the manuscript, or submitting the manuscript for publication.

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Authors and Affiliations

  1. Neurosciences, Novartis Biomedical Research, Basel, CH-4056, Switzerland

    Derya R. Shimshek, Stefan Zurbruegg, Anna Neuhaus & Fabrizio Gasparini

  2. Immunology, Novartis Biomedical Research, Basel, CH-4056, Switzerland

    Anna Schubart

  3. Diseases of Aging & Regenerative Medicine, Novartis Biomedical Research, Basel, CH-4056, Switzerland

    Nicolau Beckmann

Authors
  1. Derya R. Shimshek
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  2. Stefan Zurbruegg
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  3. Anna Neuhaus
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  4. Anna Schubart
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  5. Fabrizio Gasparini
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  6. Nicolau Beckmann
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Contributions

All authors contributed to the study design, analyzed the data, provided critical revisions for content, and approved the final version of the manuscript. D.S. and N.B. wrote the first draft of the manuscript. S.Z., A.N., A.S. and F.G. acquired the data for the study.

Corresponding author

Correspondence to Nicolau Beckmann.

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Ethical approval

Experiments were performed following the Swiss animal welfare regulations (https://www.blv.admin.ch/blv/en/home/tiere/tierschutz.html). Protocols and experiments were approved externally by the Cantonal Veterinary Office of the City of Basel, Switzerland (https://www.bs.ch/gd/veterinaeramt). The study was performed under the license number BS-2644, and prior to approval by the Cantonal Veterinary Office the experimental protocol was submitted to an ethical committee by the authorities. The ethical committee is named officially “Kantonale Tierversuchskommission” (Cantonal Commission for Animal Experiments, Federal Food Safety and Veterinary Office, Bern, Switzerland, https://www.blv.admin.ch/blv/en/home/tiere/tierversuche.html).

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Competing interests

All authors are employed by and own stocks from Novartis Pharma AG. However, the company had no influence on the decision to submit these results for publication.

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Shimshek, D.R., Zurbruegg, S., Neuhaus, A. et al. Multiparametric MRI assessment of acute neuroinflammation in a murine model of peripheral LPS stimulation. BMC Med Imaging 25, 418 (2025). https://doi.org/10.1186/s12880-025-01962-0

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BMC Medical Imaging

ISSN: 1471-2342

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