Reagents and antibodies

Lysolecithin (LPC), ketamine, xylazine, Fluoromount-G mounting medium, paraformaldehyde (PFA), normal goat serum, Triton X-100, anti-MBP chicken polyclonal antibody (Thermofisher). Anti-GFAP mouse monoclonal antibody, HEPES, Alexa dye secondary antibodies AF-488 and AF-633 were purchased from Sigma-Aldrich (Merck Group, St. Louis, MO, USA). Anti-TNF-α mouse monoclonal antibody (Santa Cruz Biotechnology). The diamidino-2-phenylindole (DAPI), probenecid (PBC), and goat anti-mouse Alexa Fluor 488) were obtained from Thermofisher (Waltham, MA, USA).

Animals

Animal experimentation and protocols were approved by the Bioethical Committee for Animal Experiments of the University of Santiago de Chile (protocol number 319/2023) in accordance with Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA). C57BL/6 mice (PN45-60) from the animal facility of the University of Santiago de Chile were housed in cages in a temperature-controlled (24 °C) and humidity-controlled vivarium under a 12 h light/dark cycle (lights on 8:00 a.m.), with ad libitum access to food and water. Animals were daily checked by the faculty veterinarian assuring mice welfare. The experimental design is summarized in Fig. 1A–C.

Fig. 1

Model of double-site LPC injection into the corpus callosum and cerebellar fibers. A Schematics of the stereotaxic set up for the microinjection of LPC showing the sites of LPC injections in corpus callosum and cerebellar white matter. Note the different references for callosal (bregma) versus cerebellar (lambda) sites and the specific coordinates depicted in the table. B Reconstitution of several bright fields (no staining) of a representative mice subjected to double-site LPC injection in corpus callosum and cerebellar fibers (right panel). Red arrows indicate the sites of injection (note the path of graphite powder left by the pipette during the surgery, see methods), while white arrows indicate focal points of demyelinated lesions at 7 days post injection (dpi). Ctx cortex, CC corpus callosum. R, C, D, V indicates rostral, caudal, dorsal and ventral, respectively. C Overview of the experimental design. Two independent experimental groups were randomly destined to LPC or PBS double-site LPC injections. Locomotor (grid strength and Rotarod tests), cardiac (heart rate test) and ventilatory (plethysmography test) functions were previously applied to assess basal levels. IF immunofluorescence. For details, see “Materials and methods” section

Double-site focal demyelination by lysolecithin (LPC) injections

Mice were anesthetized with ketamine/xylazine (K/X, 0.1/0.01 mg/g). Deeply anesthetized mice (approximately 15 min after K/X administration) were gently fixed in a stereotaxic surgery frame (Kopf Instruments, CA, USA), fitting its head parallel to the base. An embedded towel paper with ethanol 70% was used to sterilize the surgery zone. Mice hair was removed from the upper skull using a cotton swab and commercial shaving cream was applied. Shaving solution was removed and fully wiped out with a water embedded cotton swab 1 min after exposure to avoid skin irritation. Any remaining hair was removed as well. A drop of PBS 1× was added over each mice eye to avoid drying damage. Mice were treated with a carprofen solution (5 mg/kg) as analgesia (to keep mice hydrated during surgery, small PBS volumes ~ 100 µL were added subcutaneously). Then, a clean incision was made in the head skin with surgery scissors. Meninges were carefully removed with cotton fibers to provide clean access to the skull bone. To target the corpus callosum we used the following coordinates antero-posterior + 1.5 mm, medio-lateral ± 1 mm, dorso-ventral − 1.7 mm from bregma [32, 37], and to target cerebellar white matter antero-posterior + 1.7 mm, medio-lateral ± 1.5 mm, dorso-ventral − 1.7 mm from Lamba (note that the injections are bilaterally applied, as shown in Fig. 1). The craniotomy to access the brain surface at the right coordinates was performed with a Micromotor High-Speed Drill (Stoelting C., IL, USA). Then, we proceed with the intracranial injections of 2 μL of 2% LPC solution per injection site. Injections were achieved by using a Hamilton syringes (10 μL) connected to a pulled-glass pipette (any conventional capillary can be used) previously loaded with the LPC solution (2% w/v diluted in PBS solution, vortex if needed). To identify the lesion sites afterwards, we embedded the tip of the pipette with sterile graphite powder (see Fig. 1). The glass micropipette was placed right above the drill incision and stereotaxically downplaced in the corresponding Z-axis coordinate. After 3 min, the LPC solution was carefully injected 0.3–0.5 μL at a time, with a 3-min pause between injections until 2 μL (for callosal fibers) or 2.5 µL (for cerebellum) was reached (2% LPC solution in both regions). Then, another 3-min pause was made. Finally, the micropipette was carefully and slowly removed back from the mouse brain. The procedure was repeated in the contralateral hemisphere in both callosal and cerebellar regions (to achieved a total of 4 injection sites, Fig. 1). Mice in the control group were injected with vehicle (PBS) in a separate experimental group. Finally, open skin was closed with a sterile size #2 suture. After recovery, mice were treated with analgesia for 2 days (carprofen, s.c. 5 mg/kg) and checked each day following the MGS according to the experimental protocols approved by the Bioethical Committee for Animal Experiments of the Universidad de Santiago de Chile (protocol number 319/2023).

Myelin protein expression—as a measurement of (de)myelinated area—was assessed 7, 14 or 21 days after LPC injections (dpi). From a total of 42 injected mice, 4 animals died during the first week after LPC or PBS injections, and 3 mice were excluded from the study due to the lack of demyelinated areas and/or the display of mechanical lesions (see also [32]). All the animals considered in the study (n = 35) survive the entire experimental time.

Tissue preparation

Mice were anesthetized using ketamine/xylazine (0.1/0.01 mg/g) and subsequently subjected to intracardial perfusion with a 20 mL PBS 1× solution (pH 7.2). Following perfusion, the brains were carefully extracted to maintain their structural integrity. A precise transversal cut was made at the midline, leaving one hemisphere stored in a 4% PFA solution (pH 7.2) for 3 h at 4 °C, while the other half was prepared for molecular analysis and kept at − 80 °C. Once fixed, the brain tissue was sectioned into 70 µm sagittal slices using a vibratome (Microm HM 650 V, Thermo) and these slices were then placed in 96-well plates filled with 1 mL PBS 1×. Slices containing the corpus callosum and cerebellar regions were specifically chosen for subsequent immunofluorescence assays.

Immunofluorescence

For immunofluorescence analysis, the slices were incubated in blocking solution (Normal goat serum 4% and Triton X-100 0.5% in PBS 1×) at room temperature for 2 h. The sections were then incubated overnight at 4 °C in the antibody solution (Normal goat serum 2% and Triton X-100 0.2% in PBS 1×) with the myelin basic protein (anti-MBP chicken polyclonal antibody, 1:800, #PA1-10008) or the glial fibrillary acidic protein (anti-GFAP mouse monoclonal antibody, 1:2000, #SAB5201104). The slices were three times rinsed in PBS 1× (5 min each) and then incubated during 2 h at room temperature with conjugated Alexa dye secondary antibody AF-488 or AF-633 (goat anti-chicken, 1:500; Invitrogen, #A-11039). Specificity of the anti-MBP was verified in negative controls, omitting the primary antibodies. Finally, slices were washed three times with PBS 1× and mounted in Fluoromount-G mounting medium (#0100-01, SouthernBiotech) for imaging.

Confocal acquisition

Brain slices were imaged with a Zeiss LSM510 confocal microscope (Carl Zeiss MicroImaging) with the LSM510 software. Bright field images (transmitted light, see Fig. 1C) were acquired to define corpus callosum and cerebellar white matter regions. Images were then captured with a 20× (NA 0.8) objective under 488 nm excitation in Z-stack 6 µm-width. Acquired images were then reconstructed in Z-projections averaging 8 to 12 optical sections per sample (48 to 72 µm-width). Image analysis (MBP and GFAP fraction areas) was performed with ImageJ software.

Cytokine expression

To analyse cytokine gene expression we performed a three phases analysis: total RNA extraction, cDNA synthesis, and quantification via qPCR. For RNA extraction from white matter tissue (isolated cerebellar white matter and corpus callosum), we employed the TRIzol-based method optimized for this purpose. Samples stored at − 80 °C were homogenized by using 1.0 mL of TRIzol with mechanical homogenizers (D160 SCILOGEX) and/or homogenization equipment (ALLSHENG Bioprep-24R). Phase separation was achieved by adding 200 µL of chloroform, followed by centrifugation. The recovered aqueous phase was precipitated with isopropanol and washed with 75% ethanol. Purified RNA was resuspended in RNase-free water and stored at − 80 °C. From 20 µg of total RNA, cDNA synthesis was performed using the M-MLV system from Promega. DNase treatment was applied to remove potential DNA contaminants, followed by addition of Random Primer to facilitate the cDNA synthesis. The reaction involved three stages: DNase treatment, annealing of random primers, and synthesis of the first cDNA strand. The reaction was first incubated at 37 °C for 60 min and then at 70 °C for 10 min to complete the synthesis. Gene expression quantification was conducted using FastStart Essential DNA Green Master (ROCHE®). The reaction mix, comprising 2× Master mix, forward and reverse primers (10 µM), water, and cDNA, was prepared (max 200 ng cDNA per reaction). Amplification was performed in a thermocycler programmed for an initial polymerase activation stage at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C, annealing at 62 °C, and extension/reading at 72 °C. A melting curve analysis was conducted post-amplification to confirm the specificity of the products. Relative gene expression was determined using the \ΔΔC_T\ method, normalized against the reference gene 18S and compared to an untreated control. The efficiency of the qPCR reaction was validated through the generation of standard curves for each gene of interest. Additionally, we performed western blot quantification of TNF-α. Briefly, white matter was homogenized in 10 mM Tris–HCl at pH 7.4, 150 mM NaCl, 1% Triton-X 100, and 1 mM ethylenediaminetetraacetic acid (EDTA) containing protease inhibitors. Lysates were clarified by centrifugation at 8000×g (4 °C) for 20 min, and the supernatants were collected and normalized for protein concentration. Proteins were separated by 10% and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). After blocking with PBS containing 5% skim milk and 0.05% Tween 20, the membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with an HRP conjugated secondary antibody for 2 h at room temperature. Mouse anti-TNF-α (1:500, sc-133192). Horseradish peroxidase-conjugated anti-rabbit IgG antibody was used as secondary antibody (Cell Signaling Technology). Immunoreactive bands were detected using a fluorescence-conjugated secondary antibody and an enhanced chemiluminescence (ECL) system (WBKLS0100, Millipore). They were visualized on a LAS-4000 imaging system (Fujifilm). The protein bands were quantified using the ImageJ software.

Plethysmography

Whole-body plethysmography was performed in freely moving mice to evaluate their resting breathing patterns and chemoreflex function in response to hypercapnia. The experiments were carried out between 10:00 and 16:00 h at room temperature. Prior to recordings, animals were habituated to a whole-body plethysmography chamber (5L, EMKA Technologies) for 2 consecutive days. On the day of recording, mice were allowed a minimum of 2 h acclimatization to the chamber before measurement started. Respiratory flow was recorded using a differential pressure transducer, with the signal being amplified (500×) and digitized at 1 kHz. The inspiratory flow curve’s area was calibrated by injecting 5 mL of dry air into the chamber using a syringe. Throughout the 2-h recordings, mice breathed ambient air with constant inlet and outlet flows (0.75 mL/min). Chemoreflex function was examined by exposing the mice to hypercapnic concentrations of 3%, 5%, and 7%, while recording the ventilatory response and calculating the hypercapnic ventilatory response (HCVR). Data analysis involved acquiring tidal volume (Vt), and minute ventilation (VE) using the EMKA software. The hypercapnic ventilatory response (HCVR) was determined by calculating the slope of the maximal ventilatory response across different CO2 levels.

Grid strength test

The grid strength test assesses neuromuscular strength by measuring how long a mouse can hang from an inverted grid [3, 23, 25]. Briefly, mice were gently placed on a 30 × 30 cm grid with 1 cm2 mesh spaces, positioned 30 cm above a plastic containment box. The grid was carefully rotated so that the mouse faced downward. The time it took the mouse to fall (time-to-fall) was recorded. This measurement was repeated twice for each mouse, and the averaged time was registered. If a mouse jumped or climbed down the box edges, the count was stopped. After a 5-min break, the test was repeated. If a mouse hung for 600 s without falling, the test was ended, and that time was recorded as the maximum value. Mice were allowed 3 days of acclimatization to the apparatus before starting the recordings. Time-to-fall was measured on day-2 before LPC injection and then on days 5, 6, and 7 post-injection. The same mouse was tested at all time points, and then tested in the locomotor Rotarod test. Results were expressed in seconds. All injected animals (n = 4) developed an impairment in the test.

Locomotor Rotarod test

The Rotarod test was employed to assess locomotor performance, evaluating motor coordination and balance [39]. The Rotarod apparatus consists of cylinders (or rotors) with individual lanes that increase in turning speed. Rodents are placed in these lanes at secure height (Rotarod 755, IITC, Lifescience). Before beginning the recordings, animals underwent a daily acclimatization session in the apparatus for 3 days. Time-to-fall on the Rotarod was measured 5 days before LPC injection (− 5 dpi) and then at 7, 14 and 21 days post injection (dpi). During training sessions, parameters included a starting speed of 1 rpm, a maximal speed of 40 rpm and a time to reach maximal speed of 90 s. Experimental conditions consisted of a starting speed of 1 rpm, a maximal speed of 40 rpm and a time to reach maximal speed of 180 s. The same animal was tested at all time points and time-to-fall was expressed in seconds.

Urine volume

To assess the diuretic function, we used metabolic cages to collect and quantify urine volume as an indicator of uronephrological or bladder status [16]. Mice underwent testing in individual metabolic cages for 12-h sessions at night, from 8 p.m. to 8 a.m., with environment conditions set at 20–22 °C and 50% humidity. This regime was repeated for 4 consecutive days. During testing, mice has access to water and previously ground food ad libitum, and each mouse remained in the same cage throughout all sessions. The first three sessions served for mouse acclimatization, with urine volume recorded on the last day. Assessments were conducted both before model induction (basal) and at days 8–11 post LPC (or vehicle) injection.

Systolic pressure and heart rate measurement

Systolic pressure and heart rate (HR) were measured in awake mice using a tail cuff plethysmographic system, (model BP-2000 series II, Visitech, US) as described previously [2, 9]. Briefly, mice underwent a 3-day training period prior to the recording session, maintaining consistent environmental parameters: room temperature of 20–22 °C, 50% humidity, and a tail-cuff system temperature of 37 °C. During the recording session, mice were placed in a black box to restrict movement and their tails were secured with adhesive tape to facilitate pulse measurement at the base of the tail. Every session included a 15-min acclimatization period, during which three consecutive readings were not recorded, followed by 10 consecutive readings of arterial pulsatile and mean pressure captured by the BP—2000 Blood Pressure Analysis Software. Assessments were conducted before model induction (basal, − 5 dpi) and at days 7, 14 and 21 days post LPC injection.

Statistical analysis

Statistical analysis and plots were performed using Graphpad Prism 8.0 software. All data is presented as mean ± SEM. After test for normality, data were compared by using One- or Two-way ANOVA, Kruskal Wallis, or Mann Whitney Tests according to the data structure. Post hoc test is indicated when corresponded. Statistical significance was defined as p < 0.05.