Animal protocol

The research protocol that involves control and experimental groups has been previously described (López-Cervantes et al. 2021; Vázquez-Martínez et al. 2019). Briefly, young adult male Wistar rats (∼280 g and ∼2 months old) were randomly subjected to PCA or a sham surgery (Sham group). The animals were housed in individual cages at room temperature and with ad libitum access to food until full recovery. All procedures adhered to the institutional guidelines for the care and use of animals in biomedical experimentation, as well as the international ethical standards established by the National Autonomous University of Mexico (UNAM). Approval was granted by the Research Ethics Committee of the Neurobiology Institute, UNAM (protocol 081.A). The Official Mexican Regulation NOM-062-ZOO-1999/SAGARPA was also considered.

Surgery

The procedure by Lee et al. (Lee et al. 1974) for PCA was followed with certain modifications as previously reported (López-Cervantes et al. 2021; Vázquez-Martínez et al. 2019). Briefly, around 30 rats were put under anesthesia with Ketamine (ANESKET®, 1000 mg 10 ml-1, PiSA Agropecuaria, MEX)/xylazine (PROCIN®, 20 mg ml-1, PiSA Agropecuaria, MEX), and a laparotomy was performed to access the abdominal organs. The portal vein was dissected and occluded. The extreme of the portal vein was then connected to a window on the inferior portal vein that was previously obstructed with surgical clips. To optimize the survival of the PCA, the microsurgery was completed in less than 20 min to avoid compromising the survival of the animals. 24 sham-operated rats were subjected to the same procedure (until the use of the surgical clips) but without cutting any blood vessel. The animals (2–3 rats) were kept in a recovery box with controlled temperature. To prevent infections, rats received an intramuscular (IM) injection of piperacillin (6,000 IU). For analgesia, sodium metamizole (10 mg/kg IM) was administered once daily for three days. Surviving animals 24 PCA rats (~ 85% of the total operated animals) participated in the experiments.

Tissue sampling

After a 13-week postoperative period, rats were euthanized with an intraperitoneal injection of ketamine (70 mg/kg) and xylazine (8 mg/kg). Four types of experiments were conducted: (1) Tissue sampling for electron microscopy(3 sham and 3 PCA rats); (2) Ex-vivo tests using cerebellar slices for incubation with fluorescent probes (for all experiments around 10 sham and 10 PCA rats); (3) Immunohistochemical assays involving trans-cardiac perfusion to fix the organs from unresponsive animals (5 sham and 5 PCA rats); (4) Homogenate cerebellum sample for biochemical assays and western blot (~ 6 sham and ~ 6 PCA rats) (Gage et al. 2012). For fluorescent and immunohistochemical experiments, the cerebella were treated as previously reported (López-Cervantes et al. 2021). A cryostat (Leica CM3050 S) was used to obtain 7–8 consecutive sagittal sections of the cerebellum (from the vermis) at ∼30 μm thickness and a temperature of −30 °C.

Transmission electron microscopy

Cerebellar samples were processed as previously reported by Larriva-Sahd, 2006 and Paredes and Larriva-Sahd, 2010. Briefly, eight control and eight PCA rats were deeply anesthetized and intracardially perfused with 200 ml of Karnovsky’s fixative (4% paraformaldehyde, 2.5% glutaraldehyde in 0.10 M cacodylate buffer, pH 7.3–7.4. Each brain was then dissected. Then, tissue samples were post-fixed for 1 h in 1% osmium tetroxide, dehydrated, and flat-embedded in epoxy resins. Semi-thin Sects. (300–400 nm) were obtained using a Leica Ultramicrotome. Ultrathin sections measuring 70–90 nm were mounted on 200-mesh copper grids. The sections were treated with aqueous solutions of uranium acetate and lead citrate and then observed under a JEOL 1010 electron microscope.

Determination of mitochondrial parameters. Electron microscopy images were used to analyze subcellular entities identified as mitochondria, characterized by a double membrane system and cristae. The number of mitochondria, along with their area and perimeter, was quantified with image analysis software (NIH, USA). We analyzed 150–250 mitochondria in the molecular, granular, and Purkinje cell layers from 10 micrographs per group.

Mitochondria morphology was characterized according to Wiemerslage and Lee (2016). The mean area and number of mitochondria were used as indicators of mitochondrial presence. The following four parameters were considered for mitochondrial morphology quantification: mitochondrial quantity, size, interconnectivity, and elongation. The circularity was calculated using the pixel area and the estimated perimeter. The interconnectivity score, which evaluates the physical connections and fragmented status of the mitochondrial network, as well as the elongation score, defined as the inverse value of circularity, were calculated as reported by Wiemerslage and Lee, 2016 with the following formulas:

$$\:interconnectivity=\frac$$

$$\:circularity=4*\pi\:*\left(\frac^}\right)$$

Interconnectivity describes the connections between mitochondria, being an indicator of mitochondrial networking. High score of interconnectivity indicate that mitochondria have more physical connections, while lower scores signify that mitochondria are more fragmented. Elongation refers to the shape of organelle, considering the value of 1 as a circle, associated with mitochondrial fission. Values above 1 correlate to increased fusion.

Fluorescent probes

The following fluorescent probes were used in accordance with previously reported protocols:1) MitoTracker Red (100 nM) to measure mitochondrial membrane potential (ΔѰ); 2) Rhod-2 (4 µM) following treatment with NaBH4 to enhance the mitochondrial calcium signal (Dihydrorhod-2 AM) (Lee et al. 1974); 3) MitoSox (10 µM) to quantify mitochondrial superoxide; 4) dichlorofluorescein diacetate (DCFH-DA; 10 µM) to evaluate intracellular ROS (Zhang and Gao 2015). The protocol involved the following steps: Cerebellar slices (∼1 mm) from sham and PCA rats were obtained using a razor and a sagittal matrix for reference and then pre-incubated for 15 min with Krebs solution (in mM: NaCl 155, KCl 4.5, NaHCO3 1.9, CaCl2 2.4, D-Glucose 10, MgCl2 2, HEPES 5) at pH 7.4, with constant oxygenation (95% O2 and 5% CO2) at 37 °C under dim light. Each fluorescent probe was added at the final concentration specified and incubated for an additional 60 min.

After incubation, the slices were fixed in 4% paraformaldehyde for 24 h and sequentially rehydrated in sucrose solutions (from 10 to 30%). Tissue samples were cut to a thickness of 14 μm utilizing a cryostat (3050 S Leica model). Individual coverslips were washed three times with cold phosphate buffer saline (PBS). Nuclei were stained by adding propidium iodide (dilution 1:1000, Invitrogen); peak of excitation wavelength (λ ex) at 535 nm, peak of emission wavelength (λ em) at 615 nm or SYTOX Green (dilution 1:3000, Invitrogen) detection: λ ex: 504 nm, λ em: 523 nm. Fluorescence was detected with a Zeiss LSM-780 DUO confocal microscope. Fluorescent parameters were for MitoTracker Red: λ ex 510 nm, λ em: 580 nm, MitoSox: λ ex: 488 nm, λ em: 510 nm, Rhod-2: λ ex 552 nm, λ em: 581 nm and DCFH-DA λ ex:488 nm, λ em: 525 nm.

Mitochondrial dynamics

Western blotting and immunofluorescence techniques were used to evaluate specific factors involved in mitochondrial dynamics in the cerebellum of PCA rats.

Western blotting: Cerebellar homogenate sample proteins (50 µg) were separated into 7.5% −15% SDS-polyacrylamide electrophoresis gels, which were transferred to nitrocellulose membranes. Non-specific sites were blocked with 5% of milk-free fatty acids in a TBS1x solution at room temperature for 1 h. The membrane was incubated overnight at 4 °C with the corresponding primary antibody: rabbit anti-OPA1 (Abcam Cat# ab157457, RRID: AB_2864313; 1:1000), rabbit anti-MNF1 (Cell Signaling Technology Cat# 14739, RRID: AB_2744531; 1:500), and rabbit anti-MNF2 (Cell Signaling Technology Cat# 11925, RRID: AB_2750893; 1:1000) for mitochondrial fusion, rabbit anti-FIS1 (Boster Biological Technology Cat# A01932-2, RRID: AB_3081504; 1:1000), rabbit anti-DRP1 (Huabio Cat# HA500487, RRID: AB_3071572; 1:1000) and rabbit anti phosphorylated DRP1Ser616 (Cell Signaling Technology Cat# 4494, RRID: AB_11178659; 1:1000) were utilized for mitochondrial fission, and rabbit anti-GADPH (Cell Signaling Technology Cat# 2118, RRID: AB_561053; 1:1000) as loading control. Then, the membranes were washed with TBST and incubated with the secondary antibody: goat anti-rabbit IgG coupled to alkaline phosphatase (Abcam Cat# ab97048, RRID: AB_10680574; 1:3000). The membranes were scanned and analyzed using Fiji-ImageJ software.

Tissue immunofluorescence: Sagittal sections from cerebellar tissue (14 μm thickness) were washed three times with PBS 1x, pH 7.4, for 10 min per wash. The sections were subsequently blocked with PBS 1x, 0.2% Triton 100x, and 10% normal goat serum, and then incubated overnight at 4 °C with primary antibodies: rabbit anti-OPA1 (Abcam Cat# ab157457, RRID: AB_2864313; 1:250), rabbit anti-MNF2 Cell Signaling Technology Cat# 11925, RRID: AB_2750893;1:250), rabbit anti-rabbit anti-MNF1 (Cell Signaling Technology Cat# 14739, RRID: AB_2744531;1:250), rabbit anti-FIS1 (Boster Biological Technology Cat# A01932-2, RRID: AB_3081504; 1:250), rabbit anti-DRP1 (Huabio Cat# HA500487, RRID: AB_3071572; 1:250), and rabbit phospho-DRP1Ser616 (Cell Signaling Technology Cat# 4494, RRID: AB_11178659;1:250). The samples were washed with PBS three times for 5 min and then revealed with anti-Alexa 488 (Thermo Fisher Scientific Cat# A32731, RRID: AB_2633280; 1:300). Nuclei were dyed with propidium iodide (1:1000 dilution) and + RNase A (1:1000 dilution) for 15 min. The assembly was carried out with the vectashield solution. Ten representative images were taken for analysis. All images were acquired with a Zeiss LSM 780 DUO confocal microscope.

Image processing

To quantify the fluorescent signal of each assay, 3–6 representative images were taken for each layer of the cerebellar cortex, with a 63x oil immersion objective with a 2x (Granular layer) and 1.8x (Purkinje layer) optical zoom on a Zeiss LSM 780 confocal microscope. For 2D images from 14 μm thickness, z-stacking was performed according to the recommended microscope settings (0.37 μm optical sections). All images were acquired using the same intensity parameters and exposure time settings. All quantifications were performed using Image J Fiji for Image J software (NIH, USA).

Lipoperoxidation and conjugated dienes tests

The whole cerebellum was homogenized in buffer (225 mM saccharose, 10 mM Tris/HCl, 0.2% BSA, 0.3 mM EGTA; pH 7.4) using 10 to 15 passes with a DOUNCE homogenizer. Lipid peroxidation (LP) was determined using TBARS, as previously described in Vázquez-Martínez et al. (2019), to quantify malondialdehyde (MDA) and other aldehydes. Briefly, 3 mg of protein was incubated under acidic conditions and placed in a boiling water bath with 0.8% thiobarbituric acid. The pink color was extracted with a pyridine: butanol solution (15:1 v/v) and read at 532 nm. Conjugate dienes were quantified from 500 mg of protein by separating the lipid fraction with the Folch reagent (chloroform-methanol 2:1 v/v). Subsequently, the samples were dried at room temperature and reconstituted in hexane. The extract was read using a UV spectrophotometer at 233 nm (Vázquez-Martínez et al. 2019).

Cupric reducing antioxidant capacity assay

The cupric reducing antioxidant capacity (CUPRAC) assay measures the total antioxidant capacity of both hydrophilic and hydrophobic samples (Apak et al. 2008). Briefly, a cerebellar homogenate was prepared using a 7.5 mM copper (II)-neocuproine (CU(II)-Nc) reagent as a chromogenic oxidant and a 10 mM ammonium acetate solution (1:1 v/v) at pH 7. The mixture was incubated for 20 min at 50 °C, and the absorbance of the resulting CU(I)-chelate from the redox reaction was measured at 450 nm. Data were expressed as equivalents of TROLOX (Apak et al. 2008).

Glutathione peroxidase activity

Glutathione peroxidase activity was measured using a previously reported coupled reaction (Díaz-Muñoz et al. 1985). The cerebellum was homogenized in a solution containing 225 mM sucrose,10 mM Tris/HCl, 2% BSA, and 0.3 mM EGTA (pH 7.4). The samples were mixed with 50 mM K2HPO4, 10 mM EDTA, 1 nM NADPH, 1 mM sodium azide, 100 units/ml glutathione reductase enzyme, and 200 mM reduced glutathione. The final solution was carefully shaken for a few seconds, and the reaction was started with H2O2 0.042% (w/w) and read at 340 nm every minute for 5 min. The activity was calculated as micromoles of NADP + formed in the reaction (extinction coefficient of 0.00373 µM-1).

Statistics

Analyses and graphs were made with GraphPad Prism version 8.0 (GraphPad Software, SD, CA, USA). The Shapiro-Wilk and Kolmogorov-Smirnov test was used to assess normality. Data were analyzed using a student’s t-test and Welch’s t-test are presented as mean ± standard error (SEM). A significance threshold of p < 0.05 was considered statistically significant.