The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia

Neuronal cultures were prepared embryos 14.5E from C57/Bl6/J gestant mice cortices or obtained from wild type (wt) or p53-null mice (Tp53^−/−, knock-out (ko) The Jackson Laboratory; B6.129S2). The colonies were maintained at Animal Experimentation Service of University of Salamanca (USAL) in accordance with Spanish Legislation (RD53/2013). Procedures and Protocols have been approved by the research Bioethics Committee of the USAL. Neurons were seeded at 1.8 × 10⁵ cells/cm² in Neurobasal medium supplemented with 2% B27 and glutamine 2 mM (Invitrogen, Madrid, Spain) and incubated at 37 °C in a humidified 5% CO₂-containing atmosphere.

After 9–10 DIV, neurons were exposed to oxygen and glucose deprivation (OGD) induced by incubating cells at 37 °C for 90 min in an incubator equipped with an air lock and continuously gassed with 95% N₂/5% CO₂. The incubation medium (buffered Hanks’ solution lacked glucose: 5.26 mM KCl, 0.43 mM KH2PO4, 132.4 mM NaCl, 4.09 mM NaHCO3, 0.33 mM Na₂HPO₄, 2 mM CaCl₂, and 20 mM HEPES, pH 7.4) and was previously gassed with 95% N₂/5% CO₂ for 30 min. Under these conditions, oxygen concentrations in the incubation medium were 6.7 ± 0.5 lM as measured with a Clark-type oxygen electrode^47,48. When indicated the neurons were treated with a moderate subtoxic NMDA (20 μM) for 2 hours prior OGD (preconditioning condition, NMDA-PC + OGD). In parallel neurons were incubated in Normoxia (Nx) or preconditioning (NMDA-PC) such as it is described in Table 1.

Neurons were carefully detached from the plates using 1 mM EDTA tetrasodium salt in PBS (pH 7.4) and were stained with annexin V-APC and 7-AAD performed exactly as previously described⁴⁹.

Necrosis was asseses by the examination of trypan blue-staining cells as previously described^50,51. Briefly, 4 h of reoxygenation after ischemia, neuronal cultures were washed with warm (37 °C) phosphate-buffered saline (PBS; 136 mM NaCl, 2,7 mM KCl, 7,8 mM Na2HPO4, 1,7 mM KH2PO4, pH 7,4) and incubated with 0,2% trypan blue in PBS for 2 min at room temperature. Microphotographs (20× magnification; Leica) were taken for each experimental condition and viable plus necrotic (stained) cells were counted). At least two different cell cultures utilizing six separate wells were employed, such that a minimum of 7,000–9,000 neurons were counted for each data point.

Neuronal cell injury was quantitatively assessed by the measurement of lactate dehydrogenase (LDH), released from damage or destroyed cells, in the extracellular medium 4 hour after the four conditions studied (Nx, NMDA-PC, OGD and NMDA-PC + OGD). An aliquot of bathing media was combined with NADH and pyruvate solutions⁵². LDH is proportional to the rate of pyruvate loss, which was assayed by absorbance change using a Varioskan Flash (Thermo Fisher, Vantaa, Finland) spectrofluorometer. LDH levels were expressed by percentages. Control experiments have shown previously that the efflux of LDH occurring from either necrotic or apoptotic cells is proportional to the number of neurons damaged or destroyed (Koh and Choi, 1987).

Total RNA samples were harvested and purified from culture neurons (GenElute mammalian Total RNA Miniprep kit; Sigma). RT-qPCR was performed using the Power SYBR Green RNA-to-CT TM 1-Step kit (Applied Biosystems, Township, USA). Reverse transcription was accomplished through 30 min at 48 °C cycle, and PCR conditions were 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C plus 1 min at 60 °C. The following forward and reserve primers used were respectively (Thermo Scientific, Offenbach, Germany), 5′-ATTCTGCCCACCACACAGCGACA-3′ and 5′AGGGCTTCCTCTGGGCCTTCTA-3′ (p53), 5′-GGGTGTGAACCACGAGAAAT-3′ and 5′ -GACTGTGGTCATGAGCCCTT-3′ (Gapdh). The mRNA abundance of each transcript was normalized to the Gapdh mRNA abundance obtained in the same sample. The relative mRNA levels were calculated using the ΔΔCt method, and were expressed as the fold change between sample and calibrator.

Caspase-3 activity was assessed in cell lysates and according to manuacturers´ instructions via Fluorimetric Assay kit CASP3F from SIGMA and read at emission wavelength 405 nm. The method is based on the release of the fluorescent 7-amino-4-methylcoumarin (AMC) moiety. The AMC concentration is calculated using a AMC standard.

Neurons and tissues were lysed in buffer containing 1% SDS, 2 mM EDTA, 150 mM NaCl, 12,5 mM Na2HPO4 and 1% Triton X-100, (NP40: 1%NP40, EDTA diK⁺ 5 mM, Tris pH8 20 mM, NaCl 135 mM y 10% glicerol) supplemented with phosphatase inhibitors (1 mM Na₃VO₄ and 50 mM NaF) and protease inhibitors (100 μM phenylmethylsulfonyl fluoride, 50 μg/ml anti-papain, 50 μg/ml pepstatin, 50 μg/ml amastatin, 50 μg/ml leupeptin, 50 μg/ml bestatin and 50 μg/ml soybean trypsin inhibitor), stored on ice for 30 min and boiled for 5 min. Aliquots of lysed extracts were subjected to SDS polyacrylamide gel (MiniProtean®, Bio-Rad) and blotted with antibodies overnight at 4 °C. Antibodies used were anti-p53 (554157, BD Biosciences), anti-pp53 (Ser15; 9286, Cell Signaling, Danvers Massachusetts, USA and anti-cleaved caspase-3 (Asp175, 9661, Cell Signaling), anti-p21(556431, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti-PUMA (ab54288) (Abcam, Cambridge, UK), anti-lamin B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-GAPDH (Ambion, Cambridge, UK) overnight at 4 °C. After incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce, Thermo Scientific) or goat anti- mouse IgG (Bio-Rad), membranes were immediately incubated with enhanced chemiluminescence SuperSignal West Dura (Pierce) for 5 min before exposure to Kodak XAR-5 film for 1 to 5 min, and the autoradiograms were scanned. Band intensities were quantified using ImageJ software⁵³.

For co-immunoprecipitation assay, neurons were lysed in ice-cold buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% NP-40, supplemented with phosphatase inhibitors described above. After clearing debris by centrifugation, neuronal lysates (100 μg) were incubated with 1 μg of antibody for 24 hours at 4 °C followed by the addition of 10 μl of protein A-agarose (GE Healthcare Life Sciences) for 2 hours at 4 °C. Immunoprecipitates were extensively washed with lysis buffer and before being resolved by SDS-PAGE and immunoblotted with indicated antibodies. The relative protein abundances is shown in Supplementary Fig.. Full blots and gel scans are included in Supplementary Fig.. S2 S3

To fractionate nucleus from cytosol, neurons were washed with cold PBS containing 1 mM MgCl2, harvested with cytosolic buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 0.1% NP-40, v/v, 1.5 M sucrose, and protease and phosphatase inhibitors mixture, pH 7.9), triturated with a micropipette to promote cell lysis, left on ice for 30 min, and vortexed for 10 s. After checking cell lysis under a light microscope, extracts were centrifuged at 830 × g for 10 min and the cytosolic fraction (supernatant) was removed and boiled for 5 min. Lysis of the nuclei was performed by resuspending the nuclear pellet in nuclear buffer (50 mM HEPES, 1.5 mM MgCl2, 10 mM KCl mM, 0.5 mM NaCl, 1 mM EDTA, 1% NP-40, v/v, and protease and phosphatase inhibitor mixture, pH 7.9), triturated with a micropipette, left on ice for 2 hours, vortexed (10 s), boiled (5 min), and sonicated (5 min).

Neurons grown on glass coverslips were fixed with 4% (v/v, in PBS) paraformaldehyde for 30 min and immunostained with rabbit anti-cleaved caspase-3 (Asp175) (1:300; Cell Signaling Techn, Inc.), mouse anti-MDM2 (2A10, ab-16895), mouse anti-Map2 (SIGMA) antibodies⁴⁹, mouse anti-p53 (554157, BD Biosciences) and rabbit anti-MDM2 (1:500; ab38618 Abcam). Immunolabeling was detected using anti-rabbit IgG–Cy3 or anti-mouse IgG–Cy2 (1:500; Jackson ImmunoResearch; Newmarket, Suffolk, UK). Coverslips were washed, mounted in SlowFade light antifade reagent (Invitrogen) on glass slides, and examined using a microscope (Nikon Inverted microscope Eclipse Ti-E) equipped with 20× objective and a pre-centred fibre illuminator Nikon Intensilight C-HGFI and black and white charge-coupled device digital camera Hamamatsu ORCAER, or on a scanning laser confocal microscope (“Spinning Disk” Roper Scientific Olympus IX81) with three lasers 405, 491 y 561 nm and equipped with 40× and 63× PL Apo oil-immersion objective for high resolution imaging and device digital camera Evolve Photometrics. The Quantification of the average neurite length (Map2 staining) per neuron was performed using the plugin NeuronJ 1.4.0 (ImageJ, version 1.48 v; National Institutes of Health, USA). Values are mean ± SEM from 60 neurons per group measured in three different neuronal cultures. With NeuronJ, the user utilizes a computer mouse to select a starting point and an ending point. NeuronJ makes use of a minimal cumulative cost search algorithm to determine a path between the two points. The algorithm is highly resilient against varying levels of noise and neurite intensity contrast⁵⁴. The degeneration of neurites in the culture was assayed by analyzing the density of Map2-positive neurites in each four groups. Fluorescence 8-bit images were acquired as z stacks using an HCX Plan Apo CS2 63× oil objective and an inverted confocal microscope. Images were exported into ImageJ (1.48 v; National Institutes of Health, USA) in tiff format for processing. Before image analysis, a maximum-intensity projection over z-series projections spanning 2 to 4 μm was performed. Images were converted to grayscale 8-bit images and brightness/contrast was adjusted using the ImageJ “auto” function. All Map2-positive dendrites were automatically delineated using the “auto setting threshold” (default method) and “dark background” functions of ImageJ. Thresholded images were subsequently quantified as percent area (area fraction) using the “analyze-measure” function, which represents the percentage of pixels in the image that have been highlighted (% area). Values are mean ± SEM from 10–15 measurements of 3 different cultures from four different groups⁵⁴. Furthermore, the percentage of neurons with protein-staining is shown in Supplementary Fig. S1.

The animals were anesthetized by i.p. injection of diazepam (5 mg/kg, Almirall Prodesfarma, Barcelona, Spain), ketamine (100 mg/kg, Ketolar, Parke-Davis, El Prat de Llobregat, Barcelona, Spain) and atropine (0.3 mg/kg, B Braun Medical, Rubi, Barcelona, Spain) to perform orotracheal intubation and assisted ventilation (Harvard Apparatus, 683 rodent ventilator, Holliston, MA, USA). Anesthesia was maintained during the procedure with 0.5–1% sevoflurane (Sevorane, Abbott Laboratories, Madrid, Spain). To induce temporary right focal cerebral ischemia, tMCAO was carried out by using the intraluminal thread technique as originally described³⁶. Cerebral cortical perfusion (CP) was measured by Laser-Doppler flowmetry together with other physiological parameters⁵⁵. Body temperature was monitored throughout surgery via a rectal probe and maintained at 37 °C by using a heating blanket. After 60 min of ischemia, the intraluminal thread was carefully removed. Sham-operated rats underwent the same surgical procedure except for tMCAO. The surgical incisions were closed, and the rats were allowed to recover from anesthesia, and then placed back into their cages with access to food and water ad libitum. Postoperative pain was relieved by s.c. injection of 0.1 mg/kg of buprenorphine (Buprex, Schering-Plough, San Agustín de Guadalix, Madrid, Spain). Twenty-four hours after tMCAO or sham surgery, neurofunctional condition based on four tests, which total score could range from 0 (no neurological deficits) to 6 (highest neurological deficits), was examined just before euthanization⁵⁵. Afterwards, the rats were euthanized under anesthesia and seven 2 mm-thick brain coronal sections were obtained by means of a tissue slicer (Stoelting Wood Dale, IL, USA). Brain infarct size was measured (except for the 3^rd coronal section) by the 2,3,5-triphenyltetrazolium chloride (TTC) vital staining method⁵⁶ followed by morphometric analysis with correction for edema⁵⁷. The unstained 3^rd coronal sections (0.2 to −1.8 from bregma) were separed into ipsilateral and contralateral hemispheres. The cortex and striatum from each hemisphere were rapidly dissected out, flash-frozen with liquid N₂ and stored at −80 °C until used for determination of protein expression levels by western blot analysis.

IPC surgery was performed as previously described³⁵, with modifications. Briefly, the rats were subjected to 10 min of tMCAO as a preconditioning event followed by a 24 hours period of recovery before the 60 min tMCAO insult. Previous studies have shown that these preceding short ischemic insults produced no brain damage, but did induce tolerance; that is to say, a reduction in the ischemic injury induced by severe tMCAO. Sham rats underwent the same surgical IPC procedure except for tMCAO. Twenty-four hours after IPC or sham surgery, the rats were euthanized for brain TTC-staining and sampling to determine protein expression levels.

Twenty male Wistar rats (300–350 g, Charles River, Barcelona, Spain) were housed under standard environmental conditions, and fed standard chow with water ad libitum. Some rats were excluded from the study according to the following criteria: 1) CP did not drop after filament gliding (no ischemia), n = 1; 2) CP did not recover after filament withdrawal (no reperfusion), n = 1; 3) no brain infarction in spite of a right ischemia-reperfusion pattern, n = 4; and 4) death before the 24 hours time limit, n = 1. Four rat groups were established after exclusions: Sham (n = 2), IPC (n = 2), Sham + tMCAO (n = 5), and IPC + tMCAO (n = 4). Experiments were conducted in compliance with the legislation on protection of animals used for scientific purposes in Spain (RD53/2013) and the EU (2010/63/EU). Protocols were approved by the Animal Experimentation Ethics Committee from IIS Hospital La Fe.

Supplementary Figures