Mgso4

MgSO₄, phenylephrine, chloral hydrate, PTZ, sodium fluorescein and TCA were purchased from Sigma Aldrich (St Louis, MO, USA) and all were made daily in sterile lactated Ringer's solution except MgSO₄, which was ready-to-use. Texas red dextran was purchased from Invitrogen (Life Technologies, Grand Island, NY, USA) and made daily in sterile lactated Ringer's solution.

During the isolated heart experiments, after thoracotomies performed under ketamine/xylazine anaesthesia, the brain was removed and placed into a silicone containing petri dish, filled with 0–4 °C Krebs solution (composition in mmol: 110 NaCl, 5.0 KCl, 1.0 MgSO₄, 1.0 KH₂PO₄, 5.0 glucose and 24.0 NaHCO₃, obtained from Sigma-Aldrich, St. Louis, MO, USA). The solution was equilibrated previously with a gaseous mixture of 5% CO₂, 10% O₂ and 85% N₂ at pH 7.4. Basilar arteries were isolated with microsurgical tools (Fine Science Tools GmbH, Heidelberg, Germany). The arteries were equally cut into 4 mm long rings, which were then mounted in an isometric contraction measurement system (DMT-510, Danish Myotechnology, Aarhus, Denmark). The Ca²⁺-free Krebs solution was changed to Ca²⁺-containing one (composition in mM: 110 NaCl, 2.5 CaCl_2, 5.0 KCl, 1.0 MgSO₄, 1.0 KH₂PO₄, 5.0 glucose and 24.0 NaHCO₃, obtained from Sigma-Aldrich, St. Louis, MO, USA). Before every experiment, a normalization protocol was performed, by stretching the preparations with 1.5 mN force, which was increasing evenly every 15 s until the calculated intraluminar pressure reached 13.4 kPa. The experiments were then performed at this stretch level. Contractile responses for KCl (6–66 mM), serotonine (1 nM–10 μM) and angiotensin II (1 nM–100 μM) have been recorded.

In this study, the monocrotaline model was used, where a single injection of monocrotaline would induce pulmonary hypertension, within 3 to 4 weeks, in rats [26 (link),27 (link)]. To minimize the number of animals used in this study, we performed a preliminary study to determine the lowest effective dosage of monocrotaline that induces pulmonary hypertension. Preliminary data revealed that in rats receiving 60 and 120 mg/kg of monocrotaline (Sigma-Aldrich, St. Louis, MO, USA), the RV/(LV+S) ratios were comparable, and both were significantly higher than those in rats receiving normal saline (p = 0.002 and 0.029, respectively). These data indicated that 60 and 120 mg/kg of monocrotaline exerted similar effects on inducing right ventricular hypertrophy. Thus, we chose to use 60 mg/kg of monocrotaline in this study. In addition, in this study, we administered a daily injection of 100 mg/kg MgSO₄ (Sigma-Aldrich, St. Louis, MO, USA), as our previous data revealed that 100 mg/kg MgSO₄ attenuated the adverse effects of sepsis [44 (link)].

ENO1 activity was measured in the reaction buffer containing 50 mmol/L Tris‐HCl (PH 7.5), 100 mmol/L KCl, 25 mmol/L MgSO4, 1.3 mmol/L ADP (Sigma, MO, USA), and 0.15 mmol/L nicotinamide adenine dinucleotide (NADH, Sigma, MO, USA). In addition, 0.6–1 U of pyruvate kinase and 0.9–1.4 U of lactate dehydrogenase (Sigma, MO, USA) were added to the reaction solution. After adding 1.9 mmol/L 2‐phosphoglycerate (2‐PG, Shanghai yuanye Bio‐Technology, Shanghai, China) and appropriate amount of ENO1 (1 μg of purified rENO1 or 10 μg of cell lysates), the absorbance in 340 nm as a measure of NADH was recorded at room temperature every 1 min for 10−30 min on a Microplate Photometer (Thermo Fisher Scientific, MA, USA).

B. adolescentis strain E 298 b (Variant c) (DSMZ no. 20086) was purchased from DSMZ. The culture medium was prepared according to the DSMZ website and B. adolescentis was grown in an anaerobic workstation (Gene Science AG300). B. adolescentis was cultured anaerobically in sterilized DSMZ Medium 58 containing casein peptone (tryptic digest, Sigma-Aldrich), yeast extract (Sigma-Aldrich), meat extract (Sigma-Aldrich), bacto soytone (BD), glucose (Sigma-Aldrich), K₂HPO₄, MgSO₄ × 7 H₂O, MnSO₄ × H₂O, Tween 80, NaCl, cysteine-HCl × H₂O (Sigma-Aldrich), salt solution and 0.025% resazurin (Sigma-Aldrich). The purity of cultures was monitored by Gram staining and the c.f.u. was counted by plating serial dilutions on agar plates.

A sterile working field was set up for the sectioning procedure. This is to avoid any contamination of the tissue being processed for the sectioning. All tools required for this procedure were sterilized following clinical protocols and placed within reach of the experimenter to prevent delays. The sections were prepared using a vibratome (VT1200; Leica Biosystems). The sectioning chamber was filled with ice-cold preparation medium containing Hibernate-A Medium (Lot No. 1994548; Gibco) supplemented with 13 mM d-glucose (Lot No. SLBX3638; Sigma-Aldrich), 30 mM N-methyl-D-GLucamin (M2004; Sigma-Aldrich), and 1 mM GlutaMAX (Lot No. 1978435; Gibco), saturated with carbogen (95% O₂ and 5% CO₂) for 10 min before the sectioning of resected tissue. Millicell inserts (No. PICM03050; Millipore) were placed in each well of a six-well culture dish with 1 ml of growth medium containing Neurobasal l-Glutamine (Lot No. 1984948; Gibco) supplemented with 2% serum-free B-27 (Lot No. 175040001; Gibco), 2% Anti-Anti (Lot No. 15240-062; Gibco), 13 mM d-glucose (Lot No. RNBG7039; Sigma-Aldrich), 1 mM MgSO₄ (M3409; Sigma-Aldrich), 15 mM Hepes (H0887; Sigma-Aldrich), and 2 mM GlutaMAX (Lot No. 1978435; Gibco).

Hydroxy-terminated poly(dimethylsiloxane) (PDMS, viscosity of 65 cSt), acryloyl chloride (≥97% purity), poly(ethylene glycol) diacrylate (PEGDA, M_n = 575), trimethilolpropane ethoxylate triacrylate (ETPTA, M_n ~428), trichloromethane (CHCl₃, ≥99.5% purity), dichloromethane (CH₂Cl₂, anhydrous, ≥99.8% purity) tetrahydrofuran (THF, anhydrous, ≥99.9% purity), triethylamine (Et₃N, ≥99.0% purity), K₂CO₃ (anhydrous, ≥99.0% purity), MgSO₄ (anhydrous, 99.5% purity), 4-Dimethylaminopyridine (DMAP, ≥99%), 2-hydroxy-2-methylpropiophenone (HMPP, ≥97% purity), and lithium bis(trifluoromethane)sulfonimide (LiTFSI, 99.95%) were purchased from Sigma-Aldrich (Taufkirchen, Germany city, state country) and used as received.