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Equipment and applied methods LPR


Applied methods

1. In vivo methods

  • measurement of blood flow in the uterine artery of the pig by an electric transducer using an electromagnetic flowmeter (RT500 Narcomatic) under  general anaesthesia
  • measurement of porcine uterine intraluminal pressure/indirect uterine contractile activity (Bridge Amp AD Instruments) under  general anaesthesia
  • biotechnology methods of reproduction in the sheep: synchronization of ovulation (intravaginal suppositories) and acceleration of the breeding season (melatonin implants) for experimental purposes
  • mid-ventral laparotomy of the pig and sheep: ovariectomy, hysterectomy, induction of ischemia in the uterus of the pig by ligation of the uterine artery, intravital collection of uterine samples, implantation of catheters to blood vessels of mesometrium for blood collection and/or infusion of biologically active substances e.g. hormones under  general anaesthesia
  • implantation of catheters into blood vessels of the head and neck in the pig, sheep and rabbit under  general anaesthesia
  • long-term collection of cerebrospinal fluid from the third brain ventricle in sheep
  • long-term blood collection from jugular veins of the pig and sheep
  • continuous monitoring of blood oxygen saturation, pulse rate data, carbon dioxide detection and visual respiration indicator using pulse oximeter with carbon dioxide detector (Nonin Medical Inc. 9847V) under general anaesthesia

2. Ex vivo methods

  • the study of the contractile activity of the superficial veins of the head in the pig (frontal, nasal and facial veins)
  • the study of the contractile activity of isolated arteries of the female reproductive tract in the pig (the uterine artery)
  • the study on the isolated head of the pig, sheep, rabbit perfused with autologous blood
  • the study on the isolated pig’s ovary and uterus, testicle of the pig and rat perfused with autologous blood

3. Tissue incubations

  • pig’s hypothalamus explants

4. Analytic methods

4.1  Molecular biology methods

Expression on the mRNA level: Real Time PCR (qPCR) or Real Time PCR with TaqMan probes (qPCR TaqMan) and on the protein level: Western blot analysis (WB):

  • factors associated with production of cerebrospinal fluid: VEGF and its receptors: Flt-1, Flk-1 and co-receptor NRP-1, aquaporin-1,  aquaporin-4, claudin-2 (qPCR, WB) in the ovine choroid plexus
  • tight junction proteins restricting the passage of molecules via the intercellular path: transmembrane proteins: occludin, claudin-1, JAM-1 and cytosolic (scaffolding) proteins: ZO-1, ZO-2, ZO-3 and afadin-6 (qPCR, WB) in the ovine choroid plexus
  • transport proteins: transtyretin (TTR) in the ovine choroid plexus (qPCR)
  • lipopolysaccharide receptor complex: toll-like receptor 4 (TLR4), CD14 and MD-2 in the ovine choroid plexus (qPCR)
  • cytokines and their receptors and chemokines: TNFα, IL-1β, IL-6, TNFRI, TNFRII, IL-1R1, IL-1R2, IL-6R, gp130, MCP-1 in ovine choroid plexus (qPCR)
  • metalloproteinases and their tissue inhibitors: MMP2, MMP3, MMP9, TIMP-1 and TIMP-3 (qPCR) in the ovine choroid plexus
  • hypoxia marker HIF-1α in in the porcine uterus (qPCR, WB)
  • biological clock elements: Bmal 1, Clock, Npas2, Per 1-3, Cry 1-2 (qPCR TaqMan, WB) in the hypothalamus of domestic pig
  • nuclear receptors associated to the functioning of molecular biological clock: Rev-erb α, Rev-erb β, Ror α, Ror β, Ror γ (qPCR TaqMan) in the hypothalamus of domestic pig
  • antioxidant enzymes in the testis and epididymis of male deer and bison: EcSOD, GpX 4, GpX 5 (qPCR), SOD-1, Cat (qPCR, WB) and SOD-1, SOD-2 in porcine uterus (qPCR)
  • leptin receptor (Ob-Ra i Ob-Rb) in the choroid plexus and hypothalamus of domestic pig (qPCR, WB)

4.2 Immunoenzymatic (ELISA) method:

  • LPS Binding Protein in sheep
  • IL-6 in sheep

4.3 Radioimmunoassay (RIA) in plasma, tissues and body fluids:

  • thyroid hormones: T3 and T4 (free and total)
  • melatonin
  • hormones of reproductive system: estradiol, estrone, progesterone, testosterone, oxytocin, prostaglandin F2α
  • protein hormones: prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH), leptin, activin A in pig

4.4 Labeling hormones with radioisotope

  • oxytocin, GnRH, FSH, LH, prolactin, β-endorphin iodination (J125)

4.5 Immunohistochemical localization (with fluorochrome):

  • c-Fos protein (early response gene product) in porcine brain
  • NADPH-diaphorase (histochemical method) in the blood vessels of porcine brain
  • nitric oxide synthases, iNOS, eNOS in the porcine uterus and periovarian vascular complex and in rat’s large intestine
  • endogenous hypoxia marker (HIF-1α) in porcine oviduct and uterus
  • antioxidant enzymes SOD-1 and SOD-2 in porcine female reproductive tract and mice’s large intestine
  • Vascular Endothelial Growth Factor (VEGF) and its receptors Flt-1, Flk-1 in the ovine choroid plexus
  • antiapoptotic protein cFLIP in porcine female reproductive tract
  • apoptotic cells with TUNEL assay (Terminal Transferase dUTP Nick End Labeling) in porcine uterus and rat’s large intestine
  • dopamine hydroxylase (DβH) in porcine head vessels (facial, nasal and frontal vein) and peri-ovarian vascular complex
  • leptin receptor in porcine hypothalamus and choroid plexus
  • lipopolysaccharide receptors complex: CD14 and TLR4 in choroid plexus in sheep

4.6 Optical density measurments:

  • immunoreactivity measurement with Axio Vision Rel. 4.8.2 program

Equipment

1. Equipment for in vivo, ex vivo experiments:

  • Inhaled anesthetic apparatus for large animals (KOMESAROFF MINI-KOM)
  • Cardiomonitor (TEMED)
  • Pulse oximeter with carbon dioxide detector (Nonin Medica Inc.)
  • Set for experiments on isolated organs (UNIPER, Up-100, Hugo Sachs Elektronik-Harvard)
  • Set for measurement of isometric tension isolated blood vessels or muscle tissues (HSE F30, type 372, Hugo Sachs Elektronik-Harvard)
  • Infusion pumps
  • Peristaltic pump (Elphan, ZALIMP)
  • Electromagnetic transducers for blood flow measurement (Flowmeter Narcomatic RT500)
  • Surgical drill (Esculap)

2. Western blot and nucleic acids analysis workplace:

  1. Mini PROTEAN system with PowerPac basic power supply (electrophoresis system and wet transfer system, Bio Rad)
  2. Hematological roller (Chemland)
  3. Horizontal electrophoresis system Wide Mini Sub Cell (Bio Rad)
  4. Laminar flow chamber (Polon)

3. Equipment for Radioimmunoassay:

  • Gamma Counter (LKB)
  • Liquid Scintillation Counter (Beckman)
  • Radiometer with probe Rust-3

4. Basic lab equipment:

  • Vacuum pump BioVac 225 (OmegaLab)
  • Spectrophotometer (Beckman)
  • Stereoscopic microscope
  • Vortex (Biosan)
  • Shaker (Biosan)
  • Incubation chamber with shaker (Edmund Buehler)
  • Magnetic stirrer (Biosan, IKA WERKE)
  • Heated dry block  (OmegaLab)
  • Water bath
  • pH-meter (HANNA Instruments)
  • Mini centrifuge (Biosan, StarLab)
  • Centrifuge with cooling (MPW, Beckman)
  • Electronic scales (RADWAG, MEDICAT)
  • Ultra low freezer (SANYO)
  • Fume cupboard (Polon)
  • Ice maker (Scotsman)
  • Manifold Machine (pipettor) Hamilton Microlab 1000