The phenotyping module at the Czech Center for Phenogenomics houses a comprehensive collection of tools for the physiological and morphological assessment of experimental mice and rats in a controlled SPF (specific pathogen-free) environment. Our experienced staff offer a wide variety of standardized tests and services, including those of IMPReSS (International Mouse Phenotyping Resource of Standardised Screens), mandated by our active partnership in the International Mouse Phenotyping Consortium. Notable is our capacity for conducting comprehensive phenotyping pipelines, providing a wide breadth of clinical information per experimental animal, and thereby minimizing overall animal usage. Our mission is to support the preclinical research and development community with service of the highest professional standard.

Histopathology

Veterinary Pathologists: Ivan Kanchev, MVSc, DVM, and Peter Makovicky, PhD

The histopathology unit is dedicated for macroscopic and microscopic analysis of pathological alterations occurring in the postnatal period of mouse models. The unit is comparing gross morphology and microscopic differences between wild-type and gene-engineered mouse models. A major task is to screen for set of pathologies connected to specific genetic status in the postnatal period using the tools and techniques of the classical and molecular pathology.

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    Gross Morphology and Tissue Processing

    Full mouse/rat necropsy with organ isolation. A complete necropsy is performed to detect and record abnormal macroscopic alterations in internal and external organs. Provided in this service are a standardized scoring table using phenotype quality ontology (PATO) terms, images of any significant gross findings, and a written report prepared by a veterinary pathologist. The following organs are fixed, trimmed, processed and embedded in paraffin blocks: adrenal gland, heart, mammary gland (F), skin, thymus, brain, kidney, ovary (F), small intestine, thyroid, epididymus (M), large intestine, pancreas, spinal cord, trachea, esophagus, liver, prostate (M), spleen, urinary bladder, eye, lung, seminal vesicles (M), stomach, uterus (F), gall bladder, lymph node, skeletal muscle and testis (M). Additional organs can be processed by request.   

    Organ sampling and trimming. Individual organs can be processed. Unless otherwise specified, organs are processed according to the Revised guides for organ sampling and trimming in rats and mice, published in 2003 and 2004 by the Registry of Industrial Toxicology Animal-data (RITA) and the North American Control Animal Database (NACAD) groups (Exp Toxic Pathol 55: 91-106, Exp Tox Pathol 55: 413-431, and Exp Tox Pathol 55: 433-449).

    Tissue processing. By using a state-of-the-art automated vacuum tissue processor (Leica ASP6025), we are able to process and paraffin-embed specimens with the highest levels of reproducibility and quality. Where applicable, decalcification to remove mineral from bone or other calcified tissues is performed prior to processing to paraffin. If frozen sectioning is required, we embed tissues using a standard manual protocol and Optical Cutting Temperature (OCT) compound.

    Adult lacZ wholemount staining. Adult mouse tissues containing a lacZ reporter are scored for the presence of lacZ staining which is distinct from either nonspecific staining observed in wildtype control mice, or is too faint to score as present. Included in the survey are qualitative expression scoring of 105 distinct tissues, representative images for positive staining tissues, and anatomical description of those images.

    Sectioning and Staining

    Sectioning. Standard paraffin sections and frozen sections can be cut using a microtome or cryostat respectively. Users can select thickness options, section plane, and number of sections per block. 

    H&E staining. The standard primary staining procedure for all histological workflows is the hematoxylin and eosin (H&E) stain. For reproducible results with rapid turn around time, especially with large orders, we use an an automated stainer (Ventana Symphony).

    Special stains. The special stains are used to differentiate various biological constituents, including lipids, carbohydrates, amyloid and connective tissue and therefore are an informative methodology for scoring and monitoring many pathologies, including fibrosis, steatosis, amyloidosis etc. In research, the special stains represent an underutilized complement to standard immunohistochemistry and in many cases, can offer a cost-effective alternative. We currently employ the following special stains, and can perform additional stains upon request:

    PAS, PAS+Diastase, PAS+Alcian Blue, Alcian Blue pH 2.5, Mucicarmine, Reticulin, Elastic fibres, Giemsa, NASDCL, Massons Trichrome, Jones Methenamine Silver, Grocott Silver Impregnation (GMS), Pricrosirius Red, Pricrosirius Red Trichrome, Congo Red, Methyl Green – Pyronin, Chromotrope 2R – Analine Blue, and AZAN.

    Immunohistochemistry

     CCP-Validated Antibodies. Immunohistochemistry to detect the following epitopes can be provided upon request with options for chromagen or fluorescence detection: caspase 3 (mouse), CD3 (mouse), CD31 (mouse), CK19, pan-CK (rat), HMW-CK (mouse, rat), cyclin D1 (mouse), F4/80 (mouse), glucagon (mouse), glutamine synthase (mouse), insulin (mouse), Ki67 (mouse), Pax5 (mouse, rat), PCNA (mouse), sSMA (rat), vimentin (mouse, rat), eYFP (mouse). Through use of the automated Ventana Discovery ULTRA, immunohistochemistry is performed with superior quality and reproducibility.

    Antibody Validation. Requests for immunohistochemistry using any other antibodies will first require successful completion of our antibody validation pipeline. Within this pipeline, staining procedures will be optimized and antibody specificity will be assessed.

     

    Slide Scanning

    Scanning slides has become essential for modern automated image analysis workflows and data sharing, as well as allowing the secure archiving of important histological specimens. Our Zeiss Axioscan.Z1 is capable of both brightfield and fluorescent slide scanning (up to nine parallel fluorescence channels), and is even able to scan histotopograms (double-sized slides).

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    In Situ Hybridization

    In-situ hybridization can be performed upon request. Both fluorescence and chromogenic (DIG) detection is supported. Notable is the opportunity to support reproducible large-scale in-situ hybridization studies through use of the automated Ventana Discovery ULTRA platform.

    Analytical services

    Analysis of H&E slides. Identification of alterations in organ architecture, cell architecture and/or subcellular alterations. Alteration/lesion staging and grading (where applicable).

    Analysis of special stains. Determination of matrix alterations. Scoring of cell type and density. Scoring of cellular chemical components.

    Analysis of immunohistochemistry. Proliferation scoring. Apoptosis scoring. Customized scoring.

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    Histopathology Services – Request Form

    Use this form for requesting histopathology services. Therein you can choose any combination of service categories (necropsy, processing, sectioning, staining etc.) noting that combinations (workflows) and bulk orders attract discounted pricing. Upon receipt of your submission, we will provide an itemized pro-forma invoice and defined work agreement for your approval (for orders above 5000CZK incl VAT).

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    Tissue processing

    Leica ASP6025 The most modern single retort vacuum tissue processor

     

    Slide staining

    Ventana Symphony Automated H&E stainer and coverslipper

    Ventana Benchmark Special Stains Automated slide stainer for special stains

    Ventana Discovery ULTRA Automated stainer for immunohistochemistry and in situ hybridization

     

    Microscopy and analysis

    Carl Zeiss Axio Imager.Z2 motorized microscope imaging station, capable of both brightfield and fluorescence capture, Z-stack acquisition, tile acquisition and deconvolution.

    Leica DM3000 Semi automated high-throughput brightfield microscope system equipped with state-of-the-art color camera.

     

    Slide scanning

    Carl Zeiss Axio Scan.Z1 Combined brightfield and fluorescence slide scanner with ability to also scan histotopograms. Automated scanning of up to 100 standard slides and 50 histotopograms. Equiped with ultra-fast LED fluorescent module and 7 different excitation/emission filters.

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Embryology

Head: Kallayanee Chawengsaksophak, Ph.D.

The embryology unit can be used to systematically examine and score embryonic and organ development relative to established morphological and molecular milestones. They can identify the time window and likely causative factors leading to embryonic death, as well as identify any phenotypic heterogeneity.

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    Embryology Services

    Whole embryo and placenta isolation/dissection. Conceptuses can be dissected at all half day time points except for 4.5 d.p.c. (days post coitum). In the near future we will also offer full day time-points. Implantation and resorption sites are documented. Conceptuses can be supplied fresh, frozen or fixed, or can be further processed and analyzed through additional CCP services.

    Embryonic organ dissections. Embryos can be further dissected in order to obtain organs/tissues of interest. Examples include genital ridge (10.5-11.5 dpc), embryonic gonad (12.5 dpc onward), pancreatic bud (14.5 dpc) isolations, and para-aortic splanchnopleura (PSp) (8.5-9.5 dpc).

    3D imaging. Optical projection tomography (8.5-15.5 dpc) or microCT imaging (13.5-18.5 dpc) of embryos offers high-resolution assessment of embryonic gross anatomy.

    Staining for ß-galactosidase activity. Transgenic embryos and placentas carrying a lacZ expression construct can be used for whole-mount ß-galactosidase staining. The whole-mount stained embryos can be later sectioned in order to further resolve expressing regions. From 12.5 dpc, potential problems with penetration and the increasing presence of endogenous ß-galactosidase activity often make it preferable to section or dissect embryos prior to staining.

    Primary cell line derivation

    Embryonic stem cells (ESCs). Embryonic stem cell lines can be isolated from blastocyst outgrowths. Cell lines are routinely karyotyped, assessed for morphology and gender, and stained for alkaline phosphatase. Further analyses such as immunostaining for pluripotency markers and assessment of pluripotency by embryoid body formation or contribution to chimeras can be arranged upon request.

    Embryonic fibroblasts (MEFs) can be readily isolated from mouse embryos 9.5 dpc and older.

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    Immunohistochemistry and in-situ hybridization

    Expression of RNAs or proteins can be detected using in-situ hybridization or immunohistochemistry respectively. Both can be performed either in whole mount or on sections. We can also perform combined or double whole-mount in-situ hybridization and immunohistochemistry.

    Primary cell line derivation

    Apart from MEF and ESC isolations which are offered as standard services, the embryology unit can also assist in deriving many other primary cell lines of embryonic origin including epiblast-derived stem cells (EpiSCs).

    Histochemical visualization of embryonic skeletons.

    From 12.5 dpc onwards, the mouse skeleton can be visualized using the histochemical stains alcian blue (which stains the cartilaginous skeleton) and alizarin red (which stains mineralized tissues). Photodocumentation included.

    Visualization of the fetal vasculature using India ink injections

    India ink is injected into a branch of the vitelline vein of the yolk sac in embryos from 12.5 dpc and older. The three dimensional structure of the fetal vasculature is visualized following fixation and clearing. Photodocumentation included.

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Biochemistry and Hematology

Head: Karel Chalupský, Ph.D.

Biochemical and hematology phenotyping is based on robust primary screening developed under Eumorphia and EMPReSS, a European Mouse Phenotyping Resource for Standardised Screens. Phenotyping investigation includes also newest development in INFRAFRONTIER and IMPC consortia.

These tests comprise primary phenotyping pipeline. The Biochemical and hematology unit currently sets up examinations of various blood metabolites, ions, hormons, and enzymes of genetically modified mice. These data link changes of these parameters to metabolic and functional abnormalities of specific organs such as liver, kidney, and gastrointestinal tract.

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     Clinical Chemistry

    The following analytes can be measured. Each individual test requires 5 μl plasma, serum, or urine with an extra 10μl per sample needed for machine dead volume.

    Inorganic Analytes. Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic Phosphorus (P), Iron (Fe), Magnesium (Mg), unbound-iron binding capacity (UIBC), Bicarbonate (HCO3),

    Organic Analytes. Urea, Uric acid, Cholesterol (Chol), HDL- Cholesterol (HDL), LDL- Cholesterol (LDL), Triglycerides (TRIG), Non-esterified Fatty Acids (NEFA), Glucose (GLU), Total Protein (TPRO), Albumin (ALB), Creatinine (Cr), Lactate, Conjugated and Unconjugated Bilirubin, Ferritin, Transferrin, C Reactive Protein (CRP),

    Enzymes. a-Amylase, Aspartate-Aminotransferase (AST), Alanine-Aminotransferase (ALT), Alkaline Phosphatase (ALP), Hydroxybutyrate Dehydrogenase (HBDH), Lactate-Dehydrogenase (LDH), Lipase, Creatine Kinase (CpK)

     

    Hematology

    Complete Blood Count. Our hematology test requires 25 μl of EDTA-whole blood per sample. The parameters that are measured are red blood cell count (RBC#),  white blood cell count (WBC#), platelet count (PLT), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), mean platelet volume (MPV).

    Complete Blood Count with Differentials. This requires the same amount of EDTA-whole blood per sample and measures all the parameters in the standard test, but also includes absolute and differential cell counts for neutrophils (Neu#, Neu%), lymphocytes (Lym#, Lym%), monocytes (Mon#, Mon%), eosinophils (Eos#, Eos%), and basophils (Bas#, Bas%).

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    Multiplex Immunoassays

    The Bio-Plex multiplex reader employs magnetic bead-based immunoassays to simultaneously measure the levels of up to 100 different biomolecules in a single sample. Samples can range from serum and plasma to tissue culture supernatants. We currently offer measurements for the following molecules, and can design custom assays for new molecules.

    Adiponectin, Amphiregulin, Amylin, Betacellulin, CD40L, Cortisol, C-Peptide 2, Eotaxin, EPO, Estradiol, Exodus-2, Fractalkine, bFGF, G-CSF, GM-CSF, Ghrelin, GIP, GLP-1, Glucagon, HGF, ICAM-1, IFN-γ, IGFBP1, IGFBP2, IGFBP3, IGFBP5, IGFBP6, and IGFBP7, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, sIL6-R, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17a, IL-17F, IL-18, IL-20, IL-21, IL-22, IL-23p19, IL-25, IL-27p28, IL-31, IL-33, Insulin, IP-10, KC, Leptin, LIF, LIX, MCP-1, MCP-5, M-CSF, MDC, sMet, MIG, MIP-1α, MIP-1β, MIP-2, MIP-3α, MIP-3β, PAI-1, Pancreatic Polypeptide, PDGF-BB, Progesterone, PYY, RANTES, Resistin, TARC, Thyroid Hormones T3 and T4, TIMP-1, TNF-α, sTNFR, and VEGF

     

    Metabolite Quantification – HPLC

    These assays currently include the measurement of amino acids, pterins, bile acids, barbiturates, and ROS adducts (malonylaldehyde and dihydroethitidium). Further Tests can be prepared on demand.

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    Clinical Chemistry

    Use this form to obtain a quotation for clinical chemistry requests.

    Hematology

    Use this form to obtain a quotation for hematology requests

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    Beckman Coulter AU480

    Biochemical analyser

    Mindray BC 5300 Vet

    Hemoanalyser

    BioRad 200 Luminex

    Multiplex

    Agilent 1260 Infinity

    HPLC

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Bioimaging

Head: Jan Prochazka, PhD

The bioimaging unit is dedicated to comprehensive morphological and functional characterization of animal models by whole-body imaging systems in vivo and ex vivo.

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    MicroCT

    Our microCT in vivo scanner provides sensitive and high resolution 3D imaging based on X-ray projections with voxel size 9 – 35um. Our imaging set up is suitable either for in vivo or ex vivo imaging. In vivo scans can provide comprehensive 3D visualization of bones and other mineralized tissues like teeth, but also quantification of total body fat mass and lean mass (alternative to DEXA analysis). MicroCT also provides fast imaging mode for in vivo visualization of cardiovascular system and kidneys after application of contrast agents.

    Ex vivo scanning mode can be used for higher resolution imaging or use of contrast agents for imaging soft tissues (liver, kidney, hearth, neuronal tissue) or imaging fixed embryos (E9.5 – E18.5) with use of appropriate contrast agents (Iodine, PTA). For complete microCT analysis software post-processing of projection data is necessary, especially 3D reconstruction and further segmentation for visualization of morphological phenotypes. Data can be presented as static images and/or as animated 3D reconstruction movies.

    Software tool box: ITK-snap, 3D-slicer, CTvox, CTanalyser, Imaris

    figure1

    Whole skeleton microCT scan is routinely used for unbiased morphological analysis of mouse mutant lines

    Body composition analysis from 3D microCT data. Mineralized tissues like bones are in green, lean tissues like organs, intestine and muscles are in yellow and all fat tissue is in purple. Volumetric parameters can be quantified and statistically evaluated.

    Figure 6. 3D image of fixed embryo after Iodine contrasting protocol

    3D image of fixed embryo after Iodine contrasting protocol

    Imaging of contrasted soft tissues is also possible with microCT. Liver were fixed and contrasted with Iodine, bile duct structure can be visualized based on differential Iodine sequestration in liver parenchyma and bile ducts.

    Segmentation of teeth within the mouse skull. Such an approach can provide important information about tissues/structure morphology within the context of the whole skull or body.

    Radiography (X-ray)

    2D radiography is the best for fast analysis of mineralized/hard tissues, with combination of rotation stage can be also combined in 3D projection. This imaging modularity is mostly recommended as background image for other modularities (luminescence/fluorescence). Combination of optical imaging and X-ray is very suitable for anatomical annotation of fluorescence or luminescence signals.

    X-ray imaging is used for anatomical annotation of tumour position discovered by fluorescence imaging.

    Whole body fluorescence imaging

    Our device is very suitable for imaging of reflected fluorescence signals which can be used for in vivo or ex vivo imaging. Even though the ex-vivo imaging provides better signal resolution, we focus mostly on application of in vivo approach in most cases. In vivo fluorescence imaging provides unique opportunities for visualization and quantification of pathological processes like inflammation, kidney function or tumour progression in longitudinal analysis.

    The ability of cells to form tumours was visualized by detection of red fluorescent protein (RFP) expressed in inoculated cell line.

    In vivo bioluminiscence detection

    Our device includes highly sensitive camera for detection of emitted photons based on enzymatic activity like luciferase or peroxidase. This approach is suitable for in vivo imaging with emphasis on high sensitivity. This imaging is frequently used for inflammation, tumour progression, metastasis or cell homing experiments. We have successfully published our imaging protocol in the study of genetic regulation of DSS colitis. We offer our knowledge for screening of potential role of given genes in the gut regeneration processes by non-invasive imaging setup for longitudinal monitoring of healing process.

    Whole body detection of luciferase reporter. Luciferase provides very high sensitivity with out non specific background, which make is suitable for less intense processes or detection of small cell populations.

    Optical projection tomography (OPT)

    Optical projection tomography is based on screening samples from multiple angles in fluorescent or transmitted light and subsequent 3D reconstruction from projection data. This imaging approach is very suitable for early stage embryos, tissues and organs. Samples need to be cleared first by special clearing protocols. For this purpose, we are preparing protocols for 3D imaging of LacZ stained samples, whole-mount in situ hybridization samples, fluorescent proteins presence or whole mount immunologically stained samples. For basic morphology analysis we provide rapid clearing BABB based protocol and detection of endogenous autofluorescence. Final data can be presented as static images and/or as animated 3D reconstruction movies. Software tool box: ITK-snap, 3D-slicer, Imaris

    Tissue clearing and large Z-stack optical sectioning

    We can offer our experience with tissue clearing and whole-mount imaging protocols employed predominantly for cre dependent fluorescence reporters (R26mT/mG) since a standard data can be presented as volume rendered static images or animated 3D reconstructions.

    The tooth germ from a E14.5 embryo, suitable reporter has been used, tissue clearing protocol followed by large Z-stack optical sectioning with confocal microscope. Dataset was 3D reconstructed and 3D rendered in Imaris.

    Histological tomography

    In collaboration with histology unit (Dr. Peter Makovicky) we can offer unique 3D visualization from serial histological sections. In this procedure, the entire organ or embryo is sectioned on histological slides, processed for hematoxylin or any other histological staining, every single section is digitalized and then 3D reconstructed in Voloom software. Datasets can be also processed in Bitplane Imaris for state of the art quantification analysis.

    Whole adult kidney reconstructed from 500 serial histological sections, reconstructed in Voloom and segmented in Imaris software package.

     

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    HREM (survey potential interest)

    We are surveying interest of the scientific community in application of HREM (high resolution episcopic microscopy) in research projects. HREM is a novel 3D imaging method based on episcopic screening of serially sectioned specimens in fully automatized manner (embryos, organs). In principle, the device generates thousands of digitalized serial sections which are subsequently 3D reconstructed. The data can be presented as volume rendered static images or animated 3D reconstructions.

     

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    Bruker In-vivo Xtreme

    Optical and X-ray small animal imaging system for high sensitivity luminescence, fluorescence, radioisotopic and radiographic imaging.

    Bruker Skyscan 1176

    High performance in vivo micro-CT scanner for preclinical research.

    Optical projection tomography

    Custom assembly based on Wong et al, 2013.

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Neurobiology and Behavior

Head: Agnieszka Kubik-Zahorodna Ph.D.

Neurobehavioural tests using transgenic animal models make it possible to understand genetic mechanisms underlying neurological and psychiatric disorders including, but not limited to, anxiety, schizophrenia, mood disorders, and Parkinson’s disease. We employ a number of tests to examine motor abilities, cognitive functions, emotion, sensory processing as well as neurological, gait, auditory, and vision impairments in transgenic mice.

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    Animal emotionality and affect

    Open Field test evaluates animal overall motility triggered by exploratory drive in a new environment. It is also used as an initial screen for general anxiety elicited in a well-lit, open unprotected space. This fully automated test is based on video tracking system.

    Elevated Plus Maze, Light/Dark Box are other tests used to evaluate animal general anxiety which are based on approach-avoid conflict. In both tests, animals are motivated to explore a new environment in search for potential food, shelter, or mating opportunities, at the same time avoiding well-lit unprotected areas (open arms, light compartment), in favour of safer ones (closed arms or dark compartment). Both tests are fully automated and based on video tracking system.

    Novelty Induced Hypophagia (NIH) test measures suppression of food intake in response to exposure to a novel anxiogenic environment. NIH is one of the few animal tests of anxiety that are responsive to chronic antidepressant treatment, which reflects time window of therapeutic antiderpressive drugs’ efficacy in clinic. That is a reason why NIH serves as excellent model to study neural mechanisms of the antidepressant response on anxiety -related behaviour.

    Forced Swim Test (FST) and Tail Suspension Test (TST) both measure animal despair in un-escapable situation and are the most widely used tools in animal models of depression. Although TST and FST share a common theoretical basis, there are many differences between them. Therefore they could complement each other providing reliable unsophisticated screen of depressive-like phenotype.

    Social Defeat (SD) paradigm is the most frequently used ethologically relevant model of chronic stress. Repeated exposure to social defeat stress results in robust depression-like phenotype manifested in anhedonia, anxiety, and social-avoidance behaviours. SD therefore is an excellent substrate for studding the neurobiological basis of stress-related affective disorders.

    Cognitive functions

    Cued and Contextual Conditioning are based on classical Pavlovian fear conditioning, an associative learning test routinely used to study biological basis of fear, learning, and memory. Contextual, but not cued, fear conditioning is regarded as hippocampus-dependent. Although this statement is true in most of experimental designs, animals can compensate for hippocampal damage. To overcome this limitation, we also employed Context Discrimination test where intact hippocampus is critical.

    Barnes Maze test uses natural tendency of animal to avoid well-lit, open, unprotected spaces and it is applied for testing spatial learning and memory. Test is fully automated and based on video tracking system.

    Novel Object Recognition (NOR) is based on natural preference for novelty in rodents. It evaluates animal’s exploration of a novel object as a measure of working memory and attention. NOR is particularly attractive because it requires no additional appetitive or aversive reinforcement and minimum habituation and training. NOR task can be used to study short-term memory, intermediate-term memory, and long-term memory via manipulation of the retention interval.

    Spontaneous Alternation. Rodents show strong tendency to alternation between arm choices on successive trials in Y-maze. Spontaneous alternation test is a robust and quick test of exploratory behaviour and spatial working memory. Test also requires no extensive training or external reinforcement.

    Neuromotor abilities

    Beam Walking and Rotarod. Animal sense of balance and motor coordination can be evaluated in Beam Walk test and Rotarod, which also allows assessing motor learning abilities.

    Grip Strength measures maximal muscle strength of forelimbs and hind limbs on an automated grip strength meter. The test can indicate neuromuscular abnormalities.

    Gait Analysis is based on a fully automated analysis of video records of animal foot prints. Gait analysis provides not only information about motor coordination but also detailed kinematic description of animal gait. The measured parameters are animal pace, speed, foot print area/length, a number of distinct contact area, toe spread, gate angle, paw pressure, body-foot spacing, and many others. The test can be used to studding models of Amyotrophic Lateral Sclerosis (ALS), pain/arthritis, Parkinson’s disease, muscle injury model or spinal cord injury.

    Sensorimotor gating

    Acoustic Startle Acoustic Startle Reflex (SR) is an automated analysis of startle reflex in response to acoustic stimuli. The test assesses sensorimotor processing by measuring both afferent sensory information transmission and efferent motor response. The test can also serve as a primary screen for hearing impairment. The lack of sufficient sensory gating mechanism is thought to lead to an overflow of the sensory stimulation and disintegration of the cognitive functions. SR paradigm is therefore largely used to assess the effects of putative anti-psychotics and to explore possible genetic and neurobiological mechanisms of psychosis-related behaviour.

    PPI Prepulse Inhibition (PPI) is attenuation of startle response magnitude by pre-exposure to non-startling stimulus. PPI provides operational measurement of sensorimotor gating reflecting the ability of an animal to successfully integrate relevant and inhibit irrelevant sensory information. Impaired PPI is observed in schizophrenia as well as in other neuropsychiatric disorders. 

    Pain sensitivity

    Hot/Cold Plate is automated measurement of the latency for paw licking or the first observed response, e.g. jump, in response to heat or noxious cold stimulus. The response in the hot/cold-plate test is supraspinal. The modified hot/cold-plate test with dynamic plates (temperature is slowly increased/decreased from non-noxious to noxious levels) allows to measure thermal allodynia.

    Tail Flick is automated measurement of time for tail flick reflex following the exposure to a heat stimulus (IR heat beam). It is an easy and quick test to assess rodent nociception.  The Tail Flick reflex belongs predominantly to the spinal reflexes.

    Plethysmometer measures inflammatory oedema in the animal paw. It is applied to research on rheumatoid arthritis or the central development of oedema.

    von Frey Test uses locally applied blunt ended filament to animal plantar area of paw until paw withdrawal/filament bend. This is mechanical test derived from clinical procedure to assess mechanical allodynia.

    IntelliCage

    IntelliCage is state-of-the-art equipment that allows studying animal’s cognitive processes (aversive/appetitive conditioning, memory, taste aversion and many others) and activity in social group home cage housed animals. During an experiment (days) animals are not disrupted by human presence with sole exception of bedding change when necessary. The equipment allows automated cognitive and behavioural screening of animals living in social groups with minimum experimenter contact.

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Immunology


Head: Milan Reinis, PhD

The Immunology unit is taking part in the revealing of the etiopathogenic mechanisms of the immunological diseases using the transgenic mouse model. Generally, immunological disorders comprise the immune deficiencies, allergies, inflammatory diseases and cancerogenesis as well. The immune system functions are reflected in changes of the immune cell subpopulations and their products as cytokines, chemokines and other similar substances. The pathological changes can be registered in the afflicted organs, in responsible immune organs as well as in blood. We are able to evaluate the immune system functions by use of various immunological methods.

Metabolism

Head: Karel Chalupsky, PhD,  Advisor: Ondrej Seda, MD, PhD

Cardiovascular function

Head: Benoit Piavaux, PhD.

The cardiovascular screen performs assessments of structural and functional characteristics of the cardiovascular system and screens for the presence of pathologies in various mouse and rat models. State of the art equipment allows us to perform these measurements in conscious mice and rats in a high throughput manner, which is the optimal setting for screening of animal mutant lines generated within the IMPC consortium. To further cardiovascular characterization we perform more detailed measurements which do not allow for a high throughput format.

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    Primary screening
    Transthoracic echocardiography in conscious non-sedated mice or anaesthetized rats:

    • morphological parameters: systolic and diastolic left ventricular interior diameter, interventricular septum and left ventricular posterior wall thickness, gross cardiac and aortic arch abnormalities assessment
    • functional parameters: respiration rate, heart rate, ejection fraction, fractional shortening

    Electrocardiography in conscious unrestrained mice and rats

    • assessment of rhythm abnormalities using classical 3 lead acquisition

     

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Lung function

Head: Benoit Piavaux, PhD.

At the lung-function unit we aim to offer complete and custom services ranging from screening of lung-function phenotypes in naïve (transgenic) animals to the complete screening of lung-pathophysiology in models of pulmonary diseases.

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    Assessment of lung-mechanics without challenge

    For the full assessment of lung mechanics without challenge, mice will be anesthetized, tracheotomised (end-point measurement) or intubated (repeated measurement) and attached to Scireq’s Flexivent FX and ventilated. We consecutively apply a series of different perturbations. The response from these perturbations is then analyzed by the software to calculate the lung mechanical parameters as described below. Measures are repeated until at least 4 valid measurements are obtained for every perturbation.

    The measurements with tracheotomy give more accurate results, compared to the results obtained with intubation, because leakage of air can be much better controlled (ligature around the trachea) and shorter tubes (less resistance) can be used with a tracheotomy. However, as the trachea can’t be closed after tracheotomy, this measurement is an end-point measurement. Measurements with intubation can be performed more than once in the same mouse.

    Deep-Inflation. During this perturbation lungs are inflated until the tracheal pressure reaches 30 cm H2O. Inspirational capacity, the volume of air which can enter the lungs, can be calculated from this pertubation. This parameter can be affected by any pathology which reduces the size of the airway lumen (e.g. airway wall thickening or edema) but also by parameters affecting the elasticity of the lungs (e.g. emphysema or fibrosis). Also the weight of the mouse has an influence on this parameter.

    SnapShot. The SnapShot is a sinusoidal perturbation of the airway pressure. The response of this perturbation is fit to the very basic ‘single compartment model’ of the lungs and airways. In this model the lungs are represented as an elastic balloon, giving the compliance, while the airways are represented as a tube, which confers resistance to the air going in and out of the lungs. The following parameters are calculated from this model: C: Dynamic compliance: a measure for the elasticity of the lung tissue. This will be typically affected in diseases like emphysema or fibrosis. E: Dynamic elastance: The elastance is the inverse of the compliance (E = 1/C) and thus represents the same phenomenon as the dynamic compliance. R: Resistance: a measure for the resistance the air encounters to enter and exit the lungs, which is typically increased in cases of airway narrowing and bronchoconstriction, typical landmarks for asthma.

    Quick-Prime-3 and Prime-8. Both perturbations are multi-frequency perturbations. The Quick-prime-3 takes 3 seconds while the prime-8 takes 8. As the prime-8 perturbation is longer it allows for a more accurate determination of the lung mechanics parameters. However in mice with diseased lungs and thus reduced oxygen exchange in the lungs this perturbation can be too long, causing a drop in oxygen in the lungs and consequent breathing reflexes of the animal. In these animals the Quick-prime-3 perturbation is a better choice as the oxygen levels will not drop as severely. The response to these perturbations is fit to the ‘constant phase’ model of the lungs. The advantage of this model is that one can make a difference between the resistance encountered in the central large airways and in the lung tissue (small airways and alveoli). This is interesting in disease where the central airway is not particularly affected while the small airways are, like in case of emphysema and fibrosis. The following parameters are calculated: Rn: Newtonian resistance: This parameter represents the resistance the air encounters in the central airways. Tissue damping (G): The tissue damping is a measure for the air resistance in the small airways and alveoli. Tissue elastance (H): The tissue elastance is closely related to the dynamic elastance calculated in the ‘single compartment model’ from the SnapShot perturbations and is also a measure for the elasticity of the lung and is thus influenced by the same disorders as the dynamic elastance as emphysema and fibrosis.

    PV-Loops. For the pressure-volume (PV) loops the lungs are inflated in small steps of 4 cm H2O and then left in that ventilator position for 1 second before the next step until 30 cm H2O is reached after which they are deflated in the same small steps. The advantage is that, because of the slow maneuver, lungs can reach a state of quasi static equilibrium. The compliance measured this way is thus a better measure of the true compliance of the lung. However the question is if this measure is truly very relevant, as the lung will not reach this state of quasi static equilibrium in normal breathing. Due to its slow nature, PV-loops can also help to characterize lungs in fibrosis models, in which the response is usually slowed down so much that they can’t be characterized accurately with other perturbations which are relatively fast and dynamic. PV-loops can also be helpful for translational research as they are performed in humans under ventilation. We only use this perturbation to calculate the static compliance. Full analysis of these curves is not included in the standard phenotyping but can be performed upon user’s request or the curves can be sent to the customer so that they can analyze them themselves. The following parameter is obtained from PV-loops: Cst: Static compliance: As explained above this is a measure for the elasticity of the lung in a quasi-static equilibrium.

    Assessment of lung mechanics with challenge

    As for the full assessment of lung-function without challenge, we can perform this measurement as end-point measurement, with tracheotomy, or as repeated measures, with intubation. The challenge is given by aerosol which is generated in-line with the ventilation.

    The choice between end-point and repeated measures is more critical for these experiments, as administration of the challenge compound (typically methacholine), will cause increased pressure in the lungs during ventilation and during the perturbations. To maintain integrity of the lungs and insure that we have no pressure leakage from the tube we will have to stop the registration of the dose response curves once the pressure reaches 30 cm H2O when using intubation in a survival measurement. This should not be an issue for baseline measurements in which we do not expect the pressure to increase a lot, but could be a problem if airway hyper-reactivity is already established. In case of doubts, please feel free to contact us, so that we can establish the best strategy to suit your needs.

    With the challenge one is usually interested in the peak response after each dose of methacholine. Therefore we can only use 1 type of short perturbation to measure the response, which means that the following options are available:

    SnapShots. Is the shortest perturbation we can apply and thus the best choice to characterize the peak response. However the amplitude of the SnapShot is relatively high and we can’t limit the pressure during the perturbation. We can reduce the amplitude in the higher doses, to avoid reaching the 30 cm H2O pressure limit. However this will require an additional mouse to find the right amplitude. The parameters we can measure from these perturbations are the parameters from the ‘single compartment model’ R, E and C as described above.

    Quick-Prime-3. This perturbation is slightly longer, but the maximal amplitude of the input signal is much lower, which means that we can go to higher doses of methacholine without reaching the 30 cm H2O lung integrity pressure limit. As we are using the prime wave we can measure the parameters from the ‘constant phase model’: Rn, G and H; but also the parameters from the ‘single compartment model’: R, E and C.

    Mouse Models of Pulmonary Diseases.

    Asthma Models.

    Severe OVA-Induced Asthma. This is model is one of the oldest models of asthma which gives rise to a severe form of allergic asthma in the mouse with a strong Th2 type response due to the use of alum during the sensitization phase. A schematic representation of the model is shown in figure 1. Considering the robustness of the model and the fact that it is one of the best characterized models of asthma, it is a good choice for many studies. Its weaknesses are the exaggerated Th2 response and the lack of extensive airway remodeling. Also, in some cases, the phenotype could be too strong to see effects of interventions on the model.

    Lung1

    Figure 1: Schematic representation of the severe OVA-induced asthma model as used by the Czech Centre of Phenogenomics.

    Mild OVA-Induced Asthma. This model, which is also driven by OVA as model allergen, gives rise to a much milder form of allergic asthma, with much milder Th2 inflammation and less severe development of airway hyper-reactivity. A schematic representation of the protocol is shown in figure 2. This model can be useful in cases that the severe model is too strong, due to high sensitivity of the used mouse strain for example. As for the severe model its Th2 response is still exaggerated and there is a lack of extensive airway remodeling.

    Lung2

    Figure 2: Schematic representation of the mild OVA-induced asthma model as used by the Czech Centre of Phenogenomics.

    Short House Dust Mite Model of Asthma. This model uses a natural aero-allergen which also causes asthma in humans and animals. It is generally milder than the OVA driven models but the phenotype more closely mimics the pathology of allergic asthma as observed in human asthma, with a much milder Th2 inflammatory component and a more extensive airway remodeling. The protocol is shown in Figure 3.

    Figure 3: Schematic representation of the short house dust mite asthma model as used by the Czech Centre of Phenogenomics.

    Long House Dust Mite Model of Asthma. This model induces a more severe asthma phenotype compared to the short house dust mite model. The airway remodeling is more extensive, so it is a good choice if airway remodeling is your area of interest. A schematic overview is shown in Figure 4.

    Lung4

    Figure 4: Schematic representation of the long house dust mite asthma model as used by the Czech Centre of Phenogenomics.

    Emphysema Model

    Elastase-Induced Model of Emphysema. The elastase model of emphysema is a fast and simple protocol to establish emphysema. It consists of just 1 oro-pharyngal instillation of elastase after which progressive emphysema is established. Despite the fact that it has a completely different etiology as the natural disease in humans it has shown to be a powerful tool to study the development of the disease once it is established. A mild form of emphysema can already be observed 3 days after the administration of elastase and will progress to GOLD stage 2 within 3 weeks of elastase treatment.

    Pulmonary Fibrosis Model

    Bleomycin-Induced Model of Pulmonary Fibrosis. The bleomycin model of pulmonary fibrosis is one of the most used pulmonary fibrosis models in rodents. As for the emphysema model it consists of only 1 oro-pharyngal application of the compound to establish the disease. A significant fibrosis can be observed 3 weeks after administration of Bleomycin. In contrast to the emphysema model this model is not a true chronic and progressive model and some recovery from fibrosis is observed in the mouse after this 3 week period.

    Acute Lung Injury.

    LPS-Induced Model of Acute Lung Injury. Acute lung injury is a common problem in intensive care patients under ventilation, as the mechanical stress of the ventilation exacerbates mild pulmonary infections to a level in which they become a serious health issue for the patient. We are not equipped for infectious studies, but acute lung injury can be induced in mice by instillation of LPS. It will cause an acute lung injury like disease, which is strongest 24 h after LPS administration and resolves within 1 week.

     Complementary Phenotyping Services

    Histopathology. Tissues from subject to lung function assessments can be fixed, processed, embedded, sectioned and stained upon request.

    Serum IgE levels can be determined using ELISA.

    Immune Cell Typing. A standard FACS procedure using 2 cocktails of antibodies allows differentiation between: Total T cells, αβ T cells, CD4+ αβ T cells, CD8+ αβ T cells, γδ T cells, NKT cells, NK cells, B cells, monocytes, granulocytes, eosinophils, CD4+ CD25+ regulatory T cells, CD4+ CD44hi CD62Llo T cells, CD4+ KLRG1+ T cells, CD8+ CD44hi CD62Llo T cells, CD8+ KLRG1+ T cells, KLRG1+ NK cells, IgD+ B cells, Ly6Chi I-A/I-Elo monocytes and Ly6Clo I-A/I-Elo monocytes.

    Inflammatory Cytokines. Multiplex bead assay can be used to accurately measure the following inflammatory cytokines:

    Panel I: IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17a, Eotaxin, G-CSF, GM-CSF, IFNg, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1a, MIP-1b, MIP-2, RANTES, TNFa, VEGF.

    Panel II: IL-16, IL-21, IL-22, IL-25/IL-17, IL-28B, EPO, Exodus-2, Fractalkine, MCP-5, MIP-3a, MIP-3b, TARC

    Panel III: IL-20, IL-23, Il-27, IL-33, MDC, TIMP-1

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    Lung Function in Mice

    Requests for studies relating to lung function in mice.

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Vision

Head: Barbora Antosova, MSc,  Advisor: Zbynek Kozmik, PhD

The Vision Unit is dedicated to the detection of various abnormalities in mouse vision. The current equipment of the unit enables us to analyze abnormalities in eye morphology and eye physiology.

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    Eye Screen

    Optical Coherence Tomography. This non-invasive imaging procedure is used to examine the posterior part of the eye (retina and retinal blood vessels). An anaesthetised animal is placed on a platform and the spectral domain OCT, integrated with confocal scanning laser ophthalmoscopy (cSLO), is used to produce a detailed cross-sectional image of the retina and retinal blood vessels. This approach enables us to detect and analyze a wide range of mouse retinal pathologies, including changes in retinal thickness and layering, and in retinal vessel number or localisation. It is also possible to assess dynamic processes like edema formation or retinal degeneration.

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    Virtual vision test. This behaviour test provides a non-invasive functional analysis of visual performance in mice. The OptoMotry© system uses the tracking of optokinetic head and neck movements, that are reflexive in the mouse for the screening of functional vision. In this test, a mouse stands on an elevated platform in the epicenter of an arena surrounded by computer monitors, and a camera images the behavior of the animal from above. With the changing of threshold of spatial frequency, contrast, and motion of the grating, we are able to determine the visual acquity („clarity of vision“) of the tested animal. The advantage of this test is that animals with no previous exposure to the task can be tested and the measurements can be repeated regularly. This method can provide a powerful test of visual performance in gentically modified and pharmacologically treated mice.

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    Electroretinography. The electroretinogram (ERG) is a non-invasive diagnostic test to evaluate the function of various retinal cell populations in response to a light stimulus. An anaesthetised animal is placed on a heated table and ERGs are obtained using active electrodes placed on the cornea, a reference electrode in the mouth, and a ground needle electrode placed subcutaneously at the basis of the tail. The ERG can provide important diagnostic information on a variety of retinal disorders, and can also be used to monitor disease progression.

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    Figure5

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    Histology. Standard histological staining with hematoxylin and eosin (H&E stain) can be performed on paraffin or frozen sections of embryonic or adult mouse eyes. This primary histological staining provides basic information about eye morphology.

    Immunohistochemistry. Immunofluorescent (immunohistochemical) detection of specific lens and retina markers can be performed on paraffin or frozen sections of embryonic or adult mouse eyes. Analysis of specific lens or retinal markers can provide more detailed information about cell types present in these tissues and extend findings from H&E staining.

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    Cerebral Mechanics OptoMotry

    Virtual reality system for rapidly quantifying a variety of OKT (optokinetic tracking) thresholds in untrained and unrestrained small rodents.

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    Electroretinography apparatus

    Heidelberg Engineering Spectralis

    Multimodal imaging platform for spectral domain optical coherence tomography

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Hearing

Head: Jiri Lindrovsky, PhD,  Advisor: Prof. MUDr. Josef Syka, DrSc.

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    Auditory Function

    Auditory Brainstem Response (ABR). To assess the lowest intensity of sound perceived by an animal the hearing thresholds are measured. An animal in ketamine-xylazine anesthesia is stimulated by simple clicks or by pure tones of various intensity and frequency while electrophysiological response is being recorded via a needle electrode placed subdermally at the top of the animal’s head. The signal of obtained Auditory Brainstem Responses (ABR) represents a sum of activity of brainstem neurons responsible for sound processing starting at the level of auditory nerve, through cochlear nucleus and superior olivary complex, up to the inferior colliculus.

    Acoustic Startle Reflex (ASR) and Prepulse Inhibition (PPI). Startle reflex is a skeletal muscle twitch in response to an unexpected stimulus. In case of the acoustic startle reflex (ASR) the startle reaction is elicited by a short pulse of white noise or a short tone that must exceed certain intensity, around 60 – 80 dB SPL, in order to be efficient. Reflexive body movement of the animal is registered by a sensitive plate of an accelerometer on which the cage with animal stands during the experiment. The ASR amplitude and latency reflect the processing of higher sound intensities, however, the ASR can be significantly suppressed if a weak sound stimulus is played shortly (tens of miliseconds) before the startling pulse. This effect is known as prepulse inhibition (PPI) and allows testing more delicate and variable sound stimuli then ASR alone. The advantage of ASR + PPI method over other behavioral hearing tests is its time efficiency, since it does not require any training of the animals.

    Distortion Product Otoacoustic Emissions. Proper function of cochlea, specifically the cochlear outer hair cells, can be examined by recording of otoacoustic emissions (OAE). OAE are faint sounds emitted from the cochlea as a response to sound stimulation or spontaneously and they can be registered by a sensitive microphone placed in the ear canal. OAE are produced by outer hair cells which can actively vibrate and improve this way the frequency selectivity and sensitivity to low intensity sounds by approximately 50 dB SPL. If two tones are applied simultaneously to a healthy ear, series of difference tones arises as a result of nonlinear cochlear processing and these can be recorded as distortion product otoacoustic emissions (DPOAE). Usually the frequency of the two stimulation tones is related as f2=1.2f1 and only the most intensive distortion product of frequency fDPOAE=2f1-f2 is recorded. The method is non-invasive, however requires general anesthesia in animals because the stimulation-recording probe must not change its position in the ear during the whole recording cycle.

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