Disease Models for Comparative Anatomy Flashcards

Question

What are the cons of zebrafish as a model of cardiovascular disease?

Answer 1

Only a prototypical heart Small - functional studies challenging Behaviour less complicated Less reliable for insoluble drugs

Answer 2

Vasculogenesis and Angiogenesis Followed by remodelling and maturation of the initial vascular plexus into adult vasculature

Answer 3

The efficient transport of cells, molecules, nutrients and oxygen to peripheral tissues, and the removal of CO2 and waste

Answer 4

The formation of new blood cells from existing ones Has a role in pathology (cancer, angiogenesis is key for tumour growth and metastasis, eye disease, diabetic retinopathy etc)

Answer 5

Intussusceptive angiogenesis Sprouting angiogenesis

Answer 6

A vessel is split in two by a column of cells that separate the vessel in two length-wise

Answer 7

A tip cell sense a chemoattractive gradient and degrade the basal membrane on which it sits to reach towards that gradient Followed by stalk cells which elongate after and ultimately, the vessel becomes stabilised with the addition of pericytes for final maturation.

Answer 8

Within 2-3dpf, all major organs have been laid out, the major axial trunk vessels have been formed (vasculogenesis) and the heart is beating Up to 3 weeks of age, embryos (up to 5 days) and larvae (up to 30 days) are transparent, early pigmentation can be inhibited by adding a melanogenesis inhibitor to the water to preserve full transparency

Answer 9

Small size Passive diffusion distance of oxygen very small Embryos can survive for several days even when blood flow is disrupted or when vessels are dysfunctional

Answer 10

Zebrafish genome contains orthologs for most human genes, although because of zebrafish genome duplication, paralog pairs are present for a subset of the zebrafish genes Often the paralogs each cover part of the functions of their human ortholog This can complicate genetic studies if both paralogs have to be manipulated

Answer 11

around 70%

Answer 12

Confocal microangiography and in situ hybridisation on whole-mount zebrafish embryos

Answer 13

Invasive technique and visualises only perfused vessels

Answer 14

Transgenic reporter lines with fluorescent protein expression specifically in vascular ECs Time-lapse confocal microscopy Availability of reporter lines distinguishing between endothelial subtypes (arterial, venous, lymphatic) and endothelial subcellular structures (e.g. cytoplasmic versus nuclear or membrane-restricted)

Answer 15

The result of the injection of a contrast medium into either the blood or the lymphatic system, the enlargement of the resulting radiograph

Answer 16

Intercrosses and analysis of different vascular cell types by flow cytometry or fluorescence-activated cell sorting for further characterisation of isolated cells

Answer 17

ETS transcription factor (E26) of the ERG family that is expressed by endothelial cells (artery, vein and lymphatics ECs)

Answer 18

Originate in the posterior lateral plate mesoderm and migrate from 14 hours after fertilisation onward in 2 waves between somites and endoderm towards the midline immediately ventral to the hypochord They coalesce there to form a primordial vascular cord as a precursor of the dorsal artery and posterior cardinal vein.

Answer 19

Specialised - specified to arterial or venous cell fate at the lateral plate mesoderm

Answer 20

In the primitive vascular cord They segregate Vascular lumen formation occurs concomitantly with segregation

Answer 21

Expression analysis by in situ hybridisation for arterial markers like Notch1b, DII4, Tbx20 (hrt), gridlock and ephrinb2 and venous markers like DAB2, Ephb4, and VEGFR3 at the level of the dorsal artery and posterior cardinal vein

Answer 22

Imaging of dorsal artery/posterior cardinal vein formation and segregation via fluorescent microscopy of whole-mount reporter-line embryos or of their transverse sections

Answer 23

Establishment of the major axial vessels - Dorsal artery and posterior cardinal vein

Answer 24

Dorsal artery Posterior cardinal vein

Answer 25

Sprouting angiogenesis Bilateral sprouts emerge from the dorsal artery Migratory tip cell extend Long filopodia scanning the environment for guidance cues, grow dorsally along the intersomitic boundaries Follower proliferative stalk cells elongate the sprout and eventually form the vascular lumen

Answer 26

Primary intersomitic vessels (ISVs)

Answer 27

Intersomitic sprouts from the posterior cardinal vein Half form the trunk lymphatic vasculature Half becomes venous intersomitic vessels (ISVS), connecting to the primary ISVS

Answer 28

Tip cells connect and anastomose with the tip cell of their neighbour ISVs forming DLAV (dorsal longitudinal anastomotic vessel)

Answer 29

The tip cell position via alteration of the relative expression levels of tip or stalk cell genes

Answer 30

At 48-72hpf Helps to visually establish age of embryos

Answer 31

Within 48hpf Ex-vivo can observe it under microscope!

Answer 32

A) At 5 h post fertilization (hpf) the blastula (white) covers approximately 50% of the large yolk cell (yellow). At this stage, cardiac progenitor cells are located bilaterally in the lateral marginal zone. Atrial progenitor cells (pink) are located more ventrally than the ventricle progenitor cells (light blue). During gastrulation, the cardiac progenitor cells move dorsally towards the mid-line to end up in the anterior later plate mesoderm (ALPM). Cardiogenic differentiation is initiated in the future ventricle myocardial cells by the expression of cardiac myosins (purple) at the 12-somite stage. During mid- and late-somite stages, the myocardial tissue expands by continuous cardiogenic differentiation into more lateral regions of the ALPM by the cardiogenic differentiation of future atrial myocytes (orange; venous differentiation). Whilst the endocardial cells (light green) have already migrated from the ALPM towards the mid-line, myocardial cells follow this behaviour slightly later. When the bilateral heart fields fuse at the mid-line, they form a cardiac disc structure with the endocardial cells within the hole at the centre, ventricular myocytes at the circumference and atrial myocytes at the periphery of the disc (D). Cardiac morphogenesis transforms the cardiac disc into a cardiac tube. The endocardium forms the inner lining of the myocardial tube. (E) At 28 hpf, the linear heart tube has formed, with the venous pole located at the anterior left and the arterial pole fixed at the mid-line. (F) Cardiogenic differentiation continues at the arterial pole, and as a result new cardiomyocytes are added to this region (purple gradient). At 36 hpf, cardiac looping has started, with a displacement of the ventricle towards the mid-line, and the constriction at the position of the AV canal is first visible (F). The heart tube continuous to loop and forms an S-shaped loop (G). Ellipsoid extra-cardiac pro-epicardial cells (brown) are located near the AV canal (yellow), from where they start to cover the myocardium with an epicardial layer. The pacemaker is present in the inner curvature of the atrium near the venous pole (dark green).

Answer 33

Serial circulation system Heart with a single atrial and ventricle Pumps deoxygenated blood from the body towards the gills for reoxygenation Ventricular muscle is very trabeculated rather than compact (as seen in mammalian heart): better oxygenation deeper inside the muscle?

Answer 34

Upon myocardial infarction (MI) in adult humans, cardiomyocytes die and are replaced by fibrotic tissue which matures into an irreversible scar. In a MI model in the zebrafish (cryoinjury, CI) a transient scar is formed followed by complete regeneration This can also be seen in neonatal mice if the injury is performed during the first week of life

Answer 35

Resection - 60 day recovery - 20% of ventricle is damaged Genetic Ablation Cmlc2:cre X + tamoxifen - 30 day recovery - 60% ventricle is damaged Cryoinjury - <130 day recovery - 25-30% ventricle is damaged

Answer 36

Hematoxylin and eosin stain of the intact zebrafish heart before (A) and after about 20% ventricular resection (B) (5). b.a., bulbous arteriosus. (C) An intact ventricular apex at higher magnification, indicating the approximate amputation plane (dashed line). All images in this and subsequent figures display longitudinal ventricular sections of the amputation plane. (D) 1 dpa. The large clot is filled with nucleated erythrocytes (arrowheads). (E) 9 dpa. The heart section is stained for the presence of myosin heavy chain to identify cardiac muscle (brown) and with aniline blue to identify fibrin (blue) (5). The apex is sealed with a large amount of mature fibrin. (F) 14 dpa. The fibrin has diminished, and the heart muscle has reconstituted. (G) 30 dpa. A new cardiac wall has been created, and only a small amount of internal fibrin remains (arrowhead). (H) 60 dpa. This ventricle shows no sign of injury. (I) Quantification of healing at 0, 30, and 60 dpa. Values represent the size of the largest ventricular section (mean ± SEM; *P < 0.05); parentheses indicate the number of hearts examined (5). Scale bars, 100 μm

Answer 37

Schematic representation of transgenes used for zebrafish cardiomyocyte ablation. DTA is specifically expressed in cardiomyocytes upon 4-HT treatment. (B) TUNEL staining of ventricular muscle from transgenic animals injected with vehicle or 4-HT (0.5 mg/ml). Arrowheads indicate TUNEL-positive cardiac myocytes. (C) Myosin heavy chain (MHC) staining of ventricular sections after vehicle or 4-HT injection, shown at 7 days post-injection (dpi). (D) Mef2 staining to indicate cardiomyocyte nuclei, showing major cellular losses in 4-HT-injected animals. Scale bars: 50 μm.

Answer 38

Muscle: Light brown/orange Fibrin (blood clot): Red Collagen (scar): Blue

Answer 39

Inflammatory phase Reparative phase Regenerative phase

Answer 40

0-3 days following cardiac damage Leukocytes clear cellular debris Fibroblast-like cells

Answer 41

4-7 days after cardiac damage Collagen-rich matrix Epicardium proliferates Revascularisation

Answer 42

7-130 days post cardiac damage Cardiomyocytes proliferate Scar resorption Cardiac function restored

Answer 43

Dedifferentiate and proliferate

Answer 44

Epicardium to Mesenchymal Produces - retinoic acid - PDGF - VEGF - Extracellular matrix deposition

Answer 45

A critical transcription factor for proper mammalian cardiac development and essential for survival of the embryo.

Answer 46

gata4+ Cardiomyocytes

Answer 47

The epicardium forms a thickened layer covering the injured heart, a process involving epithelial-to-mesenchymal transition (EMT) of epicardial cells driven by FGF and PDGF

Answer 48

= Important source of progenitor cells for the perivascular cells of the coronary vasculature and intracardiac fibroblasts, = important for the proliferation, maturation and compaction of the underlying myocardium (secretion of cytokines, Retinoic acid, PDGF Similar to Myocardium, Epicardium re-express embryonic genes (wt1b, tbx18, tcf21) = Synthesis of extracellular matrix, providing scaffold for the proliferating cardiomyocytes.

Answer 49

Nuclear dynamics and proliferative capacity of cardiomyocytes during growth. Cardiomyocytes in fetal humans and mice typically have a single nucleus with a diploid genome (2n) and increase in mass through cell division (blue arrows). Human cardiomyocytes can proliferate for the first few months after birth but are believed to lose this capacity early in life. These cells typically undergo rounds of DNA replication without karyokinesis or cytokinesis, which result largely in mononucleated cardiomyocytes with tetraploid (4n) or higher DNA content. Murine cardiomyocytes can divide robustly until the first few days after birth, after which most withdraw from the cell cycle. These cells undergo additional DNA replication with karyokinesis but not cytokinesis, which results in binucleated cardiomyocytes that are diploid (2n) in each nucleus By contrast, most cardiomyocytes in zebrafish hearts are mononucleated with a diploid genome (2n) throughout life and have significant proliferative capacity

Answer 50

Fin fully regenerates 14 days after amputation Arteries are formed by vein-derived endothelial tip cells

Answer 51

Pros: - Transparent - Heart ex vivo - Beating heart - Orthologs Cons: - standard reagents and methods lacking - genome duplication

Answer 52

A fatal X-linked recessive muscle wasting disorder Progressive necrosis and fibrosis of muscle leads to loss of function and loss of ambulation by 12 CNS, Cardiac and Smooth muscle are also affected

Answer 53

Loss of dystrophin, which renders the muscle vulnerable to activity-induced damage

Answer 54

1 in 3500 to 5000 male births

Answer 55

Large deletions and duplications - majority Splice site mutations Small deletions and insertions Nonsense mutations - slight majority after large deletions and duplications Missense mutations - least common

Answer 56

Disrupt the open reading frame leading to a failure to fully translate the mRNA and produce a functional protein. Most mutations are de novo

Answer 57

Absence of dystrophin Activity induced damage Influx of calcium Hypercontraction mitochondrial damage and activation of proteases - Oxydative stress and Nitrosylation - Loss of strength Necrosis Inflammation and fibrosis - Oxidative stress and nitrosylation - Loss of strength - Failure of regeneration or Absence of dystrophin Loss of cell signalling Oxidative stress and nitrosylation Loss of strength

Answer 58

Respiratory assistance with positive pressure ventilation Appropriate drug treatment of any developing cardiomyopathy and potential prevention with pre-symptomatic treatment Corticosteroids can slow the progress of Duchenne's Muscular Dystrophy, maintain ambulation beyond 12, reduce scoliosis and maintain FVC However, progressive decline in muscle function reduces independence, so the goal of experimental therapies is to halt or reverse this progressive decline

Answer 59

Most boys died by 17 years old

Answer 60

Steroids introduced, increased longevity somewhat

Answer 61

Start of non-invasive positive pressure ventilation. Median survival 22. With spinal surgery for scoliosis, this raised to 30

Answer 62

Average is late 20s to early 30s but some patients are >40 years old

Answer 63

Paradoxical effect in DMD patients Differences between non-dystrophic and dystrophic in terms of muscle loss Side effects - weight gain, small stature, delayed puberty, mood changes, bone loss, Cushingoid appearance Various different regimes have been used. Reveragen is developing an alternative non-steroidal steroid

Answer 64

Animal models are a living, non-human animal used in the investigation of pathophysiology and development of treatments for human disease

Answer 65

Mirror the signs of human disease (phenotypic) or Be genetically similar (genotypic) And ideally should be both

Answer 66

Often relies on computer models and cell culture systems in the initial phases of drug development Play an important role in demonstrating whole body proof of concept of drug action and helping the translation from experimental studies to human clinical trial Normal animals are also used for toxicology and safety pharmacology

Answer 67

Invertebrate models - Caenorhabditis elegans (nematode) - Drosophila Vertebrate models - Zebrafish - Mammals including mice, rats, rabbits, cats, pigs and dogs

Answer 68

Mdx mouse GRMD dog DE50-MD dog

Answer 69

Mdx mouse - on C57BI10 or DBA/2J backgrounds Mdx2cv, mdx3cv, mdx4cv, mdx5cv on C57BI6 background Mdx52 Mdx/Utr-/- Mdx4cv/mTR-/- Mdx/Cmah-/-

Answer 70

Progressive accumulation of muscle fibrosis Pronounced, persistent inflammatory response Robust, ongoing muscle regeneration 'Peak' inflammatory/ degeneration phase: 6-9 months Highly sensitive to eccentric contraction-induced damage Exhibits characteristic changes in blood biomarkers Marked muscle atrophy via MRI N=6 per group permits direction of therapeutic responses >25% in most cases. Larger groups unnecessary 3-12 month data most statistically valuable, 12+ months unnecessary Muscle biopsy data can be reduced/refined, 4/8/12 vs 3/6/9/12

Answer 71

Costs involved in the breeding and care of the different models and generally proportional to size with dogs being very expensive In the mammalian models, smaller animals have fewer clinical signs. In dogs, the clinical signs are very obvious Therefore there is a trade off that generally means that mice are used to generate statistically robust experiments and dogs are used to confirm the mouse results and demonstrate reduction in clinical signs

Answer 72

While animal models can be very useful they do not necessarily predict all the possible responses when a drug is given to humans There are differences in immune system responses Some models show premature death, at birth or a bit later, which is not typically seen in duchenne's muscular dystrophy Animal models are useful to assess appropriate doses where dose ranging studies cannot really be done in humans, e.g. AAV gene therapy

Answer 73

DMD is a disease of bipeds (humans). Disease starts with the proximal muscles but eventually all muscles (except the extraocular muscles) are affected. Available mammalian disease models are quadrupeds, so the forces exerted around the pelvic girdle may be lower A large number of different dogs have been reported as affected by DMD and cover a large size range (from corgi to Rottweiler). Severity of the pathology appears proportional to body size. This is probably due to the square cube law where mass increases faster than surface area, thus putting more strain on the muscle membrane

Answer 74

Proof of concept studies - often dosed to get maximal effects, often via routes that would not be used in humans. Shows the potential of a novel treatment Translational studies - dosed at a level that translates to a possible human dose, via the route that would be used in man. Very important to have an excellent experimental design

Answer 75

A thorough lit review Experimental groups will include positive and negative control groups Animals will be randomly assigned to experimental groups Those people assaying the results will be blinded to group identity The number of animals in each group will be sufficient for the study to be statistically robust (power calculations) Statistical methods determined a priori, primary outcome measures defined Excellent records - e.g. who did what and when

Answer 76

Statistics!! Used to detect differences between treated and untreated animals BUT a statistically significant result does not directly translate to an effective therapy A statistically sig. result with a small effect size will not result in an effective therapy and experience shows that the effect size is almost always smaller in man compared to mouse

Answer 77

TREAT-NMD Advisory Committee for Therapeutics Drug dev. experts Provide guidance on the translation and development path of therapeutics programs in rare neuromuscular diseases with large unmet need

Answer 78

Inadequate randomisation and blinding Lack of reproducibility (poor records and poor reporting) Failure to confirm results in independent labs Poor understanding of the models Treatment by routes and doses that do not directly translate to man Drug approval depends on functional clinical benefit to the patients and not on one or more biomarkers Significance and effect size

Answer 79

Understand the patholophysiology of the human disease Can be used to develop and test novel therapies for DMD

Answer 80

The differences between them

Answer 81

That there is excellent experimental design and that effect size as well as the statistical significance should be used to select those therapies that advance to human clinical trial

Disease Models for Comparative Anatomy Flashcards

(106 cards)