CPPS 308 Final Notes PDF

CPPS 308 Notes for Final Lab #1: Introductions and Drug Dilutions Week 1 Assignments: Krebs Buffer and Serial Dilutions Documents that are in canvas but not on here: - Lab 1 outline - Dilutions and Solutions...

CPPS 308 Notes for Final Lab #1: Introductions and Drug Dilutions Week 1 Assignments: Krebs Buffer and Serial Dilutions Documents that are in canvas but not on here: - Lab 1 outline - Dilutions and Solutions - Pre-Lab 1 Dilutions and Solutions (with answers) Lab 1 Slides: Introduction to Research Ethics Learning Objectives 1. Describe the guiding principles of ethical use of animals in science 2. Describe the different types of animal models used in research 3. Describe the in vitro approaches/methods available for research, their advantages and limitations 4. Calculate & prepare a Kreb’s buffer 5. Understand how to perform serial dilutions Ethics of animal use in research and teaching - The use of animals for research, teaching and testing is a privilege, one that comes with important responsibilities: Ensuring that every animal is treated humanely and not subjected to unnecessary pain or distress Work within the accepted standards for experimental animal care Good experimental design, limiting number of animals, and selecting appropriate animal models all support the 3 R’s. Marshall Hall's Principles (Monamy 2000) key points : 1. No experiment should take place if the necessary information could be gained by · Don't do it if observation. you do not have to 2. Only experiments that would result in the fulfilment of clearly defined and attainable · Reduce repetition if aims ought to proceed. possible 3. Unnecessary repetition of an experiment must be avoided — particularly if reputable physiologists had been responsible for the original experiment. 4. All experiments must be conducted with a minimum of suffering. 5. All physiological experiments should be witnessed by peers, further reducing the need for repetition. CCAC: Canadian Council on Animal Care (1968) ↑ The CCAC’s system of oversight ensures the ethical use of animals, optimal national standards for animal care, animal welfare and the quality of animal-based science. - Assessment and Certification on a Canadian level - Standards and Guidance but - Three R’s Alternatives · decentralized - Education and Training Institutions have so responsibilities - Decentralized organization (responsibility rests on institutional Animal Care Committees; ACC) 3Rs TENET ex. Reduce Controls/groups - check stat significance = 7 - Replacement: If there is any way to do the research without using animals, it should be done. examples : Kolken rat sim labs , etc. , - Reduction: Use the minimum number of animals to obtain scientifically valid data. - Refinement: Procedures must minimize distress and pain. Enhance animal well- being. examples : Housing rats with a friend/wheel , humane Limitations and criticism: endpoints non-invasive teguniques , 1. Underlying premise is that the use of animals for science is acceptable. 2. Does not provide special consideration (exemption) to certain animal species (e.g. Animals with least public support for their use in science: primates and companion animals).  3. Conflicts between each “R”s (e.g. Reducing numbers and minimizing pain). 4. Conflicts between the Three Rs and goals of certain type of research (pain and stress research). Ethics review: - A process that allows for ongoing critical evaluation of the ethical, scientific and welfare issues which is essential in any system that regulates the use of animals in research and testing. - This process of ethical review involves evaluating: whether individual scientific projects justify the use of animals (harm- benefit assessment) consideration of practical issues relating to how animals will be used. application of the 3Rs (experimental design, animal housing, husbandry and care related issues such as provision of staff training and assessing competencies Ethics Review process: - All proposed use of animals for research, teaching, testing and display must receive ethical review, and be approved by the University Committee on Animal Care and Supply (UCACS), before the animals are obtained and the work initiated. Ethical review of animal use protocols is conducted by the UCACS Animal Research Ethics Board (AREB). a) Chair: (faculty member, appointed by the Vice-President (Research), not directly involved in the management of the institutional animal facilities b) Researchers experienced in animal care and use (15) c) Animal Resources Centre Administrative Staff and Veterinarians (2) d) Institutional Non-user of Animals (1) e) Community Representatives (2): at least one humane society representative from the Saskatoon SPCA or Saskatchewan SPCA f)Technical Staff Representatives (2) g) Student Representatives (2) CCAC = federal UCAS = University AREB = Lab level (day to day functioning) Animal Model:  - an animal that is an accidental or deliberate model of human disease. Such models are experimental living systems that are used to study disease mechanisms and provide insight into possible therapies (Segens Medical Dictionary, 2012). - Why use animal models? - allow testing hypotheses in vivo (account for complexity of biological systems) - greater and easier control over the environment (genetic makeup, diet, temperature, lighting, etc.) - shorter life span - allow identifying and describing relationships (causal or correlational) between genetic, biochemical, cellular, and behavioral processes Classifications of animal models: 1. Induced: a condition or disease is experimentally reproduced by the scientist Give the Model a disorder a. Examples: i. producing diabetes using the chemical streptozotocin to damage the insulin producing cells in the pancreas ii. producing a stroke model through surgery iii. partial hepatectomy to study liver regeneration 2. Spontaneous (genetic) models: often called "natural" models - include naturally occurring animal diseases or conditions that correspond to the same diseases or conditions in humans Born this way a. Examples: i. an animal model of diabetes. Many hundreds of animal strains/stocks with inherited conditions have been characterized and conserved. The Jackson Laboratory holds one of the largest repository of these valuable animal models in mice ( http://www.jax.org/). ii. a nude mouse (with a genetic mutation that causes a deteriorated or absent thymus, resulting in an inhibited immune system (reduced # of T cells) 3. Transgenic models: a condition is induced in an animal by manipulation of the animal's genes. A foreign DNA may be inserted (“knock-in”) in their genome, or the animal’s genes are replaced or removed ("knock-out" models) a. Example: eNOS KO mice (hypertension) 4. Negative models: some animals are resistant to a particular condition or disease, and ! therefore are useful to study mechanisms of resistance to disease. a. Examples: Immune Negative = Not test Gonna get it =  i. The failure of gonococcal infection to develop in rabbits after an experimental treatment that induces the disease in other animals. 5. Orphan models: refer to diseases for which there is no human analog, they present & exclusively in the species studied a. Examples: i. Bovine spongiform encephalopathy ("mad cow disease”) onlyonea se ii. CWD (Chronic Wasting Disease) in deer In Vitro Experiments: allow researchers to investigate the physiology and pharmacology of ! various tissue samples in a controlled environment without the complications of an intact animal model. - Isolated live tissues (muscles or organs) - Aorta rings still - Ileum sections might need to use - Perfused kidney an animal or - Perfused heart might not - Perfused arteries (mesenteric, etc.) - Cell cultures - Cellular components - Microorganisms - Biological molecules Advantages: highly controlled experimental setting/environment: controlled changes in perfusate, oxygen, drugs and other factors quick results, high throughput, reproducible data results are not affected by regulatory body systems (hormones, reflexes, enzymes) reduced use of live animals (more ethical) Disadvantages: Difficult extrapolation of results to humans Physiological solutions (buffers): Artificially prepared solution to keep isolated tissue alive under experimental conditions. - For isolated organ experiments, it is necessary to use a certain physiological solution (buffers) of different ionic concentrations (substitute to the tissue fluid). Isotonicity (equal tension or osmotic pressure) Nutrition Buffer for drugs  - Extracellular Fluid (ECF) composition: ↑ NadCI & Ca Cations: sodium (Na+, 136-145 mEq/L), potassium (K+, 3.5-5.5 mEq/L) K ↓ calcium (Ca2+, 8.4-10.5 mEq/L) Anions: chloride (Cl-, 99-109 mEq/L), hydrogen carbonate (HCO3-, HCO3- 26 mM) Glucose Water - Physiological solutions should be: Should be freshly prepared distilled water Ionization, tone > low : ↑ - pH adjusted to 7.35-7.45 - > high : "muscle activity , ↓ acidity Temperature 37°C (for mammalian tissues) ↓ activity Aeration 95% O2 and 5% CO2 (blow : high cooks: CaCl2 should be added last as it as a high tendency to precipitate – important! why is my only in specific buffers ? ↳ cofactor cell membrane , stabilization , regulates ion channels prevents Calcium , precipitation ATP stabilization , osmotic balance ~ What is aeration it the same? and why is = Prolongs pH balance, provide Of , helps stir specificallyCon solution Summary: - The Canadian Council on Animal Care (CCAC) regulates the case and use of the animals for research and teaching nationally, whereas the institutional animal care committee is responsible for ethical use of animals at a given institution. - 3R’s: replacement, reduction, and refinement are the principles considered for ethical use of animals for research and teaching. - Animal models of human diseases are classified as: induced, spontaneous, transgenic, negative, and orphan.  - Alternative approaches in research include using in vitro methods: isolated tissue experiments, cell culture, microorganisms, cell components, and biological molecules. - For isolated organ experiments, physiological solutions (buffers) are used; they act as a substitute to the ECF. Procedures promoting the 3 Rs in research: Good examples 1. Replacement: If there is any way to do the research without using animals, it should be done. Computer simulations Cell culture methods Less sentient animals 2. Reduction: Use the minimum number of animals to obtain scientifically valid data. Reduce controls Thorough literature review Appropriate animal model Experimental Design Power Analysis 3. Refinement: Procedures must minimise distress and pain. Enhance animal well- being. Appropriate Training Appropriate anaesthesia Non-invasive techniques Housing & husbandry Humane endpoints Lab #2: Report Writing and Graph Creation Week 2 Assignments: Graphing assignment and Writing an Experimental Protocol Documents that are in canvas but not on here: - Lab 2 GraphPad PRISM software instructions - Lab 2 LabChart Instructions - Pre lab 2 lecture questions - Lab 2 graphing exercises - Lab report grading criteria Slides: Lab 2 How to Write a Lab Report ↓ Refine Objectives: log does not take in time - What is the Dose (concentration) response curve What are the characteristics of the dose-response curve Graphing exercise (due in class) Learn how to successfully write a laboratory report in this course. General Guidelines Marking Criteria Learn what the Experimental Protocol is and how to write one. Experimental Protocol Report (due in 2 weeks) - By the end of this Lab, you will be able to: The Dose (concentration) response curve and its characteristics Understand how to use Power Lab software Plot Log dose-response curve graph using Prism software  Understand an experimental protocol Log Dose Response Curve: - Because a drug effect is a function of dose and time, such a graph depicts the dose-response relationship independent of time & includes four features: 1. Potency (ie, EC50) - How much drug is in there What is recorded 2. Slope on X axis ? Dose 3. Maximum effect (ie, Emax) is recorded What 4. Threshold dose (i.e, lowest dose to cause response) axis ? response on y - conc'n to reach max at max concentration Y ↓ concentration encentration brate of change/sensitivity to drug - Seach bump is a lofold change PowerLab Data Acquisition System: - A data acquisition system developed by ADInstruments comprising hardware and software and designed for use in life science research and teaching applications (Records bio signals) - It is a tool or recording device that acquire, store and analyze data. For example, It allow us to acquire biological signals (i.e., contraction, blood pressure, Heart rate). - What are the required equipment ? - - What Lab chart acquisition system is used for? It is used in pharmacology and physiology lab in particular to record and analyze biological signals from human, animal subjects or isolated organs. - Figure below shows how signals are detected by the transducer then sent to the acquisition unit and amplified. - Converts units from analog to digital SLIDES 10-22 HOW TO USE LABCHART AND GRAPHPAD PRISM Laboratory reports: TITLE PAGE: - list author’s names, - Group Name (TUES 1, WED5, FRI4) - appropriate title of the experiment (not the lab topic) INTRODUCTION (about the topic): - 2-3 paragraphs about the research topic. Provide enough background information on the researched topic to explain it clearly. - You can use various resources, but your writing has to be original, not a copy from any other resource. Paraphrase and include references. AIM/OBJECTIVES: - State the purpose of doing the experiment. MATERIALS AND METHODS: - Describe the protocol you were following when conducting the experiment. Include enough details so another researcher can repeat the procedure in the same way and replicate your results. - Examples: Composition of the used buffer solution, temp. and pH of the solution, type of aeration, organ bath volume, final bath concentrations of drugs, basal tension applied to the muscle, final bath concentrations of the agonist, nature of concentration-response curve (cumulative), washes given to the tissue, incubation time for the unknown drugs, etc., type of software used for analysis and graphing.  - However, do not include common laboratory techniques (e.g., how the solution was pipetted, how the dilutions were made, etc.) RESULTS: - Brief paragraph of the results obtained. - Should be written in the past tense. - Include graphs to represent your analyzed data (not raw data) - Example: CRC graphs, indicate the EC50 values, graphs for calculation of pA2.. No interpretations of results DISCUSSION: - Provide a brief interpretation of your results (what do these results mean)? - Discuss the significance and implications. - Answer the questions specific to each lab assignment (found at the end of each lab protocol). CONCLUSION: - Provide a brief summary of your results and your interpretations. - No new information should be introduced here. REFERENCES: - List published sources in alphabetical order. Note: the lab protocol is not a published resource. - Three references minimum. - APA format - for both in-text citations and the reference list. Experimental protocols: - Comparable to a laboratory report, but describing a future (planned) experiment, and therefore is a creative assignment. - You will be given a hypothetical experiment, - your task is to write an experimental protocol (2-3 pages). - An experimental protocol is a detailed plan of conducting a scientific experiment (e.g., grant application, ethics approval) - Experimental protocol should contain the following: 1. Title page: Your Experiment Title and Authors’ Names; 2. Introduction / Background; 3. Aims/Objectives: the purpose of the experiment; 4. Hypothesis(-es): the expected outcomes of the research project experiments. 5. Materials and Methods: detailed description of materials and procedures, including:  Description of subjects used in the experiment (species, strain, age, supplier, etc.) The description of treatment groups (and control groups), number of subjects per group. All procedures, methods, tools, procedures. 6. References: published work used (peer-reviewed journals, books, reputable online resources) - SLIDES 32-34 AND 37 ARE AN EXPERIMENTAL PROTOCOL SAMPLE Variables: a characteristic that varies! Types: ○ Independent variable: manipulated by the experimenter,e.g. the doses or the What you dis concentration - X axis ○ Dependent variable: measured (or observed)by the experimenter,e.g., blood S pressure, contraction... etc. - Y axis What you see Data collected during investigation Controls: - The group that is used as a standard for a comparison in an experiment. The control may be “no treatment”,” placebo” or positive control similar to the treatment group (control all other factors,i.e., age, gender, age...etc) to isolate the effect of the treatment. - The group that was used as a standard for a comparison in an experiment. The control may “no treatment”, EXAMPLES: DMSO Wort Drug  Pump i placebo Surgery in no clip Summary: In physiology labs, lab reports can be informally structured (answers to questions) In pharmacology labs: formal reports, must follow certain guidelines and requirements. Reports are due 14 days after each module goes live on Canvas (check the exact dates in the syllabus). Experimental protocol is a detailed plan of conducting a scientific experiment, often used in writing grant applications and research ethics applications. · Bath concentration = 1000m) = 1000 Don * 11 ↳ Always trying to find C looon) Im = ↳ = (drug conch)(volume drug) = concentration of bath i drug (volume bath ↳ Add to next calculatedCa - serial dilution make 1 : 1000 final volume : In · comm stock I0mM = 0 0mM Final concentration. = · 1038 (0 amm)(1mL) 01m) caVa ·. = 0. = lamm - 58 44g/mel NaCl : 18mm. · · How much in 200m) 0. 115m x 5844gx0 2). mol. = 1. 389 W Lab #3: Rat Aorta 1 and Drug Profiling Lab Week 3 Assignment: Drug Profiling Lab Report Documents that are in canvas but not on here: - Aortic ring dissection video - Appendix A (exporting labchart) - Sample lab report (chocolate) - Lab 3 dilution exercise - Drug profiling lab report instructions PART 1: Review research ethics and physiological salt solutions; discuss animal models used in research What is a physiological salt solution: A solution of a salt or salts that have the same solute concentration (isotonicity) as tissue fluids or the blood - Examples: Ringer, Krebs Wants to mimic physiological (most relevant) - isotonic, Tyrode etc. - Purpose: Maintain isolated tissues/organs in a viable state during an experiment. Solutions that have the same tonicity will result in no net flow of water across the cell membrane. Physiological solutions (buffers): Artificially prepared solution to keep isolated tissue alive under experimental conditions. - For isolated organ experiments, it is necessary to use a certain physiological solution (buffers) of different ionic concentrations (substitute to the tissue fluid). Isotonicity (equal tension or osmotic pressure) Nutrition Most nutritious aspect of Krebs = glucose Buffer for drugs - Extracellular Fluid (ECF) composition: Cations: sodium (Na+, 136-145 mEq/L), potassium (K+, 3.5-5.5 mEq/L) calcium (Ca2+, 8.4-10.5 mEq/L) Anions: chloride (Cl-, 99-109 mEq/L), hydrogen carbonate (HCO3-, HCO3- 26 mM) Glucose on rest for Water ad major are the put  MID ↑BMFRHYFHT MSMI What is the chemical composition of physiological salt solutions: Muscle relaxation Pr excitation & Muscle Isotonicity& (ICF) Nerved Muscle net flow /ECF) (ICF) Glucose = Sugar-food Physiological solutions Should be freshly prepared distilled water pH adjusted to 7.35-7.45 ○ At lower pH: decreases tissue tone and the effect of the drug (more ionization) ○ At higher pH: reduced acidity, improved cardiac & muscle activity Temperature 37°C (for mammalian tissues) ○ Low temperatures can reduce tone/contraction of intestine cells (takes longer!) Aeration 95% O2 and 5% CO2 - Prolongs pH level Balance ○ O2 is necessary for tissue function ○ Bubbles also help stir solutions ○ Solution should be changed often! (prolonged aeration can alter pH) CaCl2 should be added last as it as a high tendency to precipitate – important! Practice Exam ??s He noted to Ignore this 8 Reduce Refine Replace  1 = 1003 mL nx CXv = weight 1ml = 100ONL 20 68 7. 39 1379. 189 1051. 29 70. 039 433 299. 23. 899 Cava 100x15 * xsox103 C = 100m (a = 100mm = 1x15h - Vi = ? Va = Somc = 6x102 C 100 Part 2: Steps in Drug Evaluation = 0. 06NL - Objectives Describe the various aspects of pre-clinical and clinical assessment of new drugs Describe the role of regulatory agencies in assessing new drugs Drug discovery and approval: Stages: 1. Pre-Clinical (animal studies)  toxic dose Therapeutic index-effective dose 44TI = Safer drus a. Key elements of preclinical testing: Pharmacodynamics (how selective is the drug), Pharmacokinetics (t1/2, routes of elimination), Toxicology (Therapeutic index), Pharmaceutical development (what dosage forms are preferred) 2. Clinical a. Approval process: i. Regulatory agencies: Canada (Health canada), USA (FDA), Europe (European Medicines agency - EMA) ii. Regulatory bodies: 1. Clinical trials are typically carried out at multiple sites, internationally 2. Regulatory bodies may conduct site visits (inspections) a. Ensures that proper protocols are followed b. Sites not following protocol may have their data excluded b. Phases of clinical trials (APART OF APPROVAL PROCESS): i. Phase 1: Small open label or uncontrolled 1. Healthy volunteers 2. Focus on: pharmacokinetics, dosing, Safety 3. Goals: How is the drug metabolized? What is the range of doses? ii. Phase 2: medium size (N= 100 to few hundred) 1. Open label, single or double blind 2. Patients (not healthy) 3. Focus: efficacy, safety, pharmacokinetics iii. Phase 3: Large (N=several hundred to 1000s) 1. Double blind (mostly) 2. Patients (not healthy) 3. Focus: efficacy and safety 4. Goals: is the drug more efficacious than placebo? Is the drug as safe as placebo? 5. If successful will get the notice of compliance (NOC) - this means that the manufacturer has complied with all the requirements set forth by Health Canada in the Food and Drugs regulations (they are free to market in Canada) iv. https://www.youtube.com/watch?v=3Gl0gAcW8rw - overview of the drug discovery process  Same Emax right shift I , X a * X X z Not confident 8 * X D Part 3: Introduction to Smooth Muscle Pharmacology Vascular Endothelium - Functions of blood vessel endothelial cells - Have receptors: Keeps blood in a fluid state Vasodilation / vasoconstriction Stimulating / inhibiting cell proliferation Promoting / inhibiting the formation of blood clots Stimulating / inhibiting inflammation  Altering the permeability of blood vessels Angiogenesis Work together to contract / relax Endothelial-Related Mediators: - Endothelial cells release or are acted upon by mediators found in the vessel wall and blood. - Vasodilators: reduce workload of the heart by preventing smooth muscle cells from narrowing. & Nitric oxide (NO), prostacyclin, endothelium-derived hyperpolarizing factor Examples (EDHF), acetylcholine, bradykinin (BK) - Vasoconstrictors: S Endothelin 1 (ET-1), angiotensin-II, norepinephrine, thromboxane A2 Other mediators which play a role in inflammation, blood coagulation and angiogenesis Relaxation of Vascular Smooth Muscle Muscarinic = adrenergic receptor Endothelial cell = In contact with plasma  PLC ↑ pathway Smooth muscle function VS Endothelial function Endothelial Dysfunction -> If Independent drugs then Smooth muscle = still work good and endothelium = Bad Endothelial dysfunction is often described as a reduction in the relaxation of the blood vessel smooth muscle to agonist-induced endothelium-dependent relaxation. - Muscle cannot relax - Mainly caused by the depression of the NO:sGC pathway due to: Downregulation of nitric oxide synthase (eNOS) expression and activity and/or Less NO reaching the smooth muscle cells from the endothelium superoxide (often destroyed by excess superoxide production) F Reduced or excess production of other mediators released by endothelial cells. - Endothelial dysfunction is implicated in the development of a wide range of cardiovascular risk factors and conditions including atherosclerosis (Ludmer et al., 1986), heart failure (Katz et al., 1992), diabetes (Calver et al., 1992b), hypertension (Calver et al., 1992a), cigarette smoking (Newby et al., 1999), hypercholesterolaemia (Drexler and Zeiher, 1991). Evidence of Endothelial Dysfunction - In research, the presence of endothelial dysfunction can be confirmed when endothelium-dependent relaxation (to ACh) is reduced as compared to endothelium-independent relaxation (to sodium nitroprusside). - An unaltered response to direct smooth muscle relaxation (endothelium-independent) provides evidence that the smooth muscle function is normal. Experiment - Pharmacological agents used in the Aorta-2 Lab experiment:  - Phenylephrine (PE): selective α1 receptor agonist (acts directly on smooth muscle cells Vasoconstriction - Acetylcholine (ACh): muscarinic receptor agonist Endothelial-dependent vascular smooth muscle relaxation - Sodium Nitroprusside (SNP): NO donor Endothelial-independent vascular smooth muscle relaxation Drug Profiling Lab Report - Conduct the contractile CRC on the rat aortic ring using the reference drug (“gold standard”). - Conduct the contractile CRC using the test your NCE compound (use Histamine in ObSim). - Prepare the graphs and compare the results. In your assessment analysis: compare efficacy, potency, and the threshold dose of the NCE based on the data your obtained. - Discuss what other experiments (pre-clinical and clinical) should be conducted to draw conclusions whether the NCE may be an alternative therapy for hypertension. - Your pharmaceutical company has received a compound, the potential therapeutic for patients with hypertension. Lab #4: Reproduction - Placenta Week 4 Assignment: Placenta Quiz - Canvas Documents that are in canvas but not on here: - Lecture Recording - Bovine placenta and uterus Demo Early pre implantation Development: S needs to implant humans Implantation In Implantation – essentially means to become embedded and establish a maternal-fetal contact. - in humans but less in other species Problems of pregnancy frequently originate in the pre and peri-implantation period. Implantation - ~5-9 days post- fertilization and is complete by 10-12 days. Process where embryo becomes sticky, invades and becomes encapsulated within the uterine wall. Hormonally primed uterus and blastocyst communicate - uterine receptivity (20-24)  End result of complex interactions between the hormonally primed uterus and the mature blastocyst. Uterine receptivity – period of endometrial maturation when the embryo can implant e.g. ~days 20-24 of menstrual cycle. - 9-10 days post Cytotrophoblast cells 4 single make the ↳ Develop by fusing to synctiotroproblast ↳ multi-nucleated ↳ enable invasion into uterine chemical wall win response to messeng s convert endometrium into also decidra me following pregnancy ↳ is shed ↳ made be of invasion (Not in non- Invasive species) Embryonic cells termed trophoblasts are responsible for invasion. In response to invasion of trophoblast and release of chemical messengers from blastocyst, endometrium transforms into the Decidua (the endometrium of pregnancy.) Decidua = thick area that sheds post pregnancy Above event occurs 9-10 days post fertilization The Development of the Placenta - roughly 2 weeks post fwertilization Placenta is comprised of both fetal trophoblastic tissue and decidual tissue. As trophoblast invades, cavities are formed that will fill with maternal blood as maternal arterioles become breached. Fingerlike portions of chorionic tissue (villi) then penetrate these cavities. Chorionic Villi = major functional units of placenta make chorion together 42 WKS post Placenta is BOTH trophoblastic tissue a decidual & Cytotrophoblast = fully encapsulated Syncytiotrophoblast makes up the chorion ~ source of tissues for explaint cultured stem blood of Source cells Y yolk sac is relevant for stem cells  Placenta Classification- Based on Chorionic Villi Distribution - Diffuse – chorionic villi located over the entire uterine luminal epithelium e.g. pig and horse. Pig: diffuse placenta is velvety in appearance with closely spaced chorionic villi. Microscopic Horse: organized as microzones of villi named microcotyledons. differences non-invasive chorion = pink ↳ Invasive band In above: pink = placenta top two on right side = ruminants *eutherian = placenta organisms - Cotyledonary – Lobes (Cotyledons) of chorionic vascularized villous trophoblasts and uterine endometrial structures termed Caruncles come together like a “spot weld” e.g. ruminants. The combined structures are called Placentomes. They grow over time and the number varies significantly - Further branching of the villi helps the trophoblast fit into the caruncular tissue e.g. fingers into a glove associated - ↳ are very closely  Bothmom feta sociation ↳ caruncle is in mm Non invasive - Zonary – an invasive band of the chorion surrounds the middle of the fetus e.g. dog and cat. - Discoid – a disc-like structure of chorion interacting with maternal tissue e.g. higher primates and rodents. band -just a Band is divided into the TZ = transfer zone and the PZ = pigmented zone  like Human - ~ maternal side grooves divide into cotyledons. Side being shown is the maternal side Human Discoidal Placenta: the Chorionic villi is the emake up majority functional unit. Floating villi do the majority of gas exchange. Anchored villi go into the maternal wall and anchor fetus to mom if they do not work Xpre-eclampsia can happen The Placenta: no blood mixing - Each villus will eventually become filled with fetal capillaries -exchange between maternal and fetal blood, but with trophoblast the physical barrier between the two (no mixing). - Fetal capillary system becomes linked to the fetus via circulatory system of umbilical cord.  O ↓ Becomes Umbilica as Immunofluorescence of anchoring Villi first examination of the placenta Immunofluorescence and histology of Floating villi  · Red caby" for ange only Blayes gas · Green Placental Function during Pregnancy Functions partially or completely accomplished throughout pregnancy: ○ Transfer of Oxygen and Carbon Dioxide (Respiratory). ○ Fetal Nutrition (Gastrointestinal). ○ Excretory Functions -water balance, pH regulation (Renal). ○ S Hormone/Enzyme Production (Endocrine). ○ Immunologic Functions (relatively uncharacterized). Passive immunity: pass ↳ on immunity through placenta (ruminants do not do this). Only present in invasive placenta envasivecana do it Invasive , can Umbilical Cord Usually about 50 cm long, 1- 1.5 cm in diameter. Has two arteries, one vein. ~ Not same in Some ○ Arteries - bring waste and deoxygenated blood back to placenta. * Reverse ?○ Vein - bring nutrients and oxygenated blood to the fetus. frompults  Loose connective tissue – Wharton’s Jelly allows the cord to be rubbery and flexible. - allow movement & blocks in prevents blood flow Placental Transport Simple diffusion – gases (e.g. O2), simple molecules (drugs). Facilitated diffusion – glucose; a carrier system operates with a chemical gradient. Active Transport – essential amino acids, water soluble vitamins; maintain higher concentrations in fetus than in mother. Receptor-mediated Endocytosis – immune bodies (IgG), some protein, fat and ↳ viruses. Imumoglobuling viruses , Leakage – intact cells (e.g. Rh sensitization) toxic substances like thalidomide, S RARE booze, drugs, etc can cross the placenta ↳ The Placenta Covid, zika , Bacteria Is responsible for health of pregnancy ○ synthesizing and secreting hormones into maternal blood to control or effect maternal metabolism for the fetus’ benefit. Maintains the uterus in the proper physiological condition so that the fetus can remain inside the uterus for the appropriate amount of time ○ via Progesterone production Hormonal Environment of Pregnancy The Feto-Placental Unit (FPU) By the end of 1st trimester, fetal endocrine system is developed - influences placental function. Fetus (HPA axis) and placenta are key FPU elements - decidua and maternal sources of hormones can be considered contributors to the unit. The FPU largely controls the endocrine events of pregnancy ○ failure of the unit may be responsible for pregnancy loss ~25-40% failure after implantation. contact o maternal In direct I Placenta: Endocrine Function hCG blood Human chorionic gonadotrophin - glycoprotein secreted by syncytiotrophoblast. ○ beta subunit of protein is the basis of the pregnancy test (hCG excreted in urine). Detectable 1 day after implantation with a peak at 60- 90 days. Maintains the corpus luteum (leftover follicle) until placenta takes over progesterone synthesis. ↳ makes progesterone hCG - major contributor to development of gonads and adrenals in the 1st trimester e.g. Masculinization. Placenta: Endocrine Function hPL Human placental lactogen = Chorionic somato-mammotropin. Produced from the syncytiotrophoblast; peaks in the last few weeks of pregnancy. Has an anti-insulin effect: impairs maternal glucose uptake Increases glucose for fetal utilization. Fetal growth 3 allows fetus to utilize ↳ glucose high in sa be lots trimester of growth  Helps prepare mammary glands for lactation. Placenta/Fetus: Endocrine Function Steroids Placenta is an incomplete steroid producing organ, ie. Cannot synthesize steroids de novo - obtain precursors from the fetus and/or mother to convert to appropriate steroids. Steroids only drop at birth #De novo = from Scratch Y maternal circulating cholestera Placenta/Fetus: Endocrine Function Steroids Progesterone: formation in placenta depends on maternal circulatory cholesterol. ○ Maintains pregnancy after ~7 weeks gestation. ○ Uterine muscle relaxation due to progesterone from the venous blood draining the placenta. Do not want preterm labour ○ Local immune effect: inhibiting T-cell mediated tissue rejection. ○ Stimulates milk gland development in mammary glands. Estrogens: both placenta and fetus are needed in the biosynthesis of estrogens, particularly estradiol and estriol. ○ Estrogens stimulate myometrial growth. Muscle growth Y Hypertrophy  ○ Derived from fetal androgens, in particular, Dehydroepiandrosterone sulphate (DHEA-S) produced mainly in the fetal adrenal gland (4androgen precursor Fetal Adrenal Gland Is the major fetal endocrine component of the FPU. Gland has a definitive and fetal zone in the cortex. Fetal zone is 80% of the organ during gestation and secretes primarily androgens. DHEA is primary Fetal androgens enter the placental circulation: ○ precursors for estradiol and estriol. Steroid Biosynthesis: Important - fetal adrenal gland RED = EXAM makes estrogen ↳ lipid YAndrogen The Basis of Experimentation is a Question! What, When, How, Why...? Once questions and knowledge pertaining to question(s) are collated, a hypothesis can be formed for testing as well as objectives defined for experimentation. of protein Need model systems for experiments to tackle hypotheses: expression ○ Tissues preserved and sectioned for in situ assays (immunofluorescence). Y Not alive ○ Cell lines: established immortalized or primary cell cultures.take from placenta to grow> In Vitro - ○ Animal Models e.g. mice, rats, guinea pigs, etc we are very knowledgeable on mice and rats ○ Explant cultures: pieces of tissue for experiments ex vivo. Ex vivo = alive ○ Trophoblast stem cells. ○ More coming!! Using Cell Lines for Experiments trophoblasts derived from tumours ↑ - 8 his wound D Y filled wound V could also use an explant HTR8-SVneo for studying: - H=human, T=trophoblast, 8=8th clone, V=viral gene for immortality, neo = antibiotic to select JAR = from tumours On the left - CDH1 = stained for cadherin, blue = lose cadherin Methods and Models for Assessing Placental Function https://videocast.nih.gov/watch=14256 NIH Human Placenta Project Meeting. Select #4 Video: Current Methods for Assessing Placental Development and Function and Their Limitations – Dr. Yoel Sadovsky, University of Pittsburgh School of Medicine. Starts at 2:10:19 and ends at 2:42:42. Overall seminar is about 32 minutes. Some of it will be quite technical but it is meant to give you an idea of the breadth of methods and models used or in development to study the placenta. A 10 min timed quiz (MCQ and T/F) will be based on this ~30 min video presentation as well as some of the lecture material. The quiz will be available only on Canvas on October 4th, 2024 beginning at 8:00 AM. You must complete it before 11:59 PM that day. Demo of Cow Placenta Dissection See Panopto in the Course Site for Video. It is not mandatory to view it as it is only for your interest since we cannot do this in the lab this year. It is usually quite popular for students to see this placenta. Final exam questions will be from this lecture only, not the NIH or Dissection videos.  Lab #5: Rat Ileum Week 5 Assignment: Ileum Lab Report Documents that are in canvas but not on here: - pA2 value basic Pharmacological Principles - Isolated ileum experiment instructions - Ileum Mock Data - Ileum Calculations GI smooth muscle: Specifically the ileum which is the last ⅓ of SI - Mucosa/submucosa = SA of internal ileum including plica and cilia & - Circular smooth muscle = rings of muscle - squeeze and contract in presence of ACh, increases luminal pressure, contribute to contractility of luminal space to churn and move and mix - Longitudinal muscle = lead to contraction that shortens ileum, works alongside circular to induce peristalsis. - ACh = parasympathetic NS activates muscle directly, agonises muscle via muscarinic cholinergic receptors causes contraction, in contrast to the aorta which causes relaxation. Ex. atropine and verapamil = antagonists with different mechanisms to counter ACh effects Three cell layers a) Mucosa/submucosa b) Circular smooth muscle (luminal pressure) c) Longitudinal smooth muscle (muscle shortening) Dominant parasympathetic tone a) ACh produces contraction by activating muscarinic cholinergic receptors b) Cholinergic antagonists have sig. inhibitory effect on ileal motility/contractility  ↳ Agonist ↳ Antagonist SEE recorded for the answer Types of Antagonism: Competitive: - Binds to same site as agonist causes direct competition for binding at muscarinic cholinergic receptor - 1:1 ratio either antagonist is bound or agonist is bound - nu Atropine - need to increasingly add more agonist in order to regain effect: potency increase then agonist must increase in order to cause 100% response Noncompetitive: - Can be bound via active or allosteric site and will not compete with agonist to bind - Antagonist binding reduces effect of agonist without affecting the binding the agonist - Emax is depressed - cannot restore no matter what because effect is being blocked independent of agonist - Adding even more agonist will cause a larger depression of Emax Physiol. antagonism: - epinephrine raises arterial pressure through vasoconstriction mediated by A1-adrenergic receptor activation, in contrast to histamine, which lowers arterial pressure. - There are several substances that have antihistaminergic action despite not being ligands for the histamine receptor. - Same thing as noncompetitive antagonist but it does not bind to the receptor that the agonist is binging too - Influence the efficacy of the drug - - Decline is not due to antagonism of receptor but antagonism somewhere else that is countering the effect - Competitive: antagonist binds to the same site as the agonist (no receptor activation) Emax can be reached with increased agonist concentrations EC50 increase with increasing concentrations of a competitive blocker Ewaxlookslike is in - Non-competitive: Agonist and antagonist can be bound to the receptor simultaneously (active site or allosteric site); antagonist binding reduces or prevents the action of the agonist with or without any effect on the binding of the agonist (Neubig et al. 2003 Pharmacol Rev). Emax depressed (decreased efficacy of the agonist) In some cases, EC50 increase with higher concentrations of a non-competitive blocker. ↓ Emax ↑ EC50 3tilt down - Physiological: agonist and antagonist produce opposite effects on a tissue via different receptors and mechanisms. Emax depressed (decreased efficacy of the agonist) & In some cases, EC50 increase with higher concentrations of a physiological blocker  - E.g.: bronchoconstriction produced via a cholinergic muscarinic receptor agonist can be antagonised by bronchodilation produced by an adrenergic β2 receptor agonist competitive non-competitive pEC50 Value: - pEC50: left to right increase potency = the amount of agonist neede to define an effect \ what dose is needed? - High pEC50 = low EC50 - high potency to crease EC50 effect causes a lower EC50 value as less of agonist is required - indicates the potency of an agonist, but not it’s efficacy Efficacy = maximum effect of an agonist (Emax) Potency = amount of agonist needed to produce a defined effect (EC50) pEC50 = -(logEC50) - A high pEC50 = a lower EC50 which indicates a greater potency as less of the agonist is required to achieve half the maximal response - A low pEC50 = a higher EC50 = lower potency - A high pEC50 indicates a greater potency , because less of the agonist is required to achieve half the maximal response. Potency is determined by an agonist’s affinity (binding ability) and intrinsic activity. pA2 Values - Used to determine the relationship between two drugs and is used /relevant only in the case of competitive antagonism - Concentration of antagonist needed to bring double agonist back to baseline ? - pA2 determines the important relationship between two drugs "competing" for effect on the same receptor. It is the concentration of antagonist when double the agonist is required to have the same effect on the receptor as when no antagonist is present. - The two drugs are "competitive" if increasing or reducing one drug decreases or increases the effect of the other, respectively (Neubig et al, 2003). Schild plots (Regression): - Degree to which one variable predicts the influence of another drug - tools of prediction - Method of classifying different types of receptors and agonists - If two tissues make same PA2 then there is a direct relationship between agonist and antagonist that were combined and we can jump tissues to see if the relationship is the same in other tissues - Competitive drug is atropine - -log of antagonist on the x axis - Agonist characterized on y axis log CR-1 or DR-1 - Slope of -1 = competitive antagonism 1:1 ratio - Need at least 3 EC50 values in order to make the graph - Nonlinear = noncompetitive or physiological - No PA2 if not competitive - pharmacological method of receptor classification. Can be used: to determine if agonists act on the same receptor. (Eg. the same concentration of the antagonist will antagonize both agonists with the same pA2 value.) to identify if the same receptor is present in other tissues (Eg. When two different tissues produce the same pA2 value with the same agonist-antagonist pair.) to distinguish between competitive and other types of antagonism. - Plots require at least three EC50 values of the agonist obtained in the presence of three different concentrations of the antagonist. Straight line with a slope of -1 indicates a competitive antagonism. pA2 value can be obtained at the intercept with the X axis  Non-linear plot indicates a non-competitive or physiological antagonism. No pA2 value can be obtained Lecture Summary - Physiological is verapamil and competitive is atropine - Two types of smooth muscle layers coordinate contraction and motility of intestinal > - peristalsis/smortening muscles: circular and longitudinal. ↳ luminal Pressu e - Dominant parasympathetic tone: Ach produces contraction via activation of cholinergic muscarinic receptors. - Types of antagonism studied: competitive, non-competitive, physiological. - PEC50 value is used to describe agonist potency. X axis - pA2 value is determined by constructing the Schild Plot. And it is a useful tool in receptor classification or agonist-antagonist relationship classification. Objectives of the lab: - Identify the unknown blocker by testing the competitive vs. physiological antagonism on cholinergic receptors in the isolated ileum preparation by constructing CRCs. - Create a Schild Plot and determine pA2 value of the competitive antagonist. Materials: - Acetylcholine: muscarinic agonist (will cause contraction of the intestinal smooth muscle) - Unknowns: a) Atropine: muscarinic receptor blocker, competitive antagonist of ACh, will cause smooth muscle relaxation  b) Verapamil: L-type Ca2+ channel antagonist, physiological antagonist of ACh, will cause smooth muscle relaxation by blocking the Ca2+ influx via L-type Ca2+ channels (preventing the excitation-contraction coupling of smooth muscle) Your Experiment - When preparing graphs, calculate percent of maximal response obtained in the control CRC for all four CRCs. 1. Control Ach CRC 2. Ach CRC in the presence of Atropine OR Verapamil (dose 1) 3. Ach CRC in the presence of Atropine OR Verapamil (dose 2) 4. Ach CRC in the presence of Atropine OR Verapamil (dose 3) - Once your data is collected, determine the potency of Ach (pEC50). Then, exchange your four EC50 values with another student group with a different unknown blocker. a. Create a Schild Plots to find the pA2 value of Atropine. b. Create a Schild Plots to find the pA2 value of Verapamil. - In your lab report include: a. Discuss competitive, non-competitive and physiological antagonism b. Discuss EC50, Emax, PEC50, and dose-ratio values c. Include your graph with 4 concentration-response curves (CRC) graphs (Parts A-D) d. Include two Schild plots with marked the X-axis intercepts (ie, PA2 values) for both atropine and verapamil. e. What type of antagonism did atropine and verapamil induce in these experiments? How do you know? f. Include calculations as an Appendix to the lab report  Lab #6: Human Cardiovascular Lab Week 6 Assignment: Cardiovascular (Informal) Lab Report Documents that are in canvas but not on here: - Experimental Procedures for CPPS 308 exercise lab - Instruction for exercise lab - Results summary excel sheet Notes from class are live so will be based off of s recording of him talking: - As a young person we generally remember stuff like grocery lists - Elderly have dementia and memory loss so they forget their grocery list Exercise: - Enhance memory and health - What is exercise…? Difficult to define: Increased heart rate and contraction of muscle, enhanced blood flow - Increase blood pressure, increased heart rate, increased vascular constriction, increased sympathetic activity… This could also come from watching a horror movie so what defines exercise. Study in the elderly is the basis of this lab: - Want the elderly to do isometric exercise - They have the group with the exercise, group without and within ½ a year they can remember their shopping list better. Better correlation between exercise and memory. - Why does this happen? - Enhanced blood flow to the brain. - Many of the elderly have hypertension conditions so they do not want to elevate blood pressure with exercise but still want to improve the blood flow. - When you exersice you suppress parasympathetic and increase sympathetic - How do you push it to the brain? - One magic word: vasodilation - Vasodilation which is caused by: (1) Neurotransmitter - ACh causes vasodilation on the vasculature because of endothelium dependent NO release a. Aorta will relax from SNP endothelium independent - not relevant here b. Aorta will relax aorta via the ACh endothelium dependent  c. Increase blood flow the shear stress through the vessel and artery stimulate the endothelium to produce NO which give rise to vasodilation. (2) Local vasodilation - I squeeze my fist, constrict my vessel, white because of no blood after you let go it is more red than the other hand because of local vasodilation a. Autoregulation is the mechanism behind this - the wonder of exercise b. Dilation of working tissue = enhanced blood flow BACKGROUND FOR LOCAL VASODILATION: How do you increase blood pressure? Increase blood flow increase pressure gradient. - Sympathetic stimulation, Beta 1 activation will trigger your heart to beat stronger and faster. At the same time sympathetic also causes alpha 1 receptor activation = vasoconstriction so how does it stimulate an increase in blood flow? Autoregulation which occurs through exercise - Exercise causes sympathetic outflow Your carotid artery and aorta respond differently to sympathetic stimulation: if you put both into a bath and put in drug the carotid artery will not move and the aorta will constrict like crazy. When you exercise, the carotid artery takes a larger dose or greater activation in order to have the same constriction which is what causes enhanced blood flow to your brain because at the same time you increase blood pressure. Increase blood pressure = increase pressure gradient. If your carotid artery does not affect by your alpha 1 innervation you don't use brain or neck during exercise but because increase blood pressure and artery does not constrict or dilate in response to pharmacological intervention or neural activation so it can maintain the latency of the carotid artery so higher pressure = enhanced blood flow to brain. Exercise = sympathetic activation = alpha 1 activation = vasoconstriction = beta 1 activation = increase heart rate = increase force of contraction = cardiac output = perfusion Beta 2 = vasodilation - And coronary artery has more beta 2 than beta 1 so coronary artery increase in diameter during exercise due to beta 2 receptor All of the above will happen if you just drop the dumbbell on the table and scare people because it is also sympathetic activation. But with exercise you get shear stress and enhanced vasodilation. In other words, all the muscles not constricting constrict and alpha 1 activation and vasoconstriction but exercised tissue has vasodilation why? Autoregulation, because metabolites like ATP (adenosine in particular is a potent vasodilator and cause local vasodilation), and also by opening the capillary bed you enhance the blood flow increase shear stress increase NO enhance vasodilation and blood circulation. Autoregulation = the more you work the more you encounter the metabolites. Types of exercise today: 1. Isometric - recommended for elderly - hold at same muscle length for 5min a. Iso = same and metric = length 2. Dynamic - Issue will be that there is increased blood pressure which is not good for individuals with hypertensive properties. High blood pressure can cause increased “pain” so dynamic can increase pain more. Increase heart rate = increase contractility = increase cardiac output = increase blood pressure = increase workload difficulty to pump blood = heart need to pump harder = increase afterload - Afterload is proportional to Pressure times Radius / 2 times Thickness - Afterload = stress on heart needed to overcome to pump - Pressure is the main contributing factor in the increase in afterload during exercise We take readings lying down because: - Gravity because blood pools to the bottom. - Increase pooling = decrease blood pressure. - Blood pressure will be exactly what the heart is dealing with when you are lying down. Lab #7: Rat Aorta 2 Organ Bath Lab Week 7 Assignment: Aorta II Lab Report Documents that are in canvas but not on here: - Aorta 2 lab assignment - Aorta mock data - Measurement of smooth muscle function in the isolated tissue bath (for methods) What are blood vessels? - Blood is transported around the body in three different types of blood vessels: a) Arteries b) Capillaries - Meeting point c) Veins - Each blood vessel is composed of three layers of tissue. From outside in they are: the tunica externa, tunica media, and tunica intima: Direct contact with the blood, Permeable to certain material, Regulate the diffusion of substances Prevent cells sticking to its wall Contract to prevent blood flow What is the endothelium? - It is selective permeable barrier between the tissues and the blood components, regulating the passage of molecules and cells, participating in local regulation of vascular tone and blood flow.  - Under normal physiological conditions, the endothelium secretes prostacyclin, endothelium-derived hyperpolarizing factor, and nitric oxide (NO) - Under pathological condition, the endothelium secretes potent vasoconstrictor substances such as endothelin. Excessive production of reactive oxygen species (unstable molecule containing oxygen), such as superoxide anion (O2−) a) Example: Excessive exposure to angiotensin II can promote local oxidative stress and inactivate NO. b) Example: hypertension, diabetes, atherosclerosis: Leaky, blood vessel damage, lose the ability to contract and relax. Endothelial-dependent muscle relaxants Ach = M3 - Acetylcholine (ACh): muscarinic agonist, stimulates M3 receptors located on & · Iono =? endothelial cells ~ Relax Be - Ionomycin: calcium ionophore (CI), facilitates the transport of calcium across the cell membranes (and store-regulated cation entry). - Bradykinin (BK): peptide and inflammatory mediator, stimulates B2 receptors located on endothelial cells (not used in the lab today) PLC - ACh and BK activate eNOS by increasing Ca2+ levels in the endothelial cell via the phospholipase C (PLC) pathway - CI activates eNOS by increasing Ca2+ levels in the endothelial cell CI Endothelial-Independent Muscle Relaxant - Sodium Nitroprusside (SNP): inorganic compound, releases nitric oxide (NO) when SNP broken down in circulation ·↓ afterlad This NO then acts directly on the vascular smooth muscle to activate soluble · CO guanylyl cyclase (sGC) Causes peripheral vasodilation via direct action on vascular smooth muscle Reduced peripheral resistance & afterload -> increased cardiac output a) Acute heart failure b) Acute hypertension  Vasodilation Inhibitors: (1) Atropine: anticholinergic, will block M3 receptor located in the endothelial cell, Atropine · ↳ Block M3 prevents ACh induced relaxation · L-Name (2) L-NAME: non-selective inhibitor of nitric oxide synthases (NOS inhibitor) ↳ Nos inhib (3) ODQ: selective inhibitor of nitric oxide-sensitive soluble guanylyl cyclases (sGC · ODD inhibitor) ↳ SGC inhib (4) NO Scavengers: inhibit activation of sGC via NO (not used in the lab today) · No scavengers ↳ SGC via No What Control of Vascular Smooth-Muscle Cell Tone? Ach - Acetylcholine released from parasympathetic neurons binds to muscarinic receptors ↳ M3 on vascular smooth- muscle cells causing vasorelaxation; modulate intracellular SNP CA2+ levels and contractile tone. ↳ Indi - Sodium nitroprusside releases NO, which relaxes vascular smooth-muscle cell via PE ↳< (constrict) the cyclic GMP– dependent and –independent mechanisms. NE - Phenylephrine is a powerful agonist as a1- adrenoceptors (on smooth muscle cells), ↳ constrict ↳ X , X2 resulting in vasoconstriction. - Pre Contracts the aorta EPI - Norepinephrine activates α adrenergic receptors. In most blood vessels, ↳ B & X norepinephrine activates postjunctional α1 receptors in large arteries, and α2 ↳ constrict ? receptors in small arteries and arterioles, leading to vasoconstriction - Epinephrine activates both α and β receptors - More punch? This disruption of endothelial function can be broadly defined as “endothelial dysfunction” Endothelial Dysfunction - Endothelial dysfunction is associated with many pathological conditions: Atherosclerosis (key during early progression) Hypertension: inflammation and oxidative stress Diabetes: oxidation of proteins, the advanced glycation end products And inflammation, triggering atherosclerosis Smoking: reactive oxygen species SARS-CoV-1 S protein(Covid): down regulation of ACE II (angiotensin-converting enzyme), mitochondrial impairment and increasing reactive oxygen species. - The reduced relaxation of the blood vessels to agonist-induced endothelium-dependent relaxation (mainly reduced endothelial nitric oxide mediated relaxation). Thus, either less NO is produced by the enzyme endothelial nitric oxide synthase (eNOS) and/or less NO reaches the smooth muscle cells from the endothelium because it is destroyed by an excess of superoxide production (oxidative stress). - What evidence would suggest Endothelial Dysfunction in the lab?  Reduced endothelium-dependent relaxation (e.g. ACh) AND Unaltered endothelium-independent relaxation (e.g. SNP) Provides evidence of normal functioning smooth muscle Lab Objectives 1. To detect the presence of endothelial dysfunction (due to hypertension) by using data collected from two samples of isolated aortic rings from rats. 2. To create concentration response curves (CRC) using acetylcholine (ACh) for endothelium-dependent and sodium nitroprusside (SNP) for endothelium-independent (acting directly on vascular smooth muscle) relaxation on the aortic rings. 3. To obtain the Emax and EC50 values of ACh and SNP. 4. To identify which sample (A or B) of aortic rings have endothelial dysfunction. Lab #8: Frog Skeletal Muscle Lab Week 8 Assignment: Frog Skeletal Muscle Lab Report Documents that are in canvas but not on here: - Frog skeletal muscle lab 8 handout - How to suture - Isolating rectus abdominis muscle from frog pharmacology experiments What controls skeletal muscle activity Skeletal Muscle Is Voluntary Muscle Controlled by the Central Nervous System - Note: several muscle fibers are innervated by a single motor neuron. ina sensoryafferent · sensory nevions Synapse · · Axons of motor neuges · motor /efferent) From cord to Spinal muscles * lateral norm : T& L-sympathetic Motor nerve = Acetylcholine = Binds to nicotinic = causes contraction Ventral horn = motor = Efferent => Dorsal Horn = back = afferent Skeletal Muscle - Frog Rectus Abdominis is an abdominal skeletal muscle under voluntary control of the Nervous System; - One of the oldest in vitro tissue preparations; - Has been used by pharmacologists to describe dose-response curves since the early 20th century.  Nicotinic ↳ Skeletal muscle causing contraction ↳ Conotropic (lon Channels) Neuromuscular (NM) Junction - Receptor at NM junction is a nicotinic muscular (Nmus) subtype of cholinergic receptor - Located on postsynaptic muscle cell (sarcolemma) - Stimulation of motor nerves releases acetylcholine (ACh) at the NM junction AchE - ACh is rapidly destroyed by the enzyme acetylcholinesterase (AChE) - Needed to = Relaxation allow for muscle relaxation - ACh has a very brief (μsec/msec) physiological duration of action on the Nmus receptor - The Nmus receptor is an ion channel, when stimulated it allows Na+ into the muscle cell resulting in depolarization and contraction - Action potential = -70mV at rest Blockers of the Nmus Receptor 1. Competitive Block A competitive antagonist binds to a receptor, but does not activate the receptor. The antagonist will compete with the agonist for the available binding sites on the same receptor.  Surmountable block – Emax can still be achieved with larger doses of an agonist. - e.g. d-tubocurarine ❖ Practice Question: What would happen to the EC50 of ACh in the presence of Tubocuarine? It would increase. More drug needed to reach EC50 2. Depolarizing Block Depolarizing agents are resistant to degradation by AChE; Depolarizing agents prevent the effect of ACh by the persistent stimulation of the Nmus, which results in the Na+ channel remaining open; In this case the muscle fibre cannot repolarize and recover resulting in muscle paralysis. e.g. succinylcholine - Phase I: Depolarizing Phase a) Intensive muscle contractions, or muscular fasciculations (twitches), occur as the muscle fibres are persistently depolarized. b) Motor end plate is unresponsive to nerve impulses or Ach stimulation. - Phase II: Desensitizing Phase 1. Flaccid paralysis. 2. Nmus receptors become desensitized, leading to a prolonged relaxation. AChE Inhibitors - Reversible AChE Inhibitor: Transiently delays the degradation of ACh released at the NM junction Potentiates the action of ACh on the Nmus receptor e.g. physostigmine, neostigmine - Irreversible AChE Inhibitor: Completely prevents the degradation of ACh Results in persistent stimulation and depolarizing paralysis of skeletal muscle Leads to death through respiratory failure e.g. tabun, sarin Your Experiment: Frog Skeletal Muscle - Objectives: 1. Analyze data from the experiment. 2. Graph two dose-response curves (one control and one in the presence of an unknown drug) 3. Identify one unknown drug acting on the Nmus receptor. - Possible unknowns: d-tubocurarine  Succinylcholine Neostigmine Summary: 1. Rectus Abdominis – abdominal skeletal muscle under voluntary control of NM Junction. 2. NM Junction – chemical synapse between the neuron and the skeletal muscle fiber, with Ach being the primary neurotransmitter. Ach activates the Nmus receptor resulting in cell depolarization and skeletal muscle fiber contraction. 3. Competitive (non-depolarizing) and depolarizing blockers are two types of agents that can result in a skeletal muscle relaxation. 4. Reversible AChE inhibitors potentiate the effect of Ach on the skeletal muscle. 5. Lab Report: analyze data, graph two CRCs, identify your unknown drug. 6. Discuss how the remaining [two] unknown drugs might influence your findings. #resible Normal Competie NOT ON FINAL Lab #9: Neurophysiology Lab Week 9 Assignment: Neurophysiology (informal) Lab Report Documents that are in canvas but not on here: - Simulation instructions OBJECTIVES - Explore the electrophysiological properties of neurons using computer simulator software. - Compare the Current Clamp and Voltage Clamp experiment techniques. - Conduct electrophysiological experiments using the simulators and generate the neuronal response outputs for the further data analysis and discussion in the report. THE PATCH-CLAMP TECHNIQUE - Developed in the late 1970s. Allows high-res recordings of whole cells, patches (small area of neuronal membrane), and even single ion channels. - Requires technical and biological background and skills from the experimenter. - Simulator programs are very useful in training electrophysiologists GENERAL PRINCIPLE - A glass pipette (with electrolyte solution) makes a tight contact with the cell membrane. This isolates a patch for the further experimentation. - Current-clamp: Currents passing through the channels in this patch are controlled, and the resulting changes in the membrane potential can be recorded. Best for recording resting membrane and synaptic potentials https://www.youtube.com/watch?v=mVbkSD5FHOw (START -> 2m39s) - Voltage-clamp: The voltage across the membrane is controlled, and the resulting ionic currents are recorded. Best for recording cell firing activity https://www.youtube.com/watch?v=8NQenLqDXsU (WATCH ON YOUR OWN FOR MORE THOROUGH EXPLANATION) Neurophysiology Lab Report: - Run simulations to find answers to the questions presented in the lab handout (instructions). Typically, it takes 2-3 hours for students to run through all 10 simulations and take notes for their lab report. - Only answers to questions in BOLD font are required for the lab report assignment. - Reminder: this is a physiology lab report: informal structure (no need for title page, introduction, methods, etc.). Only your answers to questions are required. Lab #10: Hematology lab Week 10 Assignment: Hematology (informal) Lab report Documents that are in canvas but not on here: - HemaLab procedures - Instruction for hematology lab report LEFT OFF VIDEO AT 8:37 BUT COULD WATCH ALL AGAIN: VERY TIRED SO JUST GOING TO WRITE NOTES THAT ARE PRESENT. Hemostasis - Arrest of bleeding or the prevention of hemorrhage - Involves three distinctive phases: 1) Vascular phase 2) Platelet plug formation - Without this no clot forms, Platelet formation 3) Coagulation - Part of hemostasis (prevention of haemorrhaging) Fibrin coming together Aspirin is a COX inhibitor and prevents TXA2 and Platelet activation - Suppress platelet - No fibrin formation - No coagulation (because coagulation requires platelet activation) Thrombotic event = forming fibrin 1. Vascular phase - Smooth muscle cell constriction. Close vessel, decrease blood flow, decrease blood loss - Resulting from the contraction of vascular smooth muscle cells (VSMCs) within the damage blood vessel wall Initial response To minimize blood loss Temporary constriction or even closure of damaged vessel - Involves both neural or chemical a) Neural: Vasocontraction / vascular spasms Related to a deduction in blood vessel diameter: Increases in sympathetic tone increase alpha activation Decreases in blood volume leads a decrease in transmural pressure (Also sympathetic) (difference between intravascular pressure and the tissue pressure) b) Chemicals released  Thromboxane and serotonin are released by the activated platelets Endothelin from endothelium - due to opening of vasculature causing an increase in flow and shear stress, potent vasoconstrictor. Thromboxane, serotonin, and endothelin are vasoconstrictors 2. Platelet plug formation - functional capacity of platelet, the number of platelets = increase in response. This is what platelets are for which is why you are recommended to chew aspirin because you prevent coagulation? - Process that includes 3 series of events a) Platelet adhesion b) Platelet activation c) Platelet aggregation a. Platelet adhesion - stuck to vasculature - Process involves binding of platelets to themselves or to other components (e.g. collagen of the damaged vessel wall) In response to an increase in the shearing force at the surface of platelets or endothelial cells Or in response to vessel injury - Process mediated by the binding of platelet membrane receptors with ligands such as von Willebrand factor (vWF) - Expressed by the endothelium , and subendothelial structures (collagen, fibronectin, or laminin) - vWF a glycoprotein made by endothelial cells and megakaryocytes (from bone, where platelet formed from fragment) - It is a response by the platelets to damaged blood vessel b. Platelet activation once adhered, +ve feedback causes more platelets to activate - Binding of platelet receptors to ligands triggers an exocytotic event known as the release reaction or platelet activation Activated platelets cause exocytosis of their dense storage granule contents, which include ATP, ADP, serotonin, and Ca2+. Hemostasis Platelet plug formation Activated platelets also use cyclooxygenase to initiate the breakdown of arachidonic acid to form thromboxane A2 - Molecules released by activated platelets such as ADP , serotonin, and thromboxane A2 amplify the platelet activation response (+ve feedback) - Hemostasis Platelet plug formation - When platelets flow past artificial mechanical heart valves, the shearing forces endure by the platelets can also cause platelet activate  Aspirin is a NSAID (nonsteroidal anti-inflammatory drug) that function as a cyclooxygenase inhibitor can suppresses the normal function of platelets Activated platelets also use cyclooxygenase to initiate the breakdown of arachidonic acid to form thromboxane A2 c. Platelet aggregation: - vWF released by activated platelets binds to the platelet receptor thereby activating even more platelets and allowing platelets to form molecular bridges between platelets and the subendothelial structures such as collagen - Platelet activation also induces conformational change to its membrane receptors and allowing them to have the capacity to bind fibrinogen - Coagulation factor produced by the liver - Fibrinogen forms bridges between platelets and participates in the process of platelet aggregation - The end result of platelet adhesion, activation, and aggregation is the formation of a platelet plug - covers damaged vessel 3. Coagulation Platelet phospholipid (factor 3) Coagulation is a process of blood clot formation Blood clot consists of a mesh of fibrin containing blood cells and serum - Fibrin is element to make clot ○ Thrombus is also a blood clot, but the term is usually reserved for an