Fatima's notes Physiology 1 PDF 2022-2023

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Fatima

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These are lecture notes for Physiology 1, covering topics such as Neurons, Skeletal muscle, Smooth muscle, Body fluids, and Blood. The notes were taken in the 2022-2023 academic year.

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Physiology 1 Fatima’s notes 2022--2023 Fatima’s notes 2022-2023 Physiology 1 Contents Neurons......................................................................

Physiology 1 Fatima’s notes 2022--2023 Fatima’s notes 2022-2023 Physiology 1 Contents Neurons....................................................................................................................................... 5 Properties of the different fibre types................................................................................................................. 6 Ion channels......................................................................................................................................................... 6 Different types of membrane potentials............................................................................................................. 7 Equilibrium membrane potential of Na+.............................................................................................................. 9 Skeletal muscle.......................................................................................................................... 10 Neuromuscular junction..................................................................................................................................... 11 Sarcoplasmic reticulum (L- and T- tubules)........................................................................................................ 13 Sarcomere.......................................................................................................................................................... 15 Sliding filament mechanism............................................................................................................................... 16 Muscle Contraction............................................................................................................................................ 17 Summary – skeletal muscle contraction........................................................................................ 17 Muscle fatigue.................................................................................................................................................... 18 Types of muscle fibres........................................................................................................................................ 19 Effects of training on muscle.............................................................................................................................. 19 EMG.................................................................................................................................................................... 19 Diseases.............................................................................................................................................................. 21 Neurogenic lesion/myogenic lesion................................................................................................................... 21 Smooth muscle........................................................................................................................... 23 Smooth muscle contraction................................................................................................................................ 24 Phases of smooth muscle contraction................................................................................................................ 25 Control questions - muscles............................................................................................................................... 26 Body fluids................................................................................................................................. 29 Blood......................................................................................................................................... 31 Plasma proteins.................................................................................................................................................. 31 Erythrocytes (RBC).............................................................................................................................................. 32 RBCs degradation............................................................................................................................................... 34 Hematocrit.......................................................................................................................................................... 36 MCV.................................................................................................................................................................... 37 MCH.................................................................................................................................................................... 37 MCHC.................................................................................................................................................................. 37 Leukocytes (WBC)............................................................................................................................................... 38 Thrombocytes.................................................................................................................................................... 40 Hemostasis......................................................................................................................................................... 41 Step 1 – vasoconstriction............................................................................................................... 41 Step 2 – Thrombocyte plug formation (activation of thrombocytes)............................................ 41 1 Fatima’s notes 2022-2023 Physiology 1 Step 3 – Blood clotting................................................................................................................... 42 Vitamin-K dependent clotting factors............................................................................................ 44 O2-hemoglobin dissociation curve.................................................................................................................... 47 Filtration............................................................................................................................................................. 49 Blood typing....................................................................................................................................................... 50 A-B-O blood type system.................................................................................................................................... 50 Rh-blood type system........................................................................................................................................ 52 Erythrocyte sedimentation rate......................................................................................................................... 53 Osmotic resistance............................................................................................................................................. 55 Control questions - blood................................................................................................................................... 56 Cardiovascular system................................................................................................................ 57 Cardiac cycle....................................................................................................................................................... 60 Phases of ventricular systole.............................................................................................................................. 60 Phases of ventricular diastole........................................................................................................ 61 Cardiac muscle................................................................................................................................................... 63 The conduction system of the heart.................................................................................................................. 65 Action potentials................................................................................................................................................ 69 Action potential of nodal tissue..................................................................................................... 69 Action potentials of atrial and ventricular muscles....................................................................... 70 ECG..................................................................................................................................................................... 72 Bipolar limb leads- Einthoven triangle............................................................................................................... 75 Unipolar limb leads – Goldberger augmented leads......................................................................................... 75 Unipolar chest leads – Wilson terminals............................................................................................................ 76 Electrical axis of the heart.................................................................................................................................. 76 Pathological ECGs........................................................................................................................... 78 Heart sounds, PCG.............................................................................................................................................. 78 Percussion and auscultation.......................................................................................................... 79 Innervation of the heart..................................................................................................................................... 80 The effect of parasympathetic innervation of the heart................................................................................... 81 The effect of sympathetic innervation of the heart........................................................................................... 82 The effect of ions and transmitters on the heart (lab)...................................................................................... 83 The regulation of the BP..................................................................................................................................... 84 Cardiovascular reflexes...................................................................................................................................... 86 Autoregulation................................................................................................................................................... 90 Starling Heart-lung preparation......................................................................................................................... 92 Vessels....................................................................................................................................... 94 Regulation of capillary permeability.................................................................................................................. 97 Respiratory system..................................................................................................................... 98 2 Fatima’s notes 2022-2023 Physiology 1 Static and dynamic lung parameters.................................................................................................................. 98 Obstructive respiratory disorders................................................................................................ 102 Restrictive respiratory disorders.................................................................................................. 102 Pressures in the respiratory system................................................................................................................. 105 Kussmaul breathing pattern (kissing mouth)............................................................................... 105 Cheyne-Stokes breathing pattern................................................................................................ 105 Innervation of the lungs................................................................................................................................... 107 Sympathetic innervation.............................................................................................................. 107 Parasympathetic innervation....................................................................................................... 107 Central regulation of the respiration............................................................................................................... 109 Valsalva manoeuvre......................................................................................................................................... 110 Müller manoeuvre............................................................................................................................................ 110 Lab questions................................................................................................................................................... 111 GI-TRACT...................................................................................................................................112 Innervation of the GI........................................................................................................................................ 112 Saliva................................................................................................................................................................. 113 (O)esophagus................................................................................................................................................... 114 Stomach............................................................................................................................................................ 114 HCL.................................................................................................................................................................... 115 Hormones of GI................................................................................................................................................ 115 Gastric juice production................................................................................................................................... 118 Phases of gastric juice production............................................................................................... 118 Pancreas........................................................................................................................................................... 119 Bile.................................................................................................................................................................... 120 Small intestine.................................................................................................................................................. 123 Large intestine (colon)...................................................................................................................................... 123 Vitamins............................................................................................................................................................ 125 BAO/MAO/PAO................................................................................................................................................ 125 Basal gastric secretion.................................................................................................................. 125 Maximal gastric acid secretion..................................................................................................... 125 PAO............................................................................................................................................... 126 How could we determine BAO, MAO, and PAO in the lab?......................................................... 126 Metabolism...............................................................................................................................128 Kidneys.....................................................................................................................................133 Filtration, Nephron........................................................................................................................................... 134 Clearance.......................................................................................................................................................... 136 Proximal convoluted tube................................................................................................................................ 137 3 Fatima’s notes 2022-2023 Physiology 1 Loop of Henle................................................................................................................................................... 139 Distal convoluted tubule.................................................................................................................................. 140 Collecting duct.................................................................................................................................................. 140 ADH (antidiuretic hormone/vasopressin).................................................................................... 140 ANP – atrial natriuretic peptide................................................................................................... 142 Aldosterone.................................................................................................................................. 142 Angiotensin-Renin aldosterone system........................................................................................................... 143 pH regulation............................................................................................................................146 Questions!.................................................................................................................................... 148 Keep in mind, small mistakes and misspelling may occur. 4 Fatima’s notes 2022-2023 Physiology 1 Neurons Neurons are information messengers that uses electrical impulses and chemical signals to transmit information between the brain and the rest of the nervous system. The four-compartment model of the neuron consist of the dendrites (input), the axon hillock (integration), axon (conductor) and the synapse (output). We have different types of neurons by function: Afferent (sensory) neurons Efferent (motor) neurons Interneurons (connects other neurons) Associative neurons (only CNC) In addition to the four-compartment model, the neurons also contain glial cells that can form a myelin sheath around the axons. These cells function to increase the membrane resistance (increases conduction velocity), gives electrical isolation and maintain extracellular homeostasis. The types of cells that forms the myeline sheath are the oligodendroglia in the CNS and optic nerve, and the Schwann-cells in the PNS and around most of the cranial nerves (V, VII, VIII, IX). There are Schwann cells around the unmyelinated axons as well, but they don’t form a sheath. The firing rate shows the frequency, meaning the number of action potentials of the neuron per time. Myelinated fibers have higher conduction velocity due to the excitation spreading in a saltatory movement form one node of Ranvier to the other. The node of Ranvier is the bare membrane sections between the adjacent myelinated axons sections. The membrane under the myeline sheath, between two nodes of Ranvier, contains mostly rapid K+ channels. The rapid Na+ channels are located at the membrane of the nodes of Ranvier. 5 Fatima’s notes 2022-2023 Physiology 1 What is the name of the cells that forms the myeline sheet in the CNS? Oligodendroglia (oligodendrocytes. Where are the fast voltage gated calcium channels mostly located in the neuron? Axon hillock (but is found also along the axon, and at the axon terminal). Where is the action potential generated in a neuron? From the axon hillock. Where are the fast voltage gated sodium channels found in the myelinated neuron? In the node of Ranvier. Properties of the different fibre types Ion channels The rapid voltage gated Na+-channels belongs to the voltage dependent channels. They have 2 gates, one activation (faster) and one inactivation (slower) gate. Ion flow occurs only if both gates are opened. The activation gate is closed during resting potential and opened rapidly by depolarization when the threshold is reached. The inactivation gate is closed by depolarization and makes the fast 6 Fatima’s notes 2022-2023 Physiology 1 repolarization possible. During repolarization it is reopening slowly, meanwhile the activation gate is closing more quickly. The channel is not open during entire repolarization. Blockers of voltage gated Na+-channels: Lidocaine (local anaesthetic), tetrodotoxin (TTX), Pronase Blockers of voltage dependent K+-channels: Tetraethylammonium (TEA) Blockers of Ca2+- channels: Verapamil Different types of membrane potentials Resting membrane potential is the potential difference between the inner and outer surface of the membrane in resting conditions. During this potential the Na+ concentration in the extracellular space is high, and the K+ concentration intracellularly is high. It remains so even at the peak of the action potential. This is maintained by the Na+/K+ ATPase (pump). The sodium-potassium ATPase pumps 3 NA+ outwards, and 2 K+ inwards at the cost of one ATP. In addition. The K+ channels have a passive outflow of K+. Neurons: - 70 mV Muscle: - 90 mV Postsynaptic potential are changes in membrane potential that move the cell from its resting state and is generated by ligand-gated ion channels. We have excitatory postsynaptic potentials (EPSP) which causes depolarization, and inhibitory postsynaptic potentials (IPSP) which makes the neuron less likely to generate an action potential (hyperpolarization). End plate potential (EPP) causes depolarization of skeletal muscle fibers caused by neurotransmitters binding to the postsynaptic membrane in the neuromuscular junction. One end plate potential is enough to cause contraction in the post-synaptic skeletal muscle cell (action potential, but one EPSP is not enough to evoke an action potential. AP EPSP, IPSP Fast voltage gated Na+ channels required No fast voltage gated Na+ channels Have refractory period (= no refractory period) Can be inhibited by TTX Less energy needed to evoke No refractory period Amplitude decreases with time and distance 7 Fatima’s notes 2022-2023 Physiology 1 Action potential is generated by voltage-gated ion channels → the ion currents are able to change the membrane potential, but the ion concentration itself do not significantly change. Potential changes above the threshold can evoke action potential. All or none law; if the action potential occurs, its propagated to every part of the membrane, and the amplitude of the action potential does not change with increasing stimulus intensity. How can you block the fast voltage gated sodium channels? Local anastatic drug (lidocaine), TTX Where is the fast voltage gated potassium channels located in the myelinated neuron? Everywhere under the myeline sheath. How can we inhibit the voltage gated potassium channels TEA (tetraethylammonium) How is the resting membrane potential of the neuron maintained? Na+/K+- ATPase (3 Na+ in, 2 K+ out at the cost of one ATP) Below the threshold, which ion channels should open? Ligand gated ion channels Name two excitatory neurotransmitters, and two inhibitory neurotransmitters. Excitatory: Glutamate, aspartate Inhibitory: GABA, glycine In case of excitatory synapse, which ion should inflow/outflow? The membrane gets less negative. Inflow = sodium and/or calcium (=EPSP). In case of IPSP, which ion should inflow/outflow? Membrane gets more negative. Outflow= K+, Inflow= Cl-. 8 Fatima’s notes 2022-2023 Physiology 1 Equilibrium membrane potential of Na+ The equilibrium potential is the membrane potential when there is a balance between the IC and the EC fraction of a certain flow (inflow = outflow) for one particular ion. The proportion of the ion concentrations on the two sides does not significantly change during depolarization/repolarization, the equilibrium potential remains more of less constant. How can we balance the equilibrium membrane potential of the Na +? Outward electrical gradient as well as an inwards electrical gradient. At which membrane potential can we see an outward electrical gradient for sodium? Positive or negative membrane potential? Positive membrane potential. The equilibrium membrane potential of sodium is +55mV. We set the membrane potential to + 70 mV, will there be a sodium inflow or outflow? Outflow (to get the membrane potential down). The equilibrium membrane potential of sodium is +55mV. We set the membrane potential to + 30 mV, will there be a sodium inflow or outflow. Inflow The equilibrium membrane potential of sodium is +55mV. We set the membrane potential to 0 mV, will there be a sodium inflow or outflow? Inflow The equilibrium membrane potential of sodium is +55mV. We set the membrane potential to -60 mV, will there be a sodium inflow or outflow? Inflow. In what case will there be strongest sodium inflow, at 0 mV, -60 mV, - 70 mV or -120 mV? - 120 mV. The equilibrium membrane potential of sodium is +55mV. We set the membrane potential to +55 mV will there be a sodium inflow or outflow? Net sodium flow will be = 0 9 Fatima’s notes 2022-2023 Physiology 1 Skeletal muscle The functional unit of the skeletal muscle is the muscle fibre, which varies in length depending on which muscle we are looking at. The muscle fibres are multinucleated cells that are approximately 10-100 um in diameter. The skeletal muscles fibres can be innervated by the A-alpha motoneurons. In the periphery (e. g. biceps) these motoneurons are in the anterior horn of the spinal cord, and their axons can from there reach the muscle fibres. These neurons have the biggest axons in diameter, and therefor also the highest conduction velocity. A-α motoneurons 70-120 m/s Diameter: 15 μm Myelinated axons Glia cells – Schwann cells The functional unit of the skeletal muscle function is the motor unit, which consist of the motoneuron and all the muscle fibres it innervates. Each muscle fibre is innervated by only one motoneuron, while one motoneuron can innervate numerous muscle fibres (10-1000). The higher the innervation ratio is, the higher number of muscle fibres does the motoneuron innervate. This ratio depends on the type of muscle. Motoneurons that innervates smaller muscles with finer movements has a lower innervation ratio, for the neurons that for example innervates the extraocular muscles that moves the eyeballs (3-10 muscle fibres). Other muscles like the quadriceps have numerous muscle fibres, and one motoneuron here innervates around 1000 muscle fibres, and therefore these neurons have a much higher innervation ratio. What is the motor unit potential? Sum of action potential from one motor unit. Can a muscle fibre be innervated by more than one motoneuron? No, one muscle fibre is only innervated by one motoneuron. 10 Fatima’s notes 2022-2023 Physiology 1 Neuromuscular junction The neuromuscular junction is the synapse between the muscle and the motoneuron. The action potential travels from the cell body and down the myelinated axon. The action potential moves in a saltatory movement in the myelinated axon, from one node of Ranvier to the next. This movement, that only finds place in the myelinated axons, increases the conduction velocity of the action potential. At the axon terminal, there are voltage gated calcium channels. These channels have voltage sensitive sensors and are activated by action potential. When the action potential reaches the axon terminal, the voltage gated calcium channels will open, and there will be a calcium inflow. The inflow happens due to the electrochemical gradient. Since the calcium concentration inside the axon terminal (intracellular space) is lower than of the extracellular space, there will be an inflow of calcium ions when these channels open. The calcium ions are particularly important for the release of neurotransmitters. The neurotransmitters are located inside vesicles at the axon terminal, and due to the calcium signal, the membrane of these vesicles’ fuses with the membrane of the axon terminal, causing the release of the neurotransmitters into the neuromuscular junction. 11 Fatima’s notes 2022-2023 Physiology 1 Acetylcholine is the neurotransmitter for skeletal muscles. They bind to muscle type nicotinic acetylcholine receptors. These receptors are ligand gated ion channels. When acetylcholine attaches to these receptors, the sodium channels open, and due to the electrochemical gradient, sodium flows into the muscle cell. The concentration of sodium is (10x) higher in the extracellular space than of the intracellular space. When there is a sodium inflow, the membrane potential changes positively. The resting membrane potential (RMP) of a skeletal muscle is normally - 90 mV. This potential is maintained by sodium-potassium-ATPase. This pump pumps out 3x Na+ out and 2x K+ in. When the membrane is getting less and less negative, we call this depolarization or hypopolarization, and in case of skeletal muscle we also call it the “end plate potential”. Resting membrane potential: Neuron: -70 mV Skeletal muscle: -90 mV Along the whole muscle cell membrane, we have also have2 voltage gated sodium channels, and when the end plate potential reaches the threshold, these channels open, and there will be even more sodium inflow into the cell. An action potential is evoked. This action potential can spread throughout the whole muscle fibre. When the membrane is getting more negative again, the membrane will be repolarised. When the membrane is more negative than the resting membrane potential, we call this the so-called hyperpolarization. 12 Fatima’s notes 2022-2023 Physiology 1 Sarcoplasmic reticulum (L- and T- tubules) The triad: 1 T-tubule (transverse tubule) 2 Terminal cisternae (connected to longitudinal tubules, L-tubules) Penetration of the action potential all the way through the muscle fibre from one side to the other is achieved by transmission along the T-tubules (transverse tubules), which are internal extensions of the cell membrane. When the action potential reaches the T-tubule, where we have DHP (dihydropyridine) receptors, the voltage change is sensed by these receptors. The ryanodine receptor is attached to the DHP receptors, so when the DHP receptors are activated, it triggers the ryanodine receptors to release calcium. Calcium then binds to troponin-C inside the cell. What is the name of the T-tubule and the Terminal cisternae together? Sarcoplasmic reticulum What is the main function of the sarcoplasmic reticulum? Calcium storage 13 Fatima’s notes 2022-2023 Physiology 1 Where are the DHP (Dihydropyridine) receptors situated? Function? The wall of the T-tubule, function is to activate ryanodine receptors Where is the ryanodine (calcium release channels) receptors situated? Function? Wall of the terminal cisternae, functions to release calcium Inside the T-tubule, is there intracellular or extracellular space? Extracellular space Is the calcium source for the muscle contraction in skeletal muscle intracellular or extracellular? Intracellularly, it comes from the sarcoplasmic reticulum, which is inside the cell How many calcium ions can bind to one molecule of troponin C? 4 calcium ions 14 Fatima’s notes 2022-2023 Physiology 1 Sarcomere The sarcomere is the functional unit of the muscle fibre. The optimal length of a sarcomere to exert the maximal contraction force is between 2-2.2 um. Sarcomeres are repeating units between two adjacent Z-lines and consists of thin and thick filaments. Actin filaments thin filament anchored to the Z-lines by dystrophin and supported by nebulin (as long as the actin). In the middle we have the myosin, which is the thick filament. In the middle of the sarcomere, we have the so-called M-protein or M-line. Titin comes from the Z-line to the myosin and then back, and attaches the myosin to the Z- line, meaning it is as long as the sarcomere. Bands A-band is as long as the myosin itself, contains both H-band is the length between the two actin molecules, contains only thick filaments I-band if from the end of one myosin to the next, contains only thin filaments What are the thin filaments? Actin Dystrophin Nebulin Tropomyosin Troponin I, Troponin C, Troponin T How long is the titin compared to the sarcomere itself? As long as the sarcomere! 15 Fatima’s notes 2022-2023 Physiology 1 Do we have DHP receptors in the heart? Do we have ryanodine receptors in the heart? Yes, and yes. How long is the nebulin? Function? As long as the actin, functions to support it. In case of contraction, what band(s) gets shorter? The H- and I-band gets shorter during contraction. Sliding filament mechanism During relaxation of the muscle the is no calcium signals, and we only need ATP to relax the muscle. The calcium has to be moved back from the myoplasm to the sarcoplasmic reticulum. The active transporter calcium-ATPase can be found along the L-tubule, and pumps back the calcium to the sarcoplasmic reticulum. This means that during relaxation there is no calcium in the myoplasm. The thick filament has myosin “heads and necks”. During muscle relaxation, there is no interaction between the actin and myosin, because the so-called troponin-tropomyosin complex occupies the myosin binding site on the actin filament. Troponin-tropomyosin complex consist of: Tropomyosin Troponin I (I = inhibits) Troponin C (C = binds to calcium) Troponin T 16 Fatima’s notes 2022-2023 Physiology 1 Muscle Contraction For the muscle activation we need both ATP and calcium. When troponin C binds to calcium, the conformation of the troponin C changes, and thus also the conformation of the troponin- tropomyosin complex. When the muscle is contracting, the angle between the myosin head and the myosin neck is 45 °, and during relaxation it is 90 °. The myosin head binds to the actin site, and the head tilts from 90° to 45°, and myosin can slide towards the Z-line. The change in degree of the myosin head and neck is only changes in micrometres, how is the muscle able to be shorter with cm? The myosin head is able to bind to the next actin, and the next, and so on, causing the contracting movement. Is the amplitude of the contraction higher in skeletal muscle or in smooth muscle? Smooth muscle (think about how the uterus, which has smooth muscle, is able to almost contract back completely after birth). Summary – skeletal muscle contraction The action potential of the activated neuron reaches the axon terminal where voltage gated calcium channels are located. When these channels open, there is a calcium inflow. The calcium signal causes neurotransmitter release, in this case the acetylcholine found inside vesicles in the motoneuron. Acetylcholine gets released into the neuromuscular junction (which is the synapse between the neuron and the skeletal muscle cell) and binds to nicotinic acetylcholine receptors on the cell membrane of the muscle. When these receptors are activated, there will be a sodium inflow. When the threshold is reached, the ligand gated sodium channels will also open, and there will be even more sodium inflow. The membrane gets depolarized, and which spreads throughout the muscle cell membrane through the T-tubules. In the T-tubules, the action potential reaches the DHP receptors, which trigger the ryanodine receptors to release calcium intracellularly from the sarcoplasmic reticulum to the sarcoplasm. Calcium binds to troponin C, and the conformation of the tropomyosin troponin complex changes, and consequently the troponin I cannot inhibit the interaction between actin and myosin anymore. The angle between the myosin head and myosin neck bind changes from 90° to 45° and binds to the actin – resulting in a sliding filament mechanism which causes a muscle contraction. 17 Fatima’s notes 2022-2023 Physiology 1 Muscle fatigue Muscle fatigue is a reversible decrease in the ability of the skeletal muscle to exert force in response to physical activity. We have peripheral, central, and mental (psychic) fatigue. The significance of the muscle fatigue is to protect the body from complete/final depletion. The first stage of fatigue is first when then feeling of the fatigue appears, and then later the performance decreases as response. Peripheral: Motoneurons and/or muscle fibres are responsible 1. Transmissional: lack of acetylcholine 2. Contractional: lack of energy sources, ions Central: CNS is involved, main cause is hypoglycaemia Mental (physic): passing by the point where it hurts Motivation, tolerance, attention, and focus are important factors What are the causes of peripheral fatigue? Lactic acid formation - pH↓ Hypoxia Hypoglycaemia Hyperthermia Dehydration In what order does the muscle use the energy sources - phosphocreatine, ATP, Liver glycogen and muscle glycogen? ATP, phosphocreatine, muscle glycogen, liver glycogen When there is no ATP, what will happen with the muscle? There will not be any muscle contraction, nor muscle relaxation. The muscle will turn “stiff”, and this is what we call the “Rigor Mortis”. 18 Fatima’s notes 2022-2023 Physiology 1 Types of muscle fibres Type l-A Type ll-A Type ll-B Red fibres, caused by the myoglobin White fibres, caused by the glycogen Slow-twitch muscle It is similar to Fast-twitch muscle Aerobic glycolysis both, so it is an Anaerobic glycolysis Muscle fatigue shows later “In-between” Muscle fatigue shows early Antigravitational muscle type. More lactate production Lots of mitochondria E.g., 100m sprinters and weightlifters E.g., in Marathon runners (hours) Do the numbers of muscle fibres increase when you work out the muscle regularly? No, only the SIZE increases – hypertrophy. Effects of training on muscle Hypertrophy (bigger, stronger muscles) O2 uptake can increase Function and number of mitochondria can increase Aerobic enzyme capacity can increase – low intensity long training Anaerobic enzyme can increase – high intensity interval training Lactate tolerance can increase What is muscle strain caused by? Microtrauma = sterile inflammation in the muscle. EMG EMG stand for electrical myography and can record the electrical activity of the skeletal muscle. With deep electrodes the motor neuron potential can also be detected. In addition to the electrodes, we need an AD-instruments (transducer, amplifier, filter, AD-converter, recording device). Surface electrodes (low risk of infection, less precise) Deep electrodes (high risk of infection, painful, more precise. 19 Fatima’s notes 2022-2023 Physiology 1 In EMG recordings, what does the transducer, amplifier, filter, and A/D-converter do? Transducer – converts the mechanical signal into an electrical signal Amplifier – increases the amplitude Filter – filters out parts of the noise, e.g., to examine signals between 0,5Hz and 50 Hz, we use a 0,5Hz high pass filter and a 50Hz low pass filter A/D-converter - converts the analogue signal to digital signal What is the minimum stimulus threshold? The single pulse with lowest intensity that can induce a single twitch. When does the amplitude of the contraction force stop increasing? Above a certain stimulus intensity called the maximal recruitments threshold. What happens if there is given 2 stimuli, and the second AP arrives before the muscle is fully relaxed? The force of the next contraction will be greater, a phenomenon called summation or superposition. If the stimulus frequency is increased (e. g. 8Hz) and the muscle doesn’t relax complete in between the stimulus, what will happen? The individual twitches will fuse into a single sustained contraction. If the individual twitches are still recognisable - incomplete tetanus, and if they fuse together completely – complete tetanus. 20 Fatima’s notes 2022-2023 Physiology 1 Diseases Myasthenia gravis Myasthenia gravis is a chronic autoimmune, neuromuscular disease that causes weakness in the skeletal muscles. This is because the body produces antibodies against the nicotinic acetyl receptors (N-Ach-R) in neuromuscular junction. Malignant hyperthermia The malignant hyperthermia is a very rare disease caused by a mutation of the ryanodine receptors and causes a severe reaction to certain drugs used for anaesthesia. When the ryanodine receptors get activated by these drugs it causes some muscle activation, resulting in production of heat. The body temperature of the patient raises = hyperthermia. This is the reason to why monitoring the body temperature during operation is so important. How is malignant hyperthermia treated under surgery? The main treatment for malignant hyperthermia is a drug called dantrolene. Anaesthesiologists administer this drug immediately if they suspect malignant hyperthermia. They also stop giving the aesthetic, and the surgeon ends the surgery as soon as possible. Neurogenic lesion/myogenic lesion Neurogenic lesions In case of EMG examination, in neurogenic lesion there will be spontaneous activation of the muscle. You ask the patient to relax the muscle, but there will still be some EMG signals (spontaneous activity). The motor unit potential is a giant, usually the signal less than 1 mV, but in neurogenic lesion it can be much bigger than this. No complete interference here. Myogenic lesion In the myogenic lesion the amplitude of the motor unit potential and maximal contraction is smaller than normal. There is no resting/spontaneous activity here, meaning there is an isoelectric line in case of relaxation (straight line). Complete interference is present, but with small amplitude. What is wrong if the amplitude is large, and there is no complete interference in case of maximal contraction? Neurogenic lesion 21 Fatima’s notes 2022-2023 Physiology 1 In what case do we have resting activity? Neurogenic lesion. 22 Fatima’s notes 2022-2023 Physiology 1 Smooth muscle Smoot muscle is far smaller than the fibres of the skeletal muscle. They are usually 1-5 um in diameter, and only 20- 500 um in length. Smooth muscle is composed of myocytes, which are thin elongated cells with only one nucleus. They include cell organelles (e.g., mitochondria) and filaments – actin and myosin. The arrangement of the actin and myosin filaments are not the same as of the skeletal muscle. A large number of actin filaments are attached to the dense bodies. As we can see on the drawing, a large number of actin filaments are radiating from two dense bodies, and the ends of these filaments overlap a myosin filament that is located midway between the two dense bodies. The dense body of the smooth muscle serves the same role as the Z-disks in the skeletal muscle. Some of the dense bodies is attached to the cell membrane. In the smooth muscle, most of the myosin filaments have a “side polar” cross bridges arranged so that the bridges hinges on each direction. The configuration of the smooth muscle allows the myosin to pull one actin filament in one direction on one side while simultaneously pulling another actin filament in the opposite direction. This allows smooth muscle cells to contract as much as 80 percent of their length, instead of the 30 percent as of the skeletal muscle. Types of smooth muscle (based on intercellular connections): Multi-unit: smooth muscle cells than can function separately, and there is usually no action potential here that initiates the contraction (only tonic contractions), but by a transmitter. They have no inhibitory innervation. E.g., uterus, respiratory m. (bronchi, bronchiole). Single unit: when more muscle cells form functional unit where the stimulus spreads to thew other cells by gap junctions. The contractions are usually cause by an action potential – phasic contractions. E.g., pupillary muscle (pupillary sphincter and dilator m.), arrector pili. 23 Fatima’s notes 2022-2023 Physiology 1 Smooth muscle contraction Contraction in smooth is not voluntary, and contraction can be caused by neurotransmitters of vegetative nerves, where the postganglionic fibres form terminal plexus (varicosities) around smooth muscle (vegetative ground plexus - diffuse branching of fibres). The transmitter is released into the interstitial fluid, and from there it gets to the muscle. Contraction can also be caused by hormones (e.g., adrenaline, noradrenalin), local metabolites and mechanical effects. In place of troponin, smooth muscle contains a large amount of another regulatory protein called calmodulin. It is similar to troponin, but it initiates contraction differently. Calcium concentration in the cytosolic fluid increases as a result of the calcium influx from the extracellular fluid trough calcium channels, and the calcium release from the sarcoplasmic reticulum. The calcium ions bind reversibly to calmodulin-calcium. Then, the calmodulin complex joins and activates myosin light chain kinase, a phosphorylating enzyme which phosphorylates the myosin light chain of each myosin head, also called the regulatory chain. When the phosphorylation happens, the head has the capability of binding repetitively with the actin filament and proceeding through the entire cycling process of intermittent “pulls”, thus causing a muscle contraction. In skeletal muscle contraction, all the calcium ions needed is produced by the sarcoplasmic reticulum, but in the smooth muscle the source differs. Most of the calcium ions needed for contraction in the smooth muscle, comes from the extracellular space at the time of the action potential or other stimulus. There are 2 types of action potential in smooth muscle – one with (e.g., arteries) and one without (e.g., antrum pyloricum) the plateau phase. The ratio between actin and myosin here is 15/1, and in skeletal muscle its 2/1. 24 Fatima’s notes 2022-2023 Physiology 1 Phases of smooth muscle contraction Resting phase: the myosin heads are not bound to the actin filaments (bound to ATP). Depolarization: action potential results in Ca2+ inflow to the cell, depolarization happens. Activation: The Ca2+ bind to calmodulin, activating the myosin light chain kinase. This enzyme phosphorylates the regulatory subunit of the myosin head by the cost of ATP, which activates the ATPase activity of myosin. Contraction: the phosphorylated myosin head binds to actin and the formation of cross bridges happens, ATP hydrolysis and the angle of the crossbridge changes from 90 ֯ to 45 ֯. The thin filaments (actin) are then dragged with the myosin –> sliding filament mechanism. Maintaining phase: this phase is only present in smooth muscles, and a few ATP molecules is used in this phase. Repolarization: ADP dissociates from the myosin head, and the Ca2+ level normalizes mainly by Ca2+ pump (-ATPase), 3Na+/Ca2+ exchange of plasma membrane. Relaxation: this phase starts when the Ca2+ level decreases, the myosin head binds off from actin, straightens back to 90֯ , and binds to a new ATP. Potassium is also important here! What is not present in the smooth muscle, but in skeletal muscle? No sarcomere No Z-line No tropomyosin-troponin complex What is tonic and phasic contraction of smooth muscle? Tonic – long AP with plateau phase, long lasting contraction Phasic – short-lasting, rhythmic What is the Ca2+ source in case of the smooth muscle? Both intracellularly and extracellularly. What is the innervation of smooth muscle? Autonomic (involuntary). What is the resting membrane potential of smooth muscle? (-40) – (-70) mV (unstable) 25 Fatima’s notes 2022-2023 Physiology 1 What is the duration of the action potential in smooth muscle vs skeletal muscle? Longer than 100 ms (skeletal muscle: 4-6 ms). What channels are important in contraction of smooth muscle? Ligand gated Ca2+ channels, K+ channels, voltage gated Ca2+ channels What is the supporting structure of actin in case of smooth muscle? Dens plaque. What is the phosphate source in smooth muscle? ATP. What is the function of calmodulin? To activate the MCL (myosin light chain) kinase. Control questions - muscles Can you receive signals from both the agonist and antagonist muscle at the same time? Yes, it is. Both muscles are working. What can the long-term effects of muscle training be on the human body? Increased oxygen uptake capacity Increased glucose uptake capacity Increased number and function of mitochondria Increased aerobic capacity Increased anaerobic glycolysis Increased lactate tolerance What is the function of troponin I and troponin C? Troponin I inhibit the binding of actin and myosin, troponin C binds to calcium. In case of the skeletal muscle, is the calcium signal intracellular of extracellular? Intracellular. Can the skeletal muscle be tetanized? Yes. 26 Fatima’s notes 2022-2023 Physiology 1 What is the optimal length of a sarcomere for maximal contraction force? 2-2,2 um. How long is the titin? How long is the nebulin? The titin is as long as the sarcomere. The nebulin is as long as the actin. What does the A-band contain? Both thick and thin filaments. What does the I- band contain? Thin filaments. What does the H-band contain? Thick filaments. If we add curare to a skeletal muscle, how will the membrane potential change? Curare is a non-depolarisation muscle relaxer, it doesn’t change the membrane potential, it only inhibits the nicotinic Ach receptors. Is the effect of curare reversable or irreversible? Reversable What is indirect and direct stimulation of a muscle? Direct: stimulate the muscle directly Indirect: stimulate the nerve that stimulates the muscle If you place the nerve-muscle preparation into curare solution, and stimulate it both directly and indirectly, in what case(s) will there be a contraction? Only when stimulated directly. Curare inhibits the receptors on the muscle membrane and can therefore not contract in case of indirect stimulation. If you only place the nerve into curare solution, and stimulate the muscle both directly and indirectly, in what case(s) will there be a contraction? 27 Fatima’s notes 2022-2023 Physiology 1 In both direct and indirect stimulation. Curare only inhibits the nicotinic Ach receptors on the muscle. If you increase the stimulus intensity (voltage) in the sim-muscle program, will the amplitude of the contraction increase, decrease or not change? Depends. When you reach the maximal threshold, the maximal recruitment (of muscle fibres) is also reached, and the amplitude of the contraction can’t increase more. What did we use to measure the contraction of the muscle if you stimulate the ulnar nerve? Stimulator (placed over the nerve) Ground electrode Electrode pair (2x) EMG electrode AD-setup Why did the grip force decrease in the measurement of the muscle strength? Because of muscle fatigue What are the factors that can influence the muscle fatigue? Nutrition Drug Sleep Physical state Mentality We know that the BP increases during exercise, but is it mainly the systolic or diastolic pressure that increases? Systolic, since more blood needs to be pumped out of the heart. What cells has membrane potential? What cells has action potential? All living cells has membrane potential. Only the excitable cells like the skeletal, neural, and cardiac cells have the ability to generate action potential. 28 Fatima’s notes 2022-2023 Physiology 1 Body fluids About 60% of the body weight of an adult human is fluid, a water solution with ions and other substances. Two thirds of this body water is intracellular fluid, and one third is extracellular fluid. The intracellular fluid is kept inside the cells of our bodies, while the extracellular fluid is in constant motion throughout the body. The total body water (TBW) of an average man that weights 70kg, is 42 kg (70kg/60%*100=42kg). The difference between the intracellular and extracellular fluid is the composition of the ions in the fluid. In the extracellular fluid we have a large amount of sodium, chloride, bicarbonate, and nutrients for the cells such as oxygen, glucose, fatty acids, and amino acids. The extracellular fluid also contains carbon dioxide that will be excreted trough the lungs, and waste products that are being transported to the kidneys. The intracellular fluid on the other hand contains a large amount of potassium, magnesium, and phosphate ions. Concentrations is the extracellular space (blood plasma): Sodium ion (Na+): 135-152 mmol/L Chloride ion (Cl-): 96-106 mmol/L Bicarbonate ion (HCO3-): 22-28 mmol/L Potassium ion (K+): 3.8-5.2 mmol/L Calcium ion (Ca2+): 2.2-2.8 mmol/L (50% free and 50% bound to plasma proteins) Magnesium ion (Mg2+): 1 mmol/L Monohydrogen phosphate ion (HpO2-4): 1 mmol/L (+ Glucose (fasting): 4-5.5 mmol/L, Glucose (after meal): 3.5-8.5 mmol/L) We can measure the fluid volumes by using different dilution methods: 29 Fatima’s notes 2022-2023 Physiology 1 The total body water can be measured by (heavy water) - D2O (deuterium oxide) or T2O (tritium oxide), or with antipyrine molecule. The person gets injected with the heavy water or the antipyrine. These molecules will spread throughout the body fluids, and after a while you take a blood test, and measure its concentration (since you know how much you injected). To measure the extracellular fluid, you can use the inulin dilution method (sugar molecule) or Mannitol (manit). The inulin can get into the interstitial and the intravasal fluid, but not in the intracellular fluid. So, with this method you can measure only the extracellular fluid. The plasma volume can be measured by injecting iodinated 131 I Albumin or inject Evans blue, and then measure its concentration. In case of too high and too low concentrations of sodium in the extracellular space, what do we call it? Too high: hypernatremia Too low: hyponatremia The potassium concentration in the extracellular space is higher than normal values, what is it called? Hyperkalaemia (can cause irregular heart rhythm or stop the heart) What is hypokalaemia? Why is it dangerous? Too low levels of potassium. It’s dangerous because it can cause arrythmia. Where is the concentration of calcium (Ca2+) higher? In the intracellular space or extracellular space? Intracellular space What does hypomagnesia mean? What can it cause? To little magnesium in the extracellular space, can cause irregular heart rate. What is the dominant ion in EC and IC respectively? EC: sodium, Na2+, IC: potassium K+ 30 Fatima’s notes 2022-2023 Physiology 1 Blood The blood volume in humans is 5-6L, and it consist of cellular elements and blood plasma. The blood plasma consists of mainly water (more than 90%) and inorganic and organic elements. How can you measure the plasma volume? 𝑃𝑙𝑎𝑠𝑚𝑎 𝑣𝑜𝑙𝑢𝑚𝑒 = 𝑏𝑙𝑜𝑜𝑑 𝑣𝑜𝑙𝑢𝑚𝑒 ∗ (1 − 𝐻𝑡𝑐) Plasma proteins The plasma proteins are important organic parts of the plasma and has many important functions in the blood. Proteins forms the largest mass of organic molecules in our body. Plasma proteins: 60-80 g/L (2/3 albumin, 1/3 globulin + some fibrinogen- 1-4 g/L) What is bloodserum? The blood plasma without the fibrinogen. What are the functions of the plasma proteins? Maintain the colloid osmotic/oncotic pressure They function as buffers – blood pH regulation (normal 7.35-7.45) Maintaining the viscosity 4-6 in blood) Transport (e.g., iron by transferrin, and hormones and ions by albumin) Storage Humoral immune function (immunoglobulins) Haemostasis - Blood coagulation/clotting Participated in metabolic processes Immunoglobulins IgA (for mucosal difference) IgD (on surface of B-lymphocytes) IgE (high in case of allergic reaction) IgM (big molecules, cannot pass placenta, antibodies of A-B-O blood type system) IgG (small molecules, can pass through placenta, antibodies in Rh-blood-type system) 31 Fatima’s notes 2022-2023 Physiology 1 Erythrocytes (RBC) The number, mean diameter and width distribution of RBCs provides us information about anemias and other hematological disorders. Under physiological conditions 1% of every RBCs is replaced each day by reticulocytes, immature red blood cells. These cells need 1-2 days to develop into mature RBCs. The ratio of reticulocytes to mature RBCs reflects the activity of erythropoiesis. Erythrocytes (RBC): ♀: 3.8-5.3 million / μl ♂: 4.3-6 million / μl Size in diameter: 7-8 μm Lifespan: 100-120 days Formation: In the red bone marrow Stimulating protein: Erythropoietin (produced in kidney) What stimulates the kidney to produce erythropoietin (EPO)? Hypoxia, lack of oxygen. How is the RBC’s released as from the red bone marrow? As reticulocytes (immature red blood cells). Which stain was used to determine the reticulocytes count in the lab? Brilliant cresyl blue In case of high RBC count, and low RBC count, what can be the causes? RBC low – anemia (deficiencies – iron, vitamin, erythropoietin, and renal failure) RBC high – erythrocytosis (polycythemia) – dehydration, polycythemia vera In the red blood cell counting, what materials were used in the lab? How could you determine the RBC count? For the red blood cell counting we used the Bürker’s chamber, Hayem’s solution, mixing pipette, coverslip, microscope, and capillary blood sample. To determine the blood cell count, we counted the RBCs in the small square (1/20x1/20x1/10mm). To calculate it we took the average number RBCs in one square and multiplied it with the volume of one square (4000mm), and the multiplied it with the rate of dilution of the sample = 100. 32 Fatima’s notes 2022-2023 Physiology 1 If the average number of RBCs in one small square of the Bürker’s chamber is 10, what is the blood cell count? 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑅𝐵𝐶𝑠 × 𝑣𝑜𝑢𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒 × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 10 × 4000 × 100 = 4 000 000 How could we determine the reticulocyte count? What are the steps and the materials? After staining a glass slide with brilliant cresyl blue staining, we let it dry for some minutes. On top of coverslip, we placed a small drop of blood, turned it upside down and placed it on the stained coverslip. After some minutes we applied one drop of immersion oil on top of the coverslip and then we evaluated the preparation under the microscope using the immersion lens (100x). We counted all the RBCs in one view field and looked for reticulocytes using the meander pattern. What is the normal range of reticulocytes? 0.7-1.5% of the RBCs. What can be the cause of low and high numbers of reticulocytes? Low – erythropoiesis High – increased rate of RBC production in the bone marrow, compensatory process, or a sign of bone marrow tumours producing RBCs in excess. I measurement of the mean size of RBCs, what materials were used and what are the steps? How could we measure the diameter of the RBCs? The material we used was 0.9’% (physiological) NaCl solution, glass slide, covers slip, microscope with an eyepiece graticule, immersion oil, calibration slide and capillary venous sample. After calibrating the scale of the eyepiece graticule and determining the length of one unit, we diluted one drop of blood with a larger drop physiological NaCl solution on the glass slide and placed a coverslip over it. We placed one drop of immersion oil on the coverslip and measured the diameter of 100-200 RBCs under the microscope. To determine the mean diameter of the RBCs we found the average of the measured RBCs. How could we determine the RBC width distribution? 33 Fatima’s notes 2022-2023 Physiology 1 By calculating for each group what percentage of the total number of RBCs measured belongs to that specific group. These percentages were then plotted against the diameters (Price Jones curve). Normal curve shows a symmetric Gaussian curve with a peak at the mean diameter of normal RBC (7- 8 μm) and a narrow base. RBCs degradation Red blood cells have an average life span of 100-120 days. When these cells have lived out their life span, their cell membrane ruptures, and hemoglobin is released. The hemoglobin is then phagocytized by tissue macrophages (reticuloendothelial system). The hemoglobin splits into heme and globin, and from the opened heme ring we get free iron that is recycled and transported into the blood by transferrin (plasma proteins). Biliverdin is also formed but is rapidly reduced to free bilirubin in the spleen, also called unconjugated or indirect bilirubin. Bilirubin is a bile pigment and is water insoluble. The indirect bilirubin is gradually released from the macrophages into the plasma. The indirect bilirubin strongly combines with albumin and is transported throughout the blood. The indirect bilirubin is then absorbed through the hepatic cell membrane and inside the liver cells, where is becomes conjugated bilirubin (active bilirubin). The active bilirubin is then exported to the bile canaliculi and into the intestines. In the intestines the bilirubin is converted into urobilinogen, which is highly water soluble. Some of this biliverdin (about 5%) is excreted by the kidneys into the urine, hence the name urobilinogen. Alternatively, the urobilinogen becomes stercobilinogen. The stercobilinogen Is dark in colour and gives the colour of the faces. If bilirubin is water insoluble, how can it be transported? Since it binds to plasma proteins, which is water soluble. Where does the degradation of the RBCs happen? In the reticuloendothelial system of the spleen. 34 Fatima’s notes 2022-2023 Physiology 1 35 Fatima’s notes 2022-2023 Physiology 1 Hematocrit The hematocrit is the volume proportion of the formed elements in the blood, in other words it’s the ratio of volume of blood cells to the volume of whole blood. The hematocrit depends on the RBC count (99%), Iron, folic acid, vitamin B12, Intrinsic factor, Kidney, EPO, altitude, hypoxia, WBC count, and platelet count. Under physiological conditions like pregnancy, the hematocrit level can also be low, or by anemia and blood loss. The hematocrit can be high in case of dehydration, erythrocytosis and polycythemia vera. Normal values of hematocrit (Htc): ♀: 0.37 – 0.47 / μl = 37-47% ♂: 0.41 – 0.52 / μl = 41-52% What cells contributes to the hematocrit, and how many percentages? Red blood cells – 99% White blood cells – 0.1-0.2% Thrombocytes – 0.8-0.9% How can we determine the hematocrit in the lab? What is the method and what materials is used? To determine the hematocrit level, we first filled a heparin coated capillary with blood from the punctured fingertip. After filling 4/5 of the capillary, we sealed the other end with plasticine and placed the capillary in the centrifuge for 5 minutes (10 000 RPM). To determine the hematocrit, we placed the capillary on the left side of the hematocrit chart with the bottom of the blood column on the “0“line and moved it horizontally until the top of the blood plasma column transected the “100” line. The line of the chart passing through the meeting point of the plasma and the cell column determined the hematocrit value. 36 Fatima’s notes 2022-2023 Physiology 1 MCV The MCV stands for mean corpuscular volume and shows the average size of one red blood cell. The normal range for MCV is 82-92 fl. If the RBC size is higher than 94 fl, it’s the so called macrocytosis, and if the number is lower than 80 fl, it is called microcytosis. 𝐻𝑡𝑐 𝑀𝐶𝑉 = 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 MCH MCH stands for mean corpuscular hemoglobin and shows the average amount of hemoglobin present in a single red blood cell. The normal value for the MCH is between 28-36 pg. If this number is higher, it’s the so-called macrocytic anemia, and if its lower it is called microcytic anemia. [𝐻𝑔𝑏] 𝑀𝐶𝐻 = 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 What can cause microcytic anemia? Iron deficiency. What can macrocytic anemia be caused by? Vitamin B12 deficiency (involved in DNA synthesis), intrinsic factor deficiency (produced by parietal cells, leads to vitamin B12 deficiency), folic acid deficiency (needed for DNA synthesis for the erythropoietin cells). MCHC MCHC stands for mean corpuscular hemoglobin concentration and shows the average concentration of hemoglobin in 1L of red blood cells. The normal values for MCHC are 310-360 g/L (adults). Lack of hemoglobin in the blood may indicate anemia. [𝐻𝑔𝑏] 𝑀𝐶𝐻𝐶 = 𝐻𝑡𝑐 37 Fatima’s notes 2022-2023 Physiology 1 Leukocytes (WBC) The white blood cells are a part of the body’s immune system and help the body fight against. An easy way to remember the different leukocytes is the mnemonic “Never Let Monkeys Eat Bananas”. Leukocytes (WBC): 4000 – 10 000 / μl Neutrophil granulocytes (50-70%): biggest fraction of the white blood cells (9-12 μm) o Young neutrophils (1-4%): if the number of young neutrophils is high (left shift), it could be an indication of a bacterial infection Lymphocytes (20-40%): the smallest white blood cells (6-8 μm) o T-lymphocytes: fights against other cells – cellular immune response o B-lymphocytes: produces antibodies (immunoglobulin IgA, IgD, IgG, IgE, IgM) - humeral immune response. Monocytes (2-8%): they are the biggest of the white blood cells (12-20 μm). They can leave the vessel to the tissue, here they are called macrophages and are involved in the antigen representation. Eosinophils (1-4%): they are larger than neutrophils (12-14 μm) and fights against parasitic infection and allergic reactions. They also regulate the basophil granulocytes Basophil granulocytes (0,8%): these are the smallest leukocytes (8-11 μm), and the number of these cell can be higher in case of exogenic parasite infection (small insects) or allergies. They produce histamine. 38 Fatima’s notes 2022-2023 Physiology 1 For the differential leukocyte counting, what staining do we use for the blood smear film? How long do we stain it for? When the blood film has completely dried, we dye the slide in diluted May-Grunwald staining for 3 minutes, and then we place the film directly into the mixture of deionised water and May-Grunwald solution for 1-3 minutes. Lastly, we place the blood film in the mixture of deionised water and Giemsa solution for 10-15 minutes. For the white blood cell counting, what staining is used? What is the rate of dilution of the blood sample? What square of the Bürker’s chamber is used? Türk’s solution, and the rate of dilution is 1:10. The large square is used in case of the leukocyte count (1/5x1/5x1/10=250mm). Multiply the average with 2500! The average number of lymphocytes in each large square is 4, what is the WBC count? 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑊𝐵𝐶𝑠 × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 10 000 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 4 × 250 × 10 = μl If 40% of the white blood cells are lymphocytes, and the average number of lymphocytes in each large square of the Bürker’s chamber is 4, what is the lymphocyte count? 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 − 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑡ℎ𝑒 𝑡𝑦𝑝𝑒 𝑜𝑓 𝑊𝐵𝐶 = × 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑐𝑒𝑙𝑙 𝑡𝑦𝑝𝑒 100 10 000 4000 𝐿𝑦𝑚𝑝ℎ𝑜𝑐𝑦𝑡𝑒𝑠 = × 40% = 100 μl The number of lymphocytes is 4000/ μl 39 Fatima’s notes 2022-2023 Physiology 1 Thrombocytes Thrombocytes, also called platelets, are every important in the formation of the thrombocyte/ platelet plug. They don’t have nuclei and thus cannot reproduce, but they contain actin and myosin molecules, and the contractile protein thrombosthenin. Normal range of thrombocytes/platelets: 150 000 – 400 000 / μl Size: 1.5 – 4 μm Lifespan: 10-14 days Formation: In the bone marrow (from megakaryocytes) Formation stimulated by: thrombopoietin (produced in kidney) What is it called in case of too low and too high levels of platelets? Too high – thrombocytosis Too low – thrombocytopenia In the estimation of platelet count, what material were used? What are the steps? In this experiment we used the Bürker’s chamber, coverslip, microscope, pipette, test tube and Rees-Ecker’s solution We first pipetted 0.9ml solution into the test tube and then 0.1 ml of blood, before mixing them together. The test tubes were then incubated for 1-2 hours. After incubation, the thrombocyte rich plasma was visible on the surface if the blood sample as a whitish layer. We carefully sampled this layer and placed it on both sided of the Bürker’s chamber. We placed the chamber under the microscope and counted the platelets in over ten of the large rectangles using the L-law. If the average number of platelets in the rectangle (1/5x1/20(x1/10)) of the Bürker’s chamber is 20, what is the platelet count? 𝑃𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑐𝑜𝑢𝑛𝑡 = 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠 × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑟𝑒𝑐𝑡𝑎𝑛𝑔𝑙𝑒 × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑃𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑐𝑜𝑢𝑛𝑡 = 20 × 1000 × 10 = 200 000. (Multiply the average number by 10 000) 40 Fatima’s notes 2022-2023 Physiology 1 Hemostasis All processes to prevent and stop bleeding includes the hemostasis. Step 1 – vasoconstriction The first step of the primary hemostasis is the contraction of the smooth muscle in the wall of the broken vessel. This decreases the blood flow by shrinking the lumen, making it easier to form the plug and decreasing the blood loss. Vasoconstrictors Serotonin ATP Prostaglandin-F2 (PgF2) Adrenalin (epinephrine) via alpha-1 receptors Noradrenalin (norepinephrine) via alpha-1 receptors Thromboxane A2 Endothelin (strongest one) Hormones; angiotensin2, ADH (antidiuretic hormone/vasopressin) Step 2 – Thrombocyte plug formation (activation of thrombocytes) Normal bleeding time: 2-3 minutes (less than 5 minutes) When the endothelium of the vessel is damaged, the blood will be exposed to the tissue around. The surrounding tissue is rich in proteins, especially collagen, which the platelets bind strongly to. This activates the platelets, causing them to release the content of their granules. They also change in shape, making it easier to form a plug. These granules contain molecules like serotonin, ADP, and thromboxane A2, which further stimulates the vasoconstrictions of the vessel and also activates other nearby platelets. The activated platelets bind and stick to the damaged endothelium, forming the platelet plug. To ensure this plug, the third step of the haemolysis is continued. The inactivation of the thrombocytes is caused by the vasodilators prostacyclin (PgI2) and nitric oxide (NO). If the bleeding is higher than 6 minutes, what can be the reason? Malfunction of the thrombocytes or thrombocytopenia (low level of thrombocytes). If the bleeding time is lower than normal, what can it be? Not pathological, meaning it is fine. 41 Fatima’s notes 2022-2023 Physiology 1 How would the bleeding time change in case of Von Willebrand factor deficiency? Increase to longer than 5 minutes. Step 3 – Blood clotting The platelet plug alone is not stable enough to ensure that the bleeding stops. Blood clotting is the third step of the hemostasis and stabilizes the platelet plug that is formed by fibrin. Extrinsic pathway is more likely in the body, while the intrinsic is what we saw in vitro in the lab, where we don’t have tissue factors. Normal clotting time: 5-10 minutes 1. Intrinsic pathway (In vitro, on glass slide) The intrinsic pathway starts with the activation of prekallikrein, which becomes kallikrein. Kallikrein can activate factor XII, which then becomes XIIa (active factor XII). The active factor XII has positive feedback on the prekallikrein, meaning it can activate other prekallikrein molecules to become kallikrein. The main function of the XIIa is to activate factor XI, which becomes XIa (active factor XI). These factors have protease activity, which is their way of the activation. XIa can activate factor IX, which becomes IXa (active factor IX). IXa can activate factor X together with active factor VIII, Ca2+ and phospholipids. Intrinsic tenasecomplex: IXa, VIIIa, Ca2+ and phospholipids Active factor X (Xa) can activate factor II together with active factor V (Va), Ca2+ and phospholipids. These together is known as the prothrombin complex. Prothrombin complex: Xa, Va, Ca2+, phospholipids Factor II is also called the prothrombin. When the prothrombin (factor II) is activated, it becomes active factor II (IIa), also named the thrombin. The thrombin (IIa) can activate the fibrinogen, which is the factor I. Fibrinogen activates the fibrin, which is the active factor I (Ia). All the fibrin monomers connect together by the active factor XIII (fibrin stabilizing molecule), and it becomes a fibrin polymer, securing the platelet plug. 2. Extrinsic pathway (In vivo, in the body) The only difference between the intrinsic and extrinsic factor is that tissue factor activates the factor VII, which becomes VIIa (active factor VII). VIIa together with the tissue factor, Ca2+ and phospholipid activates factor X. These together are called the extrinsic tenasecomplex. The rest of the pathway is similar to the intrinsic pathway. 42 Fatima’s notes 2022-2023 Physiology 1 Extrinsic tenasecomplex: tissue factor, VIIa, Ca2+, phospholipid Name four substances which can remove the free calcium in blood the blood, and thus prevent the blood clotting in vitro? Sodium oxalate, sodium acetate, sodium citrate and EDTA. What clotting factor doesn’t have protease activity? XIIa, Va, VIIa, IV (Ca2+), Ia (fibrin) What are the functions of the thrombin (IIa) Activates thrombocytes, fibrinogen (factor I), and factor V, VIII, XI, XIII and together with the thrombomodulin it activates protein C. What happens with the clotting time in case of liver failure? It increases. The liver produces plasma proteins, and most clotting factors are plasma proteins. 43 Fatima’s notes 2022-2023 Physiology 1 Vitamin-K dependent clotting factors The vitamin-K dependent clotting factors are produced in the liver only. The vitamin-K contributes to the gamma-carboxylation. The carboxylation is important because the calcium is needed later for the activation of different factors. Factor II (prothrombin) Factor VII Factor IX Factor X Vitamin- K antagonist Coumarin Warfarin Vitamin-K dependent anticoagulant factors Protein C Protein S In case of vitamin-K deficiency, how would the prothrombin (clotting time), INR and Quick index change? Prothrombin time – increased INR – increased Prothrombin (Quick) index - decreased If you give antagonist vitamin-K to a patient, how would the prothrombin (clotting time), INR and Quick index change? Prothrombin time – increased INR – increased Prothrombin (Quick) index - decreased If we have a blood sample in a test tube, and we add extra vitamin K, how would the prothrombin time, INR, and Quick index change? 44 Fatima’s notes 2022-2023 Physiology 1 In vitro they won’t change at all. Vitamin K clotting factors produced in the liver only! Is the active factor XII a protease? Where is it produced? No. It is produced by the thrombocytes. How could you measure the intrinsic pathway activation in the lab? By measuring the prothrombin time. First, we pipetted 100μl sample plasma on a watch glass in the 37 ֯C water bath. After some minutes, we pipetted 200ul thromboplastin-Ca2+ reagent into the plasma sample, started the stopwatch, and tried to pull out the first fibrin fibre. When the first fibrin appeared, we stopped the stopwatch. We repeated the same steps with the standard plasma. Then we calculated the results. Normal prothrombin time: 13-22 seconds How can you measure the prothrombin (quick) index? What it the normal value? 𝑃𝑟𝑜𝑡ℎ𝑟𝑜𝑚𝑏𝑖𝑛 𝑡𝑖𝑚𝑒 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑝𝑙𝑎𝑠𝑚𝑎 𝑃𝑟𝑜𝑡ℎ𝑟𝑜𝑚𝑏𝑖𝑛 𝑖𝑛𝑑𝑒𝑥 (%) = × 100% 𝑝𝑟𝑜𝑡ℎ𝑟𝑜𝑚𝑏𝑖𝑛 𝑡𝑖𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑝𝑙𝑎𝑠𝑚𝑎 Normal value of prothrombin: 70-120% The prothrombin time of the patient is 30 second, and the prothrombin time of the standard sample 15 second. What is the prothrombin index? 15 𝑃𝑟𝑜𝑡ℎ𝑟𝑜𝑚𝑏𝑖𝑛𝑒 𝑖𝑛𝑑𝑒𝑥 = × 100% = 50% 30 How can we standardize the prothrombin time? What is the normal value of INR? By using the INR (international Normalized Ratio). 𝐼𝑆𝐼 𝑃𝑇𝑠𝑎𝑚𝑝𝑙𝑒 𝐼𝑁𝑅 = ( ) 𝑃𝑇𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 Normal value: 0.8-1.2 What is the risk of higher INR and lower Quick index? Prolonged clotting time, risk of bleeding. What is the risk of lower INR and higher Quick index? 45 Fatima’s notes 2022-2023 Physiology 1 Shortened clotting time, risk of thrombosis In case of fat-malabsorption (fat cannot be absorbed in the small intestine), how would the prothrombin (clotting time), INR and Quick index change? Prothrombin time – increase INR – increase Prothrombin (Quick) index - decrease Why? Vitamin-K is fat-soluble vitamin, and in case of fat-malabsorption the fat-soluble vitamins will not be absorbed. The vitamin-K dependent factors (II, VII, IX and X) will be affected, and the clotting time will therefore be longer. If you give Warfarin or Coumarin to your patient, will the clotting time be longer, shorter, or not change? How will the prothrombin (clotting time), INR and Quick index change The clotting time will be longer. Prothrombin time – increase INR – increase Prothrombin (Quick) index – decrease In case of hypercalcemia, will the blood clotting be faster or shorter? Calcium is needed, but in case of more calcium it won’t be any change in the clotting time. 46 Fatima’s notes 2022-2023 Physiology 1 O2-hemoglobin dissociation curve Hemoglobin is the primary oxygen transporter in the blood, and functions also for Co2 transport. The hemoglobin is the most important buffer for pH in the blood system. Hemoglobin is a good buffer due to the high concentration, and because it contains a lot of histidine molecules. Histidine has an imidazole group, which can easily bind and release H+. The O2-hemoglobin dissociation curve shows the hemoglobin saturation with oxygen. Normal hemoglobin concentration: ♀: 120-160 g/L ♂: 140-180 g/L A right shift in the curve means that more oxygen pressures is needed to get the same saturation, and the oxygen affinity to hemoglobin decreases. There is a weaker connection between O2 and Hb. The reason for the right shift is hypercapnia (CO2↑), acidosis (↑H+, ↓pH), hyperthermia (increased body temperature) and increased concentration of diphosphoglycerate (↑2,3-DPG). This usually happens during exercise. A left shift in the curve means that the O2-affinity to hemoglobin is increased. The connection between O2 and Hb is stronger. The reason for the left shift is hypocapnia, alkalosis (↓H+, pH↑), hypothermia (decreased body temperature) and decreased concentration of diphosphoglycerate (↓2,3-DPG). 47 Fatima’s notes 2022-2023 Physiology 1 How does the O2-Hb curve change in case of thrombocytopenia? No change. How does the O2-Hb curve change in case of lymphoctosis? No change. How does the O2-Hb curve change in case of decreased pH? Right shift (acidosis). How does the O2-Hb curve change in case of hypothermia? Left shift. How does the O2-Hb curve change in case of hypocapnia? Left shift. How does the O2-Hb curve change in case of increased 2,3 DPG? Right shift. How could we determine the hemoglobin concentration in the lab? Drabkin’s method: osmotic hemolysis of RBCs followed by transformation of Hb molecules to cyan-hemoglobin. First step of this transformation is the oxidation of ferrous (Fe2+) ions to Hb molecules to ferric (Fe3+) ions resulting hemiglobin by potassium-cyanide. Cyan- hemoglobin is stable and its concentrations can be determined photometrically by measuring its absorbption at 540 nm. By hemoglobinnometer (photometer): by this device we compare the absoption of light having passed through a hemolysed blood sample to that of the light having passe trough a calibrated prism. Due to the transmission properties of the prism and the built in 546 nm opticcal filter, we can examine the sample with light of a wavelenght corresponding to the absorpbtion maximum of Hb. We compare the colour of the light passing through the hemolysed blood sample and the standard color. There are four scales on the right of the device, the uppermost one shows the absolute values in g/100 ml, while the next three scales show the result as percentage of the mean value. 48 Fatima’s notes 2022-2023 Physiology 1 Filtration The hydrostatic pressure of the capillaries is 30 mmHg, the colloid osmotic pressure of the plasma is 25 mmHg, the hydrostatic pressure of the interstitial fluid is 5 mmHg, and the colloid osmotic pressure of the interstitial fluid is 5 mmHg. Calculate the effective filtration pressure. Effective filtration pressure = 30 mmHg – 25mmHg - 5 mmHg + 5 mmHg = 5 mmHg. When the effective filtration pressure is positive, it means the water goes from the capillaries to the interstitium, and if its negative the water goes into the capillaries. When does the filtration increase/ decrease? Filtration ↑ (oedema) (+) Filtration ↓ (-) High BP (blood pressure) Low BP (blood pressure) Low protein intake Blood loss Histamine (increases permeability of vessels) Dehydration Low hydrostatic pressure of interstitial fluid Hyperproteinaemia Thrombosis (blood clot in vein) High hydrostatic pressure of interstitial fluid Glycoprotein outside the vessel Increase in histaminase (degrades histamine) Liver failure Massage (pressure) How does the more glycoprotein in the interstitial space effect the filtration? Filtration increases - because the colloid osmotic pressure increases. How does the filtration change in case of liver failure? Filtration increases – liver cannot produce plasma protein, the colloid osmotic pressure of the plasma decreases, and the inward force of the capillary decreases, meaning that the filtration increases. 49 Fatima’s notes 2022-2023 Physiology 1 Blood typing The plasma membrane of RBCs contains a lot of antigens, which are unique for every blood type. These are molecules that which – when introduced into the body of another individual not tolerant to them – trigger antibody production. Antigens, also called agglutinogens, are carbohydrates or proteins and are either a direct product of a gene or produced by an enzyme expressed form a gene. The most used are the ABO and Rh blood system. A-B-O blood type system The ABO blood group system is composed of four main blood types – A, B, AB, and O. The phenotypes of these blood groups are generated by three antigens, the A, B and H antigens. The alleles of the ABO coding for the enzyme variants generating the A and B antigens are codominant against the recessive O allele coding for the inactive variant of the enzyme which leaves the H antigen unchanged. E.g., when type A antigens is not present in a person’s RBCs, antibodies known as anti-A antibodies – also called agglutinins - develop in the blood plasma. When the type B antigen is not present on the RBCs, antibodies known as anti-B develops in the plasma. This means that type A blood contains anti- B agglutinins/antibodies, and type B blood contains anti-A agglutinins/antibodies in the blood plasma. This also means that type O blood contains both anti-A and anti-B agglutinins/antibodies in the blood plasma. - Type A blood contains type A antigens on the RBCs, and anti-B antibodies in the plasma. - Type B blood contains type B antigens, and anti-A antibodies in the blood plasma. - Type O blood contains neither type A or B antigens on the RBCs, but both anti-A, and anti-B antibodies in the blood plasma. - Type AB blood contains both type A and B antigens on the RBCs, but no antibodies in the blood plasma. What immunoglobulins are responsible for ABO blood type groups

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