-1MjW Sept 13th 2013 PCTH404: IN VITRO/IN VIVO (FUNCTIONAL) EXPERIMENTATION. In Vitro The important elements in functional in vitro studies include: the physiological solutions necessary to keep tissue viable; the nature of the isolated tissue preparation used; and functional pharmacological responses measured. PHYSIOLOGICAL SOLUTIONS Many different physiological salt solutions are used. The have been used for years and have often beend named after their inventor, e.g. Krebs’, Tyrode, McIlwain. Despite over 100 years of their use in in vitro experiments most of the standard salt physiological solutions are still poor substitutes for blood, but since they are completely defined they are less complex than tissue culture media which classically contain some foetal bovine serum. Composition: include the following,  Mono-valent ions: Sodium, Potassium, Chloride – essential for ionic balance, excitability and osmolarity.  Di-valent ions: Calcium, Magnesium (essential for maintaining excitation and osmolarity), bicarbonate (for pH), phosphates.  Ca++ concentration used in physiological salt solutions is usually 2.5 mM, but in blood and extracellular fluid the ionized calcium concentration is 1.0 to 1.5 mM; Ca is essential for excitation/contraction-secretion coupling  Glucose and fatty acids are essential for energy requirements varying on the tissue. It is easy to use glucose, which also provides oncotic pressure, but there is no insulin in most solutions to facilitate glucose transfer into cells while some tissues prefer fatty acids as their source of carbon for intermediate metabolism (e.g. cardiac tissue)  Oxygen – bubbled usually 100%, or 95% plus 5% CO2 (carbogen) in order to maintain pH for some solutions (K-H). However, oxygen solubility in water is low and thus carries little oxygen.  Usually contains no hormones, amino acids, proteins, fatty acids all essential to long term (days) tissue survival.  Temperature is adjustable for homeotherms (warm blooded), room temp for poikilotherms (cold blooded). Normally the bathing temperature is kept somewhat below body temperature so as to reduce metabolic needs and so preserve tissue.  PO4 or other buffer systems (essential for maintaining physiological pH); pH7.4 is physiological  a phosphate buffer system is often used.  phosphate/bicarbonate buffer systems require carbon dioxide bubbled at 5% so as to maintain pH

-2 Other buffers, e.g. TRIS, MOPS, PIPES etc., can be sometimes be toxic Many essential elements normally present in blood are absent in the physiological solutions used routinely for in vitro work. However, cell cultures require more complex solutions that have to include hormones, fatty acids, and proteins. These components are generally not considered important where growth and cell differentiation are important for culturing cells but mainly irrelevant in short-term experiments (hours). Tissue can be stored for hours at low temperatures (4°C) and still function well for acute experiments over the following hours at temperatures of above 30°C. Other nutrient solutions: Culture media Solutions for culturing cells are much more complex than simple physiological salt solutions. They contain many more of the essential constituents (hormones, fatty acids, amino acids, etc.) that are necessary and essential for growth and differentiation. However, many culture media also contain “magic” constituents such as foetal bovine serum and we have yet to determine exactly what the ingredients are in these UNDEFINED additions to culture media. Cardioplegic solutions In order to preserve tissue for experiments for a period of time after their initial use can be made of cold (< 4°C) conditions and elevated potassium concentrations. Cardioplegic solution, for example, are very complex “witches’ brew” containing, in addition to the usual ions, antioxidants and steroid hormones. Cardioplegic solutions came into use in cardiac surgery as a result of a need to preserve cardiac tissue during open heart operations, and for its transplantation. They are sometimes used when isolating tissue (after removing heart from an animal, and while preparing for cannulating the aorta or when making further surgical adjustments such as isolating ventricular wedges, etc). Use of whole blood: Whole blood obviously has all of the constituents necessary for preserving tissue function, but blood is difficult to use, because:  Coagulation has to be prevented.  Blood is easy to oxygenate, but attempting to do this often results in problematic bubbling because of the surface active properties of proteins in the blood.  Contact of blood with glass and other foreign surfaces results in the release of pharmacologically active compounds, e.g. amines (5HT) and kinins as well as the loss of platelets and other blood cells  Blood is a very good nutrient for bacteria and hence bacterial contamination is an issue.  However, blood can be used in blood gas machines, if available, or an animal can be used as an EXTRACORPOREAL source of blood (blood taken from a large artery used to flow over and thereby maintain isolated tissue which when used is returned via a suitable

-3vein. This technique is the basis of the superfusion cascade assay system (“Vane Cascade”). METHODS FOR DELIVERING PHYSIOLOGICAL SOLUTION TO TISSUE There are three main methods for delivering physiological solutions to tissue: perfusion (directly connected to the vascular system); tissue bath (sample immersed in fluid); superfusion (solution running over the tissue surface). Considerations Insufficient O2, or flow in general (“ischaemia”), to all parts of a tissue preparation is a problem with all the methods used above, but is greatest with bathed tissue because of the very limited oxygen diffusion that can only occur at the surface of the tissue that is exposed directly to the bathing solution. Oxygen only penetrates to the depth of two cell layers, or three layers of cells at most. This problem can be less of an issue with superfusion since the tissues used in such preparations are often thinner than those used in bathed preparations. The use of perfusion via blood vessels results in the nutrient solution entering into every capillary, but the oxygen carrying ability of physiological solution is very limited. However, synthetic oxygen carriers are available but expensive. When using vascular perfusion, oxygen availability depends on two physiological parameters: amount of dissolved oxygen, flow rate and vascular pressure. There are two modes of perfusion: 1) fixed volume per unit time, or 2) fixed perfusion pressure. Which of these two methods to use depends on the physiological needs of the tissue. For tissue in which auto-regulation of flow is predominant, it is physiologically more relevant to use fixed pressure and allow the tissue to take the flow it requires to fulfill its tissue oxygen needs (by auto-regulating vascular constriction/relaxation), e.g. the Langendorff cardiac preparation. If fixed flow is used, the perfusion pressure indicates the level of vasoconstriction in the perfused vessel bed. If fixed pressure is used, flow is the appropriate indicator. Oedema (tissue retention of fluids) While the oncotic requirements of tissue are partially met by having the correct concentrations of small molecules in the physiological solution, oedema in the tissue sample still occurs. This is so, even when the perfusion technique is used. A heart perfused by the Langendorff technique can steadily accumulates oedema fluid. Washout of waste products With bathing and superfusion of tissue there is little or no effective washout of the cellular waste products that accumulate in the extracellular space which is not in easy communication with bathing fluid. Perfusion is more efficient with respect to wash-out. TYPES OF ISOLATED TISSUE PREPARATIONS

-4All types of tissue have been used in isolated tissue experiments. Most tissue preparations are used in the bathed format since it requires skill and technical ability to isolate and cannulate the necessary blood vessels supplying an organ or a tissue sample. However, in some organs, access to the vascular system is very easy, e.g. the aorta of an isolated heart. Tissue preparations Typically gut, blood vessels, uterus, bronchi and similar tissue is occasionally used in the perfused, or superfused, mode but bathed tissue is most common used except for blood vessels which are easily cannulated. Cardiac tissue in the form of the whole heart is easily perfused. It is also possible to perfuse isolated segments of the heart (ventricles, atrioventricular node, sino-atrial node). Nervous tissue can be perfused but is most often examined as very thin slices. Cleary defined organs such as lung, kidney, spleen and liver are much easier to perfuse. Considerable ingenuity has been used to achieve a variety of perfused tissues including retrograde perfusion via a vein rather than orthograde perfusion via an artery . Examples of Muscle Preparations  simple excised “chunks” of tissue (e.g. guinea pig ileum)  strips of tissue, generally affixed at the ends  spirals - cut along natural axis (e.g. blood vessels)  rings – e.g. bronchi, blood vessels (circular nature)  skeletal muscle can be cannulated or naturally thin muscles (diaphragm, sartorius, extra-ocular) can be bathed Recognition has to be given to the physiology and anatomy of the tissue under study. For example, if care is not taken with blood vessel preparations the endothelium will be damaged, and thus the endogenous source of nitric oxide (NO) will be lost. Nerve/Muscle Functioning Preparations Nerve/muscle preparations depend critically on the physiology and anatomy of the neuromuscular junction that is being studied. A neuromuscular preparation is easy to achieve with many different muscles where discrete nerves and their innervations sites are readily identifiable i.e., it is easy to visualize both nerve and muscle. In smooth muscles preparations, this is not the case and nerves cannot readily be identified. Electrical Stimulation of Nerves and Muscle Direct Contact Electrodes Implanting, or even just touching a tissue preparation with an electrode, allows for delivery of current/voltage to stimulate the tissue to respond (contract, secrete, etc.). Successful stimulation depends on stimulus current (or voltage) strength and pulse width. Characteristically nerves can always be maximally stimulated at square wave pulse widths of 1.0 msec. Skeletal muscle and cardiac muscle are intermediate between the two muscle types.

-5Field Stimulation of tissue Field stimulation Is used for nerve/muscle preparations where the nerves cannot be easily visualized and separated from other tissue. It simply requires plate and/or rod electrodes to be placed around, or within, the tissue so as to create an electrical potential field across the tissue. It requires large currents (voltages) to create an adequate potential field across the tissue and depending upon pulse width and current where nerves are most easily stimulated. . Differentiating responses due to nerve stimulation versus direct tissue stimulation  as above, nerve tissue is more easily stimulated and therefore requires a shorter pulse and lower current threshold, to avoid direct tissue stimulation  tetrodotoxin (TTX) at 0.1 microMolar will completely abolish all sodium current nerve transmission. Therefore responses to stimulation that remain in the presence of TTX are due to direct tissue stimulation.  neuronal tissue is very responsive to anoxia so that temporarily decreasing oxygen can reversibly reduce neuronally dependent responses  transmitter release can be blocked with N-type channel blockers (alphaconotoxins from a venomous marine cone snail) MEASUREMENT OF RESPONSES IN ISOLATED TISSUES:  Physical – movement or contraction; NOTE: contractile activity should be measured in the most physiologically appropriate manner: isometric (keep length constant, measure change in tension) for skeletal muscle, isotonic (keep tension constant, measure change in length) for smooth muscle, or auxotonic (a mixture of the two which best represents the physiology of much smooth muscle)  Chemical - production of second messengers, status of enzymes, oxygen utilization; NOTE: isolated preparations are a poor source of material for biochemical determinations since most of the tissue in such preparations is dying from lack of nutrients and accumulation of metabolic by-products (penetration of oxygen into tissue is limited to only the outermost layers)  Electrical - measure currents with patch clamp electrode, measure intracellular potentials (e.g. cardiac action potentials) or field potentials (e.g. ECG) with contact electrodes, or indirectly e.g. photo-electric dyes (voltage sensitive dyes) can be used as an indirect measure of transmembrane potential. Such dyes located within the cell membrane and change their optical characteristics (absorption or emission) as the voltage across the cell membrane changes  Secretions - flow and nature of secretions from small or large glands. IN VIVO EXPERIMENTS The first, and perhaps most important, consideration is whether or not one has to use anaesthesia. ANAESTHESIA Anaesthesia is recognized to include:

-6 Absence of pain  Absence of movement  Reduced reflexes  Controlled conditions  Level of anaesthesia must be monitored at all times using various indicators  abolition of reflexes: righting (in rodents), blink, deep tendon, pupil status  reflex response of blood pressure and heart rate to noxious stimuli  respiration becomes deeper, laboured and diaphragmatic as the depth of anaesthesia increases  as cardiovascular depression deepens with anaesthesia tissue lose their red colour and tissue turgidity increases  a balance between enough, and too much, anaesthetic has to be maintained on the basis of clinical judgment of the overall status of the animal  it is the duty of all those working with anaesthetic drugs to thoroughly understand the pharmacology of such drugs since there are marked differences between them. These means understanding anaesthesia, cardiovascular status, pain, animal well-being, and post surgical stress and discomfort. The ideal anesthetic is:  Cheap and easy to use  Easy to control depth of anaesthesia with known effects on reflexes.  No cardiovascular effects  Long lasting  IV or IP administered Parenteral anaesthetics (generally for non-recovery surgery in larger animals and mice for recovery surgery) are most often given i.p. as the easiest route. Avoid s.c. and use i.v. only if skilled Choice of anaesthetics. Depends on accessibility on whether for recovery or non-recovery  Pentobarbital is easy to use for non-recovery procedures; relatively short duration of action (1-2 hours), 50 - 60 mg/Kg for small animals but, with surgical manipulation increases in BP and HR can occur reflecting the presence of functional pain reflexes (at least at the level of the spinal cord). A more satisfactory barbiturate is thiobutabarbital.  Ketamine/Xylazine is easy to administer, effective and reasonable duration but has a somewhat different pattern of anaesthesia. Can be used for recovery and non-recovery surgery.  Chloralose - chloral hydrate complex with glucose, given i.v. as large volume and warm (50°C), very long lasting with, paradoxically, intact reflexes despite the anaesthesia. Not for recovery experiments, and not normally used. Can be useful in neurophysiological work on reflexes since many spinal reflexes remain intact.

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Urethane (Ethylcarbamate)- decreases blood pressure and not good for the cardiovascular system, possible carcinogen, and it increases capillary permeability. Not often used, and not for recovery surgery.  Etodimate- long lasting, no cardiovascular depression, depresses reflexes  Propofol - ultra short acting, can be given by infusion for maintained anaesthesia. In human anaesthesia propofol always supplemented with halogenated hydrocarbons and /or opioid narcotics  Narcotics supplemented with other anaesthetics Consult local animal care authorities regarding the preferred drugs in your location since there are geographic and personal preferences, although these are not always pharmacologically relevant. Vapour anaesthetics (halogenated hydrocarbons) – especially for recovery. Their use requires skill and training, special apparatus, and a clear understanding of the drug, apparatus and anaesthesia. By definition all are liquids at room temperature but are vapourized in air or oxygen. Vapourization usually requires special vaporizing apparatus which because some have clinical use are very expensive. The anaesthetic concentration for halogenated hydrocarbon vapour anesthetics is in the range 0.5 - 1.5% vapor content of carry gas in air or oxygen. High concentrations (4 - 5%) are lethal, but can be useful to quickly, and carefully, induce anesthesia (within seconds to a few minutes) but overdose is very likely if concentration is not adjusted appropriately as anaesthesia is induced, and progresses to deeper and deeper states. One can adjust anesthetic level to achieve constant blood levels by monitoring alveolar gases using an IR analyzer and adjusting the vapourizer appropriately – but is an expensive procedure. One should always rely on one’s clinical skills and understanding to avoid errors. It is easy to use 100% O2 as the carrier gas but since all gases in cylinders are very dry it is useful humidify them with water (vapour). As a scientist it is useful to understand the physics of vapours and gases. Remember combustion is intense in pure O2 and so care is needed with using O2 apparatus that use high temperatures and which might give rise to combustion. Generally, intubation (if possible) of the trachea is useful to ensure adequate ventilation of the animal. Health and safety have obsessive fears about halogenated hydrocarbons and so various systems are used to scavenge the gases from the working environment. The halogenated hydrocarbons (most often isoflurane) are the preferred anaesthetics. However, since recovery from them is rapid (see pharmacology notes for anaesthetics) analgesic coverage (pre and post-surgery) with an NSAID or narcotic analgesics is appropriate in most cases. However, all analgesics have their characteristic pharmacology which varies with the species concerned and should be known by the users of such drugs. Respiratory depression with narcotic analgesics can be a concern. Endotracheal Intubation Can be an easy technique in some species, but more difficult in others, e.g. an easy species is dog, a difficult species is pig. The technique requires skill, adequate lighting, local anesthetics and suitable endotracheal tubes. With training almost all species including mice and birds can be successfully intubated.

-8Ventilation Various ventilators (or pumps), gases and gas mixtures are readily available. One useful adjuvant is nitrous oxide which cannot be given in a high enough concentration alone produces anaesthesia but 70% N2O in oxygen gives excellent analgesia that can be supplemented with a halogenated hydrocarbon at lower concentrations so as to give full anaesthesia. It is good to monitor arterial concentrations of oxygen and carbon dioxide, or alveolar gases, where possible. Good pO2 is approximately 100 mmHg or more, higher if 100% O2 is used. Good pCO2 is approximately 40 mmHg. Blood pH should be 7.4. One has to avoid “shunting”, which occurs when blood is flowing through a part of the lung that is not being ventilated. This can occur when the thoracic cavity is open to air. As result pO2 will be below the expected. Types of Halogenated Hydrocarbons  Halothane - very easy to use, inexpensive, anaesthesia easily reversed, but it is now perceived to be a health hazard to humans with respect to the suspicion of causing liver damage although the evidence for such is very weak.  Isoflurane, Enflurane, Desflurane these newer halogenated hydrocarbons are more expensive. Differences between them and halothane are limited but possibly halothane causes more cardiovascular depression Monitor Vital Signs in Anaesthetized Animals Check cardiovascular status: BP and HR should be within normal limits Check fluids use saline drip of up to 20ml normal saline/kg/hour and use urine flow as an indicator of blood volume status. Check mucous membranes which should be moist and red. Temperature: Keep temperature constant using a thermometer and heater as appropriate. Remember that while 37°C is regarded as normal for homeotherms, different animals have slightly different temperatures, with birds having the highest. Other factors to consider: Serum K and other ions: tissue damage results in the release of K ions. In addition impaired kidney function will also elevate serum potassium. An example of why this can be of important is with beta blockers which appear to be antiarrhythmic against ischaemia-induced arrhythmias in anaesthetized, but not in conscious rats. The reason is that in the acute (anaesthetized) preparation with beta blockers, there is a significant rise in serum K and it is the rise in serum K that is antiarrhythmic. SURGICAL METHODS FOR AVOIDING THE USE OF ANAESTHETICS Neural Ablation 1) Pithing: the brain and spinal cord are destroyed under short term anesthesia. As a result of pithing, BP and HR are usually low. This is a very stable preparation if given adequate fluids and appropriate temperature. The serum K has to be monitored since it can rise dramatically. Mounting the animal in the vertical position and ensuring adequate blood volume (maintaining venous return by pressure bandages) can bring pressures back to close to normal. 2) Spinal Transaction: this is possible at different levels; it results in “spinal shock” where BP and HR are low and no CV reflexes; can last for months in primates,

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hours in rats. Depending on the level of spinal transaction there can be loss of breathing. Decerebrate: the effects of decerebration depend upon the level of transection between the brain and the rest of the CNS. Transection can be made at any level, e.g. pons or medulla. Decorticate: this is the simple isolation of the cerebral cortex from the rest of the CNS. If necessary, the cortex can be removed by suction.

SURGICAL TECHNIQUES FOR IN VIVO PREPARATIONS All of the surgical techniques that had been applied in man can be applied to experimental animals, and many more that can’t be applied in man due to ethical reasons. Before utilizing any surgical technique, it is necessary to acquire full training in all of the general aspects of animal surgery and anaesthesia. This requires times and exposure to personnel trained in all of these aspects. It is insufficient to just try and “pick up” techniques from colleagues. There are many sources of information even down to the details of correct and appropriate suturing techniques to be found on the web and in books. Most academic facilities mandate, and provide, basic training. General Considerations (for animals that will recover from surgery) Use the best anaesthetic techniques possible, i.e. fluids, neuromuscular blockade and careful monitoring (more important for large animals). Use local anesthetics to alleviate pain (they also constrict local vessels and reduce blood loss/flow into area). Use NSAIDs and narcotic analgesics for the same purposes, but beware of respiratory depression effects of narcotic analgesics if too high a dose is given and that narcotic analgesics (morphine related) can cause excitation though this is to some extent species dependent. Sterile techniques are best but can be cumbersome and require specialized facilities (i.e. sterile OR). Clean (aseptic) techniques are considered by some to be unacceptable, although they can be adequate in the absence of sources of pathogens. If indications of infection occur it is vital to use sterile techniques. Avoid using antibiotics as a cover for dirtiness. The overuse of antibiotics encourages infections with resistant organisms. 70% ethanol is a very useful but incomplete disinfectant, which is not too damaging to tissues. However, it is best to sterilize instruments (e.g. autoclave) and not just clean them with a disinfectant. During recovery, use careful wound maintenance. Use wound sprays to cover damaged area. Remove sutures as soon as appropriate. A non-absorbable antibiotic, such as a neomycin, may be useful and is not likely to give rise to resistant organisms. Place wounds in an accessible site, e.g. between shoulder blades. It may be necessary to isolate surgically prepared animals to avoid cage mates picking and gnawing at wound sites. The main problem is usually infections. These can be at the surgical site, in which case improved techniques and procedures. There can be concurrent infections such as in the lungs and the kidney which can be problematic. Implants:

- 10 Insertion of cannulae for BP measurements, arterial blood flow measurements and to facilitate injections, etc. are all possible. Injections can be done via any route using mini-pumps, push-pull cannulae, etc. Devices can be inserted for telemetry (can monitor a large number of functions using wireless remote sensor), blood vessel occluders, snares, and flow probes. In the future there will be many more bio-electrical sensors for constantly monitoring electrical physical and chemical activities. It is possible to measure routinely a variety of physiological using implanted transducers which communicate wirelessly with recorders leaving the animal to behave relatively normally. For example blood pressures, ECG, breathing and many and many other functions are routinely monitored in free moving animals. Explants: Remove tissue to create disease states. Expose tissue, e.g. access to all parts of the GI system can be made possible with suitable cannulae and fistulae. Examples: Coronary Occlusion Experiment Can be used in either acutely or chronically prepared animals. Acute preparation involves implanting a coronary artery occluder and activating it so as to produce acute myocardial ischaemia while animal is anaesthetized. In the chronic preparation with electrodes and the occluder device is performed under anaesthesia, and the animal allowed to recover for a few days. Tightening of occluder is performed in conscious animals who have fully recovered from the operative procedures. Occluder devices can include mechanical snares, electrical-mechanical snares, or electro-coagulation. In mobile conscious animals we record blood pressure, heart rate, ECG and cardiac output before and after coronary occlusion. In vivo studies of the brain: Electrical recording from the brain: a variety of permanent electrodes are available (implant under anaesthesia, used when animal is conscious). These can be used to record field potentials or local electrical activity, even in single cells. Ionotophoresis/injection ports are used to apply drugs, or can use push-pull cannulae. THE SPECIAL CASE OF PSYCHOPHARMACOLOGY In order to assess the actions of psychoactive drugs it is important to be able to measure the activity, more particularly the behaviour, of animals. Behavioural pharmacology studies require particular skills and immaculate experimental design. The large subjective element in such studies, and the use of non-cardinal scales, does not invalidate the value of such experiments but does necessitate careful design and execution. Naive or trained animals can be used, each has its place. It is preferable, where possible, that blind, random, crossover and dose response experimental designs are used. Pre-treatment can be with drugs or surgery. Many drug treatments are used to model human disease conditions with varying success. Drug treatments can be systemic or discrete (and local), e.g. induction of Parkinsonism. There are many experimental paradigms available to model such conditions such as learning, memory (long and short term), aggression, anxiety, fear, pain, neuropathic pain, Alzheimer’s, Parkinsonism, etc. Pain - many paradigms for acute pain, chronic pain, pain of inflammation, neuropathic pain, from pin prick to dorsal root injury.