James Drife MD FRCOG FRCPEd FRCSEd Hon FCOGSA

• Professor, Department of Obstetrics and Gynaecology, General
• Infirmary, Leeds

The second scientist will likely pick up most or all of any ambiguities medications 101 discount calcitriol 0.25 mcg on line, grammatical errors 7r medications buy calcitriol 0.25 mcg amex, and obvious mistakes that can otherwise creep into a report medications reactions calcitriol 0.25 mcg on line. Instead medicine tour cheap calcitriol 0.25 mcg otc, as for protocols symptoms heart attack women 0.25 mcg calcitriol buy visa, a template report should be developed and maintained by staff. Use of boilerplate reports (as described for protocols) may also be an appropriate and acceptable approach. However, reports contain more detailed and varied content, although this approach is well-suited to screening study reports. Protocols and reports for each study type should be laid out, formatted, and contain the same wording as far as possible to facilitate review. If any standard wording in a document needs updating, then consideration should be given to making similar updates in other templates and boilerplates. This will also help them to identify those components of the assay that are critical so they can arrange inspections accordingly. Scientific management should be present at any presentation so they can correct any misunderstandings and answer or research any questions that the presenters cannot. It may also be useful to invite an external expert to present or train staff in specific techniques. Staff should retain records of any internal or external training courses and related certificates within their training record file. Attendance and a list of presentations given at external meetings should also be included in the training file. New staff should be introduced to tests gradually, focusing on one area or technique at a time. These can be prepared by mixing cells from negative and positive controls from a routine study in a range of proportions from 0% to 100% before slide making and staining in the usual way. In this way, for example, 10 or 20 rodent micronucleus slides can be made for each "dose level. The trained scientist/technician should sit alongside the pupil and explain key features using negative and positive controls explaining protocol criteria, what represents a readable slide/ metaphase, criteria for identification of aberrations, artifacts, and assessment of toxicity. It is useful to have a library of photographs to demonstrate examples of each that the student can help develop further if a photomicroscope is used. Then, the "student" should examine one field (or metaphase) at a time in conjunction with the trainer to ensure they can identify each aspect appropriately. Once the trainer feels comfortable with the interpretation and quantitation of events by the student, the student should be given a set of the training slides to read. It is useful during this examination to record vernier locations of key events recorded by the student so that they can be confirmed by the trainer. It will usually be necessary for the student to examine more than one set of slides before they can be assessed as competent unless they have already been trained in a similar assay. The trainer should also help with or confirm proper General Recommendations 37 use and set-up of the microscope as well as the scanning method (one field at time to avoid fatigue or travel sickness). If your laboratory is new to a technique, then it is a good idea to obtain a set of example slides from an experienced laboratory for quality comparison and training purposes. You should consider joining those relevant to your location and attending their meetings, partly to obtain the latest information on test methods and strategy, and partly to network with experts in the individual test methods who may be able to help you with assay development, training, or trouble shooting. I have outlined some suggestions in the following sections that should be considered as examples only. In this case, even slight improvements in the quality of slides will have a very beneficial effect in the long-term and are something you are liable to think about while actually reading slides. Your aim should be for every slide to be perfect, with (in the case of chromosome aberration slides) a high proportion of readable metaphases-perfectly spread, stained, and clear. In the case of the in vivo micronucleus test, cells should show good morphology with no evidence of lysis and excellent staining differentiation. Cell density should be consistent between slides and optimized to minimize scanning while avoiding overlapping. Purification and appropriate characterization of bacteria and cells are essential to confirm their utility. Bacteria and cells should never be grown beyond the exponential phase during maintenance or when grown for testing; otherwise, they can accumulate undesirable phenotype traits, will be less sensitive to mutagens, and, in cytogenetic tests, show low mitotic indices making slide reading more laborious. A little effort at the time of the test performance can greatly reduce the chance of problems arising or minimize their impact. A few suggestions (golden rules) are given here and in the individual chapters within this book: 1. Always transport it in a secondary container that helps prevent breakage if dropped. Help prevent spillage by placing it in a rack and immediately replacing the lid after usage. It is easy to lose your place when performing dilutions or dosing a long series of cultures, particularly if interrupted. Physically move the culture/plate/bottle/animal from one area to another after the addition, for example, when adding S9 mix to a series of bottles in a rack, dose them in a consistent order. Move bacterial mutation plates from the right to the left as they are plated with top agar. Move animals from their holding cage to a labeled cage with fresh bedding as each is dosed. For in vitro studies, keep an adequate number of spare cultures, spare racks of bottles, and spare plates to act as immediate replacements in case of an immediately obvious dosing error; make sufficient S9 mix to accommodate a few mis-dosings. For example, unexpected levels of liquid in bottles or in the micropipette tip will help highlight any obvious errors at an early stage. Check the morphology of cells under the inverted microscope each time you remove cultures from the incubator to confirm they are healthy. Maintain a list of supplies, suppliers, and catalog numbers that you can refer to for reordering. Staff should be trained in aseptic techniques to minimize potential microbial contamination of test systems. Additional precautions include regular thorough cleaning of laminar flow cabinets and the laboratory area. Disposable protective clothing helps minimize the chance of potential microbial contamination. To minimize potential chemical contamination when formulating, the vehicle should be prepared and aliquoted first before formulation of the test substance, including amounts used for diluting the stock test substance formulation and any samples taken for chemical analysis. Positive control substances should be prepared (or aliquoted from stock) separately and usually after test substance formulation. Positive control substances should, as far as is practical, be held separately from other test and reference substances. Please see the Formulation section (Chapter 3) for other precautions needed to ensure the safety of staff and minimize potential contamination of the facility. To a large extent, the checks mentioned in the previous section will reduce the need to repeat part of the study, but appropriate study design may also reduce the need for repeat testing. For example, although preliminary work in an in vitro study may give you a good idea of dose levels to use in the main test, toxicity tends to vary between test occasions; therefore, it is a good idea to include a slightly wider dose range by including an extra high and low dose level, for example, at least five dose levels in case of the chromosome aberration test, and then only those dose levels that achieve the targeted level of toxicity need to be subjected to detailed examination for aberrations. Similarly, in vitro mammalian cell tests will often include three dose levels of each positive control, with only that level showing the targeted level of toxicity being used for collection of results. Stock reagents added to culture medium are liable to microbial contamination if opened on multiple occasions and will themselves often support microbial growth; some organisms can even use antibiotics as a carbon source. All additions to bulk media should be added via a sterile filter after a visual check of the addition for signs of contamination. A stainless steel tray containing water should be placed in the bottom of the incubator to humidify the atmosphere. Small-volume cultures should be placed in a partially sealed food-grade bag or plastic box to reduce loss of volume during incubation. Some laboratories experience issues with compatibility of certain batches of S9 fraction or serum with their test system. Consider qualifying a particular batch of these for specific tests and reserve adequate bulk amounts if found to be suitable. This is especially appropriate if the company manufacturing the product has not tested it for compatibility in an analogous system. Any performance testing or related quality control information for these and other reagents including culture medium provided by the product supplier should be retained by the laboratory. Residual formulations (if known to be stable) may be stored until results of the test are available. For example, test substances can be identified by a code letter that is related to the test substance only in that particular study file while exact copies of the encoded results are held in each file. This is less of a problem if all the test substances belong to the same sponsor, in which case it may be acceptable (or even desirable for comparative purposes) to perform the study under a single protocol with a single report. This approach not only greatly reduces the overall workload but also reduces the number of animals used for scientific research. To simplify scheduling and reduce overtime requirements, it is often easier to always run studies of a particular type on the same day, for example, if bacterial mutation tests are performed on Tuesday and Friday, the plates can be removed on Friday and Monday morning and scored on the same day. Other procedures like maintenance, preparation of reagents, slide reading, preparation of reports, which are necessary but not time-critical, can be organized to fit around the scheduled testing. Clearly, it does not help with efficiency if each study is planned to start as soon as the test substance arrives without taking into account scheduling of other tests (as described). These default dates can then be used for the draft protocol that will be sent to the study monitor. At this point, outline dates can be agreed for the final protocol, but these should either be provisional and approximate. Hopefully, you will soon reach a point where this overtime limit is exceeded; at that point, you will need to consider hiring additional staff or equipment to automate certain tasks. A simple and very effective way of reducing the overall in-life phase of a study and at the same time reduce labor is to perform multiple aspects of a project in tandem. For example, you may be asked to perform Ames, chromosome aberration, and a rat micronucleus test on a single test substance with supporting formulation analysis. If the substance shows good aqueous solubility, then usually the same solvent can be used for each test; otherwise, and more typically, an organic solvent will be chosen for the in vitro studies and an aqueous suspending agent will be used for the in vivo studies as in the example in the following paragraph Example. A range of dilutions should then be made and tested for compatibility with the culture medium used in the chromosome aberration test. The test substance should be suspended in 1% methylcellulose at a concentration that allows the limit dose level for the micronucleus test to be achieved using an acceptable dose volume before assessing doseability, i. The low concentration evaluated will be close to the limit of quantitation or based on the planned lowest dose for appropriate assay(s) (Continued) Validation of the analytical method and formulation stability General Recommendations 43 Table 2. Homogeneity should also be established under the same conditions in the case of suspensions Formulations for the two in vitro tests should be made together and analyzed in the same run. All formulations should be stored under conditions that were previously confirmed as suitable until analysis confirms acceptability Often tests are divided into main and confirmatory tests. In this case, the two phases should be performed on the same day using the same formulation. In that way, results will be available much earlier and the costs of formulation analysis will be greatly reduced Formulation and analysis for the in vitro studies In vitro studies 2. Conditional formatting can be used in Excel to highlight values that are outside expected limits. Links from Word to Excel files are useful but should be broken once the Word document has been updated to avoid inadvertent updates later. Note that if graphs are copied from Excel into Word, they should be pasted as Pictures (paste special) to avoid carryover of spreadsheet information and larger file sizes. Slide code labels should be generated in Excel using the random function and the code should be saved electronically. Information from the electronic file can be imported into the results spreadsheet and then the table should be ordered logically. Commercial packages can handle certain aspects of tabulation and historical control information semiautomatically. The requirement is that each plate, culture, animal, formulation slide, or specimen from a study should be uniquely identifiable and traceable. When any of these items will be transferred to another location, these principles should be applied rigorously: formulation samples should be labeled with study number, test item (chemical or vehicle name as appropriate), concentration, and sampling date as a minimum and accompanied by an 44 Chapter 2 appropriate form showing sampling details that are also used for acknowledgment of receipt; in this way the form is used to document chain of custody. In other cases, it is more convenient and appropriate to generate an alpha-numeric code within a study design spreadsheet. If more than one experiment is being handled in an area at any one time and there is a potential for overlap or confusion, then a prefix may be used to distinguish between experiments. For example, if Ames tests are being performed on several days of the week and the same numbering system (1, 2, 3, etc. Note that plates and bottles should be labeled on the side rather than on the lid if there is any chance that the lids will become mixed. Note that several short-term in vivo experiments may be housed in a single room to reduce costs and save on space requirements. Note that individual facilities are likely to have their own numbering systems for animals that may, for example, help distinguish between groups. Even so, the prefix system may still be used if there is any overlap in numbering of individual animals in the same room. Animals for short-term studies can be identified by tail-marking using an indelible marker (rather than tattooing), although the number may need to be reapplied after a few days. For reference purposes, the study design spreadsheet is held in the laboratory throughout the dosing process. Stock solutions of positive controls and other reagents formulated on one occasion should be batched out. Most stock positive control solutions are sufficiently stable for 6 months if stored at approximately 220 C in darkness.

The right and left gonadal veins drain the gonads and eventually join the left renal vein symptoms jaw bone cancer calcitriol 0.25 mcg cheap. The hepatic portal vein drains the organs of the digestive tract and goes to the liver treatment deep vein thrombosis cheap calcitriol line. Veins Merging into the Inferior Vena Cava the anterior and posterior tibial veins and the peroneal vein drain the calf and the foot treatment yeast overgrowth discount 0.25 mcg calcitriol with visa. Blood flow to the skin brings oxygen and nutrients to and removes waste from skin tissue and glands medicine stick purchase calcitriol 0.25 mcg with amex. Dilation of blood vessels in the dermis occurs when survival and maximum function treatment 99213 calcitriol 0.25 mcg overnight delivery. The autonomic nervous system regulates heartbeat we are embarrassed, resulting in blushing seen in light-skinned individuals. Skeletal System Bones store and release calcium to maintain plasma rate and blood pressure. Endocrine System the bloodstream transports hormones to their target levels of calcium. During the aging process, the heart cells also accumulate collagen fibers and lipid molecules, producing stiffness and less elasticity. Calcium deposits increase and the connective tissue becomes stiff, resulting in abnormal valve function. A narrowing of the aortic semilunar valve and the bicuspid valve is especially common. Coronary artery disease is the major cause of death and heart disease in older Americans. Changes in the frequency of the electrical conduction system of the heart lead to a higher rate of arrhythmias or irregular heartbeats in older adults. Assuming no heart disease conditions, maintaining a consistent level of aerobic exercising improves the working capability of the heart muscle. In older adults, a daily walking regimen is one of the best exercises to maintain good heart performance. Cardiologists are physicians whose specialty training is in diagnosing and treat- ing the disorders of the heart. Cardiovascular technologists are allied health professionals who, under the direction of a physician, perform diagnostic examinations on patients for peripheral vascular studies and invasive and noninvasive cardiology. Electrocardiographic technicians are allied health specialists who operate and maintain electrocardiographic equipment, which records the electrical impulse of the heart. These technicians provide recorded data on heart performance for review by a physician. Lymphatic System the lymphatic system drains and returns interstitial Reproductive System Increases in blood volume to the penis maintains an fluids back to the bloodstream. The heart, blood, and the blood vessels of the body constitute the cardiovascular system. Iron and the B vitamins are provided by the digestive system for red blood cell formation. Respiratory System the respiratory system provides lungs for the exchange of oxygen and carbon dioxide with the red blood cells. The outer layer is the fibrous pericardium, made of tough fibrous connective tissue. Urinary System the kidneys filter the blood of wastes, excess electro- lytes, and water. A space called the pericardial cavity separates the epicardium of the heart from the serous pericardium of the pericardial sac. The upper chambers are the right atrium and the left atrium separated internally by an interatrial septum. Each atrium has an external appendage called an auricle, whose rough appearance is caused by the musculi pectinati. The lower chambers are the right ventricle and the left ventricle, which are separated internally from one another by the interventricular septum. Externally, the coronary sulcus, a groove, separates the atria from the ventricles. The anterior and posterior interventricular sulci separate the ventricles from one another externally. The superior or anterior vena cava receives blood from the upper parts of the body. The inferior or posterior vena cava receives blood from the lower parts of the body. The pulmonary trunk splits into the right and left pulmonary arteries, which carry deoxygenated blood to the lungs. The ascending aorta carries oxygenated blood away from the heart to all parts of the body. It is divided into the arch of the aorta, the descending thoracic aorta, and the abdominal aorta. Of the two ventricles, the left ventricle has the thickest walls of cardiac muscle because of the great distance it must transport blood. The bicuspid or mitral valve is found between the left atrium and the left ventricle. The cusps of the valves project into the ventricles by the chordae tendineae, which connect to papillary muscles in the ventricles, thus ensuring a one-way blood flow. The pulmonary semilunar valve is found in the right ventricle where the pulmonary trunk exits the heart. The aortic semilunar valve is found in the left ventricle where the ascending aorta leaves the heart. Deoxygenated blood returns to the heart from all parts of the body via the superior (anterior) and inferior (posterior) venae cavae to the right atrium of the heart. From the right ventricle the blood is pumped through the pulmonary semilunar valve to the pulmonary trunk, which divides into the right and left pulmonary arteries. The pulmonary arteries carry the blood to the lungs where it releases carbon dioxide gas and picks up oxygen. The blood is squeezed through the bicuspid or mitral valve into the left ventricle. The left ventricle pumps the blood through the aortic semilunar valve to the ascending aorta, which distributes the blood to all organs of the body. The conduction system of the heart generates and distributes electrical impulses over the heart, which cause contraction of the heart. The bundle of His branches down both sides of the septum as the right and left bundle branches, distributing the electrical impulses over the medial surface of the ventricles. Pulmonary circulation includes the deoxygenated blood that leaves the right ventricle through the pulmonary semilunar valve to the pulmonary trunk, which branches and goes to the lungs. In the lungs, carbon dioxide gas is released and oxygen gas is picked up to return to the left atrium via the four pulmonary veins. Arteries and veins have walls made of three layers: the innermost, tunica intima, made of a single layer of endothelial cells; the middle, tunica media, made of smooth muscle; and the outer, tunica adventitia, made of white fibrous connective tissue. Capillaries are microscopic vessels made of a single layer of endothelial cells with their basement membrane. They connect arterioles with venules and because of their structure, allow the exchange of gases, nutrients, and waste between blood and tissue cells. Veins have less elastic and smooth muscle than arteries but have more fibrous connective tissue. In a cardiac cycle, the two atria contract simultaneously while the two ventricles relax, and the two ventricles contract simultaneously while the two atria relax. The phase of contraction is called systole, and the phase of relaxation is called diastole. It has numerous branches named either according to the region of the body or organ it goes to or according to the bone its branch may follow. Veins are found closer to the surface of the body, and many can be seen superficially. The veins of the body converge with either the superior or inferior vena cava, the two largest veins of the body, which empty into the right atrium of the heart. Systemic circulation includes all the oxygenated blood that leaves the left ventricle through the aortic semilunar valve to the aorta and all the deoxygenated blood that returns to the right atrium via the superior and inferior venae cavae. Two are the coronary circulation that goes to the heart and the hepatic portal circulation that travels between the intestine and the liver. Explain blood flow through the heart, naming the major blood vessels entering and exiting the heart, the chambers, and valves. Compare the anatomy of the walls of arteries and veins and relate this to function. Explain how the conduction system of the heart functions and what factors may affect its rate. Write a twoto three-paragraph entry in your notebook summarizing what you learned. Create a list of things you can do to mAtchIng Place the most appropriate number in the blank provided. Mitral valve Pacemaker Visceral pericardium Right atrioventricular valve Pericardial sac Irregular folds of myocardium Appendage of atrium Ventricular contraction Bundle of His Relaxation phase fibers Project cusps into ventricle Contraction phase maintain a healthy heart. Visit the American heart Association website to learn how to recognize the warning signs of a heart attack and stroke. Study TooLs Study guide Online Resources activities for Chapter 14 PowerPoint presentations Copyright 2016 Cengage Learning. He tells his health care provider that he has experienced similar episodes of chest pain in the past, which were apparently triggered by stress. Based on his symptoms, the care provider tells Enrico that he is experiencing an attack of angina pectoris. She treats his condition, and then instructs Enrico to see his primary health care provider as soon as possible. Meanwhile, Enrico is advised to avoid stressful situations and take a small dose of aspirin daily. What underlying heart condition causes the chest pain associated with angina pectoris Now with your scalpel make a horizontal cut medially between the two auricles across the top of the heart. You will have cut the heart into two half sections, each a mirror image of the other. The easiest way to identify the correct chambers you are viewing is to note which ventricle has the thickest outermost wall. Above the left Materials needed: A dissecting kit, a preserved sheep heart, and a dissecting pan 1. As you hold the heart in your hand, notice that most of the heart is ventricles; in fact, about three-quarters of what you are holding are the ventricles. This is the external horizontal groove that goes around the heart and separates the atria from the ventricles externally. Locate the anterior and posterior interventricular sulci that separate the right and left ventricles from one another externally. It does not matter which half of your cut you are viewing because they are mirror images of one another. Above the right ventricle is the right atrium and the valve between them will be the tricuspid valve. Note how the chordae tendineae pull the cusps down into the ventricle and attach to the papillary muscles in the ventricles. Note the space in the left ventricle is smaller than the space in the right ventricle because there is so much more myocardium in the walls of the left ventricle. Less space equals more muscle, equals more pressure to pump the blood through the thousands of miles of blood vessels. If possible try to identify some of the great vessels entering and leaving the heart. Both systems transport vital fluids throughout the body, and both have a system of vessels that transport these fluids. The lymphatic system transports a fluid called lymph through special vessels called lymphatic capillaries and lymphatics. In addition to fluid control, our lymphatic system is essential to helping us control and destroy a large number of microorganisms that can invade our bodies and cause disease and even death. The lymphatic system consists of lymph, lymph vessels, lymph nodes, and four organs. Other functions are to transport fats from the digestive tract to the blood to produce lymphocytes and to develop immunities. In our bodies where blood capillaries are close to the cells of tissues, the blood pressure in the cardiovascular system forces some of the plasma of blood through the single-celled capillary walls. Most, but not all, of this fluid gets reabsorbed into the capillary by differences in osmotic pressure. Fats from the intestine travel through the lymphatic system, which delivers them to the blood when the lymph rejoins the blood at the right and left subclavian veins. Lymphatic Vessels Lymphatic vessels originate as blind-end tubes that begin in spaces between cells in most parts of the body. These vessels are not found in the central nervous system, red bone marrow, vascular tissue, or portions of the spleen. The large number of valves helps to ensure that the lymph will not backflow but go in one direction only.

The autonomic nervous system in addition uses adrenaline (also called epinephrine) as a transmitting agent medicine used to treat chlamydia 0.25 mcg calcitriol fast delivery. This is experienced when we prick our finger on a rose thorn and immediately pull away from the source of pain treatment 2 discount calcitriol 0.25 mcg buy on-line. The reflex arc has five components: (1) a sensory receptor in the skin medications gabapentin buy 0.25 mcg calcitriol visa, (2) a sensory or afferent neuron medicine 7 day box calcitriol 0.25 mcg purchase with visa, (3) association or internuncial neurons within the spinal cord symptoms your dog has worms 0.25 mcg calcitriol order fast delivery, (4) a motor or efferent neuron, and (5) an effector organ like a muscle. You have probably experienced a reflex arc when you had a physical examination and the doctor hit you below your knee with a rubber mallet. The doctor hits the patellar tendon just below the patella (the stimulus), Media Link Watch an animation on the firing of neurotransmitters on the Student Companion Website. Tracts are also found in the brain and connect parts of the brain with each other and with the spinal cord. Descending tracts conduct impulses down the cord and are concerned with motor functions. Tracts are made of myelinated fibers and therefore are classified as white matter. Its diameter varies at different levels because it is surrounded and protected by bone (the vertebrae) and by disks of fibrocartilage (the intervertebral disks). It is made up of a series of 31 segments, each giving rise to a pair of spinal nerves. It is a transparent fibrous membrane that forms a tube around and adheres to the surface of the spinal cord (and brain). This space contains loose connective tissue and some adipose tissue that acts as a protective cushion around the spinal cord. The impulse then travels to a motor neuron (response) back to the muscles that contract and your leg extends. Heartbeat rate, digestion, and breathing rates are controlled and maintained by reflexes concerned with involuntary processes. Coughing (the choke reflex), sneezing, swallowing, and vomiting are other examples of automatic subconscious reactions to changes within or outside our body. The term white matter refers to groups of myelinated axons (myelin has a whitish color) from many neurons supported by neuroglia. The gray areas of the nervous system are called gray matter, consisting of nerve cell bodies and dendrites. Because ganglia are made up primarily of unmyelinated nerve cell bodies, they are masses of gray matter. Functions of the Spinal Cord A major function of the spinal cord is to convey sensory impulses from the periphery to the brain and to conduct motor impulses from the brain to the periphery. A person may have a sense of being able to do anything physically, leading to serious injury to oneself. Amphetamines overstimulate postsynaptic neurons, resulting in muscle spasms, restlessness, rapid heartbeat, and hypertension. Athletes, like bodybuilders, can quickly increase their bulk, although this benefit has dangerous side effects. One example of a depressant is Valium, which is prescribed in low doses to relieve tension. Codeine can be prescribed by a doctor, but heroin has no legal use in the United States. Overuse can result in coma, convulsions, and respiratory problems that could lead to death. Marijuana has been prescribed for individuals with advanced stages of incurable diseases. The posterior or dorsal root is the sensory root and contains only sensory nerve fibers. The other point of attachment of the spinal nerve to the cord is the anterior or ventral root and this is the motor root. It contains motor nerve fibers only and conducts impulses from the spinal cord to the periphery (like muscles). A needle could also be inserted with an anesthetic agent to administer spinal anesthesia in this way. If a radiopaque substance is injected in this area, an X-ray can be taken of the spinal cord to detect any damage or defects in the cord. Yet spinal nerves, surrounded by the meninges, go all the way down to the end of the vertebral column. Because there is no spinal cord at the end of the vertebral canal, a needle can be inserted into the subarachnoid space in this area without damaging the spinal cord. There are 8 pairs of cervical nerves, 12 pairs of thoracic nerves, 5 pairs of lumbar nerves, 5 pairs of sacral nerves, and a single pair of coccygeal nerves. The spinal nerves are also numbered according to the order (starting superiorly) within the region. Thus, the 31 pairs are C1 through C8 (cervical), T1 through T12 (thoracic), L1 through L5 (lumbar), S1 through S5 (sacral), and Cx (coccygeal). The Nervous System: Introduction, Spinal Cord, and Spinal Nerves 245 Cervical Plexus C1­C4 Nerve supply to muscles of the neck and shoulder. Neurons are nerve cells that transmit nerve impulses in the form of electrochemical changes. Astrocytes are star-shaped cells that wrap around neurons for support in the brain and spinal cord and connect neurons to blood vessels. They form connective-like tissue rows for support and form the fatty myelin sheath on the neurons in the brain and spinal cord. Schwann cells form myelin sheaths around nerve fibers in the peripheral nervous system. A neuron is composed of a cell body with a nucleus and other intracellular organelles. Dendrites are extensions of the cell body and are the receptive areas of the neuron. An axon is a single long extension of the cell body that begins as a slight enlargement, the axon hillock. The axon may branch, but at its end there are many extensions called axon terminals. On large peripheral axons, a Schwann cell produces a fatty myelin sheath that surrounds and insulates the axon. They are found in the retina of the eye, the inner ear, and in the olfactory area of the nose. Unipolar neurons have only one process extending from the cell body, which then branches into a central branch that functions as an axon and a peripheral branch that functions as a dendrite. A nerve cell fiber has a higher concentration of Na1 on the outside than inside and a higher concentration of K1 on the inside than outside. The nerve fiber has a negative electrical charge on the inside and a positive electrical charge on the outside. When a nerve impulse begins, the sodium ions (Na1) rush in changing the inside electrical charge from negative to positive. Potassium ions (K1) move out to try to restore the resting membrane potential and the sodium-potassium pump operates to restore the original charge. The nerve impulse is a self-propagating wave of depolarization followed by repolarization moving in one direction down the nerve fiber. The all-or-none law states that if a nerve fiber carries any impulse, it will carry a full strength impulse. A synapse is an area where the terminal branches of an axon are close to but not touching the dendrites of another neuron. When an impulse reaches the axon terminals, it triggers the release of a neurotransmitter like acetylcholine into the synaptic cleft, which allows the impulse to travel across the synapse. Other neurotransmitters in the body are epinephrine or adrenaline, norepinephrine, serotonin, dopamine, and the endorphins. The Nervous System: Introduction, Spinal Cord, and Spinal Nerves 247 the reFlex arC 1. A reflex arc has five components: a sensory receptor in the skin, a sensory or afferent neuron, association or internuncial neurons in the spinal cord, a motor or efferent neuron, and an effector organ. The posterior or dorsal root is sensory and connects with the posterior or dorsal gray horn of the spinal cord. The anterior or ventral root is motor and connects with the anterior or ventral gray horn of the spinal cord. White matter refers to groups of myelinated axons from many neurons supported by neuroglia. Gray matter consists of nerve cell bodies and dendrites, as well as groups of unmyelinated axons and their neuroglia. The spinal cord is made of 31 segments, each giving rise to a pair of spinal nerves. The outermost spinal meninx is the dura mater or tough mother, the middle spinal meninx is the arachnoid mater or spider layer, and the innermost meninx is the pia mater or delicate mother. Between the dura mater and the arachnoid is a space called the subdural space, which contains serous fluid. Between the arachnoid and the pia mater is the subarachnoid space in which the cerebrospinal fluid circulates. The peripheral nervous system consists of the afferent system composed of neurons, and the efferent system composed of neurons. The autonomic nervous system is divided into the division, which stimulates, and the division, which restores activities and stimulates vegetative functions. The meninges have an outer meninx called the, a middle meninx called the or spider layer, and an inner meninx called the. The spinal cord conveys sensory impulses from the periphery to the brain (ascending tracts) and conducts motor impulses from the brain to the periphery (descending tracts). Each pair of spinal nerves connects to a segment of the spinal cord by two points of attachments called the roots. Astrocytes White matter Ganglia Nucleus Oligodendroglia Horns Meninges Tract Gray matter Microglia 1. White matter in spinal cord Search and explore Search the Internet with key words from the chapter to discover additional information. Key words might include central nervous system, classification of nerve cells, structure of a neuron, physiology of nerve impulse, spinal cord, and spinal nerves. Case study T hree high school students are being evaluated by health care providers at a drug rehabilitation clinic that serves adolescents. Upon examination, the care provider notes that Hector has a rapid heart rate and a dangerously high blood pressure. Carolyn, a 16-year-old girl, is being reassessed following a visit to the emergency room last night. When they could not arouse their son, they rushed him to the hospital where he experienced a convulsion. Do you think the drug Dante used is legal or illegal, or does it depend on the circumstances Note how the sensory dorsal posterior root of the spinal nerve enters the dorsal gray horn and how the motor ventral anterior root of the spinal nerve leaves the ventral gray horn of the cord. Test the knee-jerk reflex on your lab partner by gently tapping the patellar tendon with a small rubber mallet. Note how the extension of the knee is completely involuntary and subconscious, illustrating the performance of the reflex arc. Materials needed: Prepared microscope slides of neurons and neuroglia cells, anatomic model of the spinal cord, and rubber mallet. Study the parts of multipolar internuncial neurons, noting the following structures: the cell body with nucleus and dendrite extensions of the cell body; find axons with branches and axon terminals. Notice the small astrocytes and their stained nuclei scattered in the slide preparation. Examine an anatomic model of a section of the spinal cord with attached spinal Copyright 2016 Cengage Learning. IntroductIon this chapter is a continuation of the discussion of the nervous system that begins in Chapter 10. The brainstem controls breathing, heartbeat rates, and reactions to visual and auditory stimuli. The diencephalon includes the thalamus and the hypothalamus, which control many functions, including those related to homeostasis. The cerebrum controls intellectual processes and emotions, while the cerebellum maintains body posture and balance. The autonomic nervous system controls all the involuntary functions of the body such as regulating our internal organs and controlling glands. The special senses are part of the nervous system and include sight, hearing and balance, smell, and taste. Brain has a specific performs specific Structure enables Functions includes include Brain protects Cranial meninges Shock absorption, circulation of nutrients Coordination of muscular movements, balance Muscular movements, emotions, intelligence Temperature and pain recognition, homeostasis, thirst center, sleep patterns Consciousness, heartbeat, breathing, visual and auditory responses includes include Brainstem: medulla, pons, midbrain Diencephalon: thalamus, hypothalamus Cerebrum Cerebellum Cerebrospinal fluid allows allows controls control allow © Cengage Learning ConCept Map 11-1 the brain. The cranial meninges is the name given to the meninges that protect the brain, and they have the same structure as the spinal meninges: the outer dura mater, the middle arachnoid mater, and the inner pia mater (discussed in Chapter 10). The brain, like the spinal cord, is further protected by the cerebrospinal fluid that circulates through the subarachnoid space around the brain and spinal cord and through the ventricles of the brain. The ventricles are cavities within the brain that connect with each other, with the subarachnoid space of the meninges, and with the central canal of the spinal cord. The cerebrospinal fluid serves as a shock absorber for the central nervous system and circulates nutrients. The third ventricle is a slit between and inferior to the right and left halves of the thalamus, and situated between the lateral ventricles. Each lateral ventricle connects with the third ventricle by a narrow oval opening called the interventricular foramen or foramen of Monro. It connects with the third ventricle via the cerebral aqueduct, also known as the aqueduct of Sylvius.

Phlebitis in a superficial vein is indicated by tenderness treatment juvenile rheumatoid arthritis 0.25 mcg calcitriol visa, with or without redness and swelling along the course of the vein; pyrexia of variable degree and duration accompanies the disorder in the earlier stages but usually subsides in a few days 5 asa medications discount calcitriol line. Intra-abdominal phlebitis may be responsible for both continued pyrexia and vague treatment conjunctivitis order 0.25 mcg calcitriol with visa, but possibly severe treatment water on the knee 0.25 mcg calcitriol order with amex, abdominal pain in certain cases for which no explanation is forthcoming medications covered by medicare purchase cheap calcitriol on-line. Venous thrombosis in the pelvis, not necessarily associated with obvious femoral or popliteal thrombosis, may account for pyrexia after childbirth. Thyroiditis, an inflammatory but non-infective condition, may cause the complaint of sore throat, the thyroid itself being painful and tender. Elderly women are apt to develop a purulent form of endometritis, sometimes pyrexial, with pelvic pain, bearing-down pain, pain in the back, and a foul vaginal discharge that is often blood-stained, the condition simulating advanced carcinoma of the body of the uterus. Vesiculitis, although perhaps of local origin, is generally due to a gonococcal infection of the seminal vesicles. The complaint is mainly of hot burning pain in the rectum, aggravated by defecation; proctitis or carcinoma of the rectum or acute prostatitis is simulated. Diagnosis is established by rectal examination, the finger locating a tender swelling in the vesicles. Colitis, whether infective or ulcerative, will be suggested by a history of diarrhoea, with the passage of blood and mucus associated with more or less pain along the course of the colon, particularly the descending colon; carcinoma or diverticulitis may be simulated. The diagnosis is confirmed by endoscopy, barium enema and/or bacteriological studies. It is sometimes (but not always) pyrexial, and it may simulate other abdominal lesions such as gallstones. Glycosuria in association with pyrexia and a dull aching pain in the abdomen across the site of the pancreas may be suggestive, but the symptoms are generally too vague to be characteristic. Chronic pancreatitis should be suspected in a patient with recurrent abdominal pain, particularly if the pain or tenderness extends to the left of the midline, if gallstones are present, and if there have been bouts of overconsumption of alcohol. Repeated serum amylase estimations taken within 12 hours of an acute episode are elevated in most cases, but as more acinar and ductal cells are destroyed, these become less evident. Following acute pancreatitis, suppurative pancreatitis may occur in the second or third week with a return of fever. Familial Mediterranean fever (paroxysmal polyserositis or periodic fever) is an inherited disease of unknown aetiology that is characterized by acute episodes of self-limited fever with signs of inflammation of the peritoneum, pleura and joints. The disease occurs most commonly in patients of Mediterranean or Middle East origin, particularly in Sephardic (but not Ashkenazic) Jews, Armenians, Turks, Arabs, Greeks and, less commonly, Italians and others. Abdominal symptoms occur most often, occurring in over 95 per cent of patients, sometimes mild, but sometimes with severe localized pain that spreads over the whole abdomen, associated with abdominal distension and muscle rigidity and sometimes ileus, so that an acute surgical emergency may be suspected. Acute arthritis is less common, usually affecting one joint, the knee in most cases. Such arthritic episodes usually last for only a few days, but they may occasionally last for weeks or even months. The onset of this disorder is usually in childhood or adolescence, although it may come on at any age, with males being affected more often than females. In about 25 per cent of cases, transient inflammatory skin lesions like erysipelas occur, usually below the knees. Sarcoidosis, a chronic granulomatous inflammatory condition, may cause prolonged fever, sometimes with relatively little systemic upset. Hilar glandular enlargement on X-ray examination, erythema nodosum, and a weakly positive or negative tuberculin test are suggestive findings. Diagnosis may be achieved by bronchial and transbronchial biopsy performed at bronchoscopy in cases with an abnormal chest X-ray. In previous times, for example, untreated Addisonian anaemia was a febrile disease. Systemic lupus erythematosus may for many months present as pyrexia, often accompanied by skin rashes and symptoms relating to locomotor and other tissues. The high erythrocyte sedimentation rate and joint pains may lead to confusion with rheumatoid arthritis. Polymyositis and dermatomyositis may also be associated with fever, as may (although rarely) systemic sclerosis (scleroderma). The bite of this insect spreads the disease ­ sleeping sickness ­ by invasion of the central nervous system. The trypanosome may be identified in blood films, lymph node aspirates, more rarely bone marrow or, in the final sleeping stage, the cerebrospinal fluid. Diagnosis depends upon other factors, particularly the blood count, as in leukaemia. In agranulocytosis and aplastic anaemia, the infection is Malaria the main types of malaria are: benign tertian (Plasmodium vivax or Plasmodium ovale), in which rigors occur on alternate days with a maximum temperature of 39. The intervals between the attacks of fever vary in accordance with the time that successive generations of the various strains of parasites take to mature. A patient may be infected by one set of bites by a mosquito with a tertian or quartan ague, and subsequently become infected by other mosquitoes with either tertian or quartan parasites. Similarly, infection by two batches of parasites might result in a complicated clinical picture in which the attacks of pyrexia might be irregular or almost continuous. As a rule, a paroxysm, with its various cold, hot and sweating stages, lasts for about 8 hours. The diagnosis of malaria will be confirmed by the discovery of parasites on analysis of thick films of the blood. One remarkable feature is that malaria may remain latent for many years, particularly with Plasmodium malariae. However, considering the large number of serving men infected during the Second World War, relapses of malaria 1 year after returning home were rare. Reappearance is brought about by a general deterioration in health, or through some intercurrent illness. It is due to infection with Borrelia recurrentis being introduced by an infected louse when crushed against an abrasion or wound. Many strains are also conveyed by ticks, the disease being transmitted by their bites. The spirochaete may be identified in a blood film shortly before the febrile paroxysm but not in the intervals. Schistosomiasis this condition is widely spread through Egypt, Zimbabwe and certain other parts of Africa. Involvement of the urinary tract with Schistosoma haematobium is extremely common in these countries. As a rule, death or recovery renders such types of pyrexia of short duration, for instance in cases of pontine haemorrhage. Sometimes, however, the patient may survive long enough to come into the category of cases of prolonged pyrexia. Kala-azar (visceral leishmaniasis) There is no characteristic chart, and the pyrexia is often extreme and of a swinging, but continued, type. Great enlargement of the spleen with continued pyrexia would suggest the diagnosis, which is confirmed by discovering Leishman­Donovan bodies from material obtained by splenic or sternal puncture. This condition was not uncommon on the Mediterranean coast, and even today may be acquired on a short continental holiday, albeit rarely. There is less tendency to pyrexia in dermatitis herpetiformis, herpes gestations and erythema iris than in acute and subacute pemphigus. Pemphigus is always a serious and often a fatal malady, the skin blebs being but a local manifestation of some severe but ill-defined systemic reaction. Any diffuse inflammation of the skin, from acute psoriasis to a gold dermatitis, may be accompanied by fever. Plague and cholera Plague is also epidemic, the two best known types being the bubonic and the pneumonic. The diagnosis depends on discovering plague bacilli (Pasteurella pestis) in fluid obtained by aspirating a bubo, or from the sputum by special bacteriological methods. The pneumonic form is the more acute, and it may simulate lobar pneumonia; the bubonic form is of longer duration, with a lower grade of pyrexia. Lymphosarcoma may be associated with prolonged pyrexia, as may carcinoma, particularly if associated with sepsis, as is seen not infrequently in the bronchus and large intestine. Infection is not necessary for pyrexia to be present; high fevers are occasionally seen in primary and secondary carcinoma where no sepsis exists, and a low-grade pyrexia is common. Particularly likely to be accompanied by prolonged pyrexia are hypernephroma, hepatoma, secondary carcinoma in the liver and widespread metastatic disease. It is said that some have the art of squeezing the bulb between the fingers or between the teeth with just sufficient force to raise the temperature, but without breaking the glass. On suspicion of such a possibility, the temperature is taken ­ preferably per rectum ­ under careful scrutiny. If a factitious pyrexia is suspected, the patient may be left unattended on the first occasion; if the temperature is raised, it should then be taken again immediately under close observation. A marked difference between the two readings strongly suggests that the first reading has been faked. Cases are also recorded where patients have deliberately infected themselves to produce a fever. This is seen, for example, in Henoch­Schönlein syndrome (anaphylactoid purpura) and various skin allergies. Fever, pericarditis, pleurisy and pneumonitis may occur 2 weeks or more after the injury, and recurrences are common. Fever occurring in the treatment of pulmonary tuberculosis, for example, may be due to isoniazid, aminosalicylate or streptomycin, or to all three drugs. Antibiotics, sulphonamides, arsenicals, iodides, barbiturates and many others may be responsible. Of course, bacterial meningitis should always be borne in mind in a listless child with fever. It is as well to remember that drugs may themselves cause fever, and that the curative drug may sometimes maintain the fever it was used to cure. It is also as well to remember that products of tissue destruction may cause fever, as after myocardial infarction, pulmonary infarction, gangrene and tissue necrosis, and accumulation of blood in body cavities, gut, joints or elsewhere. Body temperature is apt to be disturbed in children by causes that would not produce pyrexia in an adult; hence a transient, irregular or recurrent rises of temperature in a child may often be of little significance. Nevertheless, if such rises of temperature persist, it is unwise to regard them as purely trivial. There are three conditions in particular that need to be specially borne in mind: (i) urinary infections; (ii) upper respiratory tract infections; and (iii) exanthemas and the infectious diseases of childhood before their specific rashes or other clinical features appear. With air travel readily available, malaria, kala-azar and any other tropical disease may be imported overnight and, with a large immigrant population from overseas, conditions such as amoebiasis, schistosomiasis and leprosy may be encountered in the ordinary outpatient clinic. Urinary infection Usually due to Escherichia coli, this is only too easily missed in a child, as there may be no symptoms attracting attention to the urinary system. The presence in the urine of leucocytes makes the diagnosis likely, although it needs culture of a clean specimen of urine to establish it. Upper respiratory tract infections these include tonsillitis, acute pharyngitis and rhinitis, and otitis media. Although symptoms usually point to the cause of the fever, in a small child this is not always so; the child has fever, is distressed and apparently in pain, but they cannot indicate to the parents where the trouble lies. Of the many other causes of fever in childhood, starvation, dehydration and acidosis should be mentioned. Tuberculosis in the lung, lymph nodes or elsewhere is much less common now in developed countries than previously, but it is still common in many parts of the world. In some cases, absence of fever in the presence of acute infection carries a bad prognosis. The quantity of pus may vary; when present in large quantities, it forms a thick grey or yellow sediment. On microscopy, pus cells (often neutrophils) are seen as rounded multinucleated bodies about twice the size of a red cell. As pus cells are, in fact, protein, dipstick testing is almost invariably positive for this substance. This test will also be positive if there are abnormally large numbers of epithelial cells in the sample so that microscopy of a urine sample is the only reliable simple test for the presence of pus. The site of the pus-producing lesion cannot be determined simply by examination of the urine. The general and specific history of the individual case is essential, although, in general, vesical lesions are often consequent upon renal lesions, particularly when these are infective. For example, when a pyelonephritis arises as a result of haematogenous spread, the initial symptoms may be those of cystitis. In time, the pyrexia, rigors and sweating attacks of pyelonephritis become manifest, together with severe loin pain on the affected side. A pyonephrosis need not necessarily present with pyuria if outflow from the kidney is blocked for one reason or another, such as a stone. The ureteric efflux may be thick and turbid, and exuding like toothpaste from the orifice if the urine production from that side is diminished. This line extends into a split as the bladder is distended and rivulets of blood are seen on the back wall of the bladder, falling down as a curtain into the area behind the trigone. The kidney that is the origin of acute pyelitis/pyelonephritis will appear larger on ultrasound examination, and the fluid in the collecting system will appear less transonic than normal urine, implying turbidity. Complete cessation of renal function is occasionally observed in acute interstitial bacterial nephritis. Cystoscopy Instrumentation of the lower urinary tract is not recommended during an acute illness as the risk of septicaemia is considerable. However, following appropriate antibiotic therapy and the resolution of gross infection, cystoscopy under further intravenous antibiotic cover may provide useful information.

It is common to only score small and large colonies for controls to ensure assay acceptance and for any positive concentrations to give an indication of potential mutagenic mechanism medications vaginal dryness order discount calcitriol line. The mean cloning efficiencies of the negative controls from the Mutation Experiments should be within the range 65À120% on Day 2 medicine website generic calcitriol 0.25 mcg fast delivery. The increases are concentration-related medicine 5 rights 0.25 mcg calcitriol free shipping, as defined by a statistically significant trend medications list buy generic calcitriol 0.25 mcg. The test item will be considered positive in this assay if both of these criteria are met and negative if neither of the criteria are met symptoms 5 days past ovulation buy calcitriol 0.25 mcg amex. Results that only partially satisfy the assessment criteria described are considered on a case-by-case basis. Positive responses only seen at concentrations of high toxicity are unlikely to be biologically relevant, but testing using other endpoints may be required to confirm this. However, this should only be taken as an indication of mutagenic mechanism, not as definitive evidence. An assay with such a high "false-positive" rate would seem to be of little value in any screening paradigm. Three hundred and three compounds were concluded to be negative (85% of the total number of compounds tested). Furthermore, only 19 compounds were positive by a mechanism that could not be related to the compounds primary pharmacological activity or were positive in other genotoxicity assays. This should be taken into consideration when comparing the performance of other in vitro genotoxicity tests and, perhaps more importantly, it is against this incidence that the performance and validation of novel in vitro genotoxicity tests should be judged. The use of L5178Y mouse lymphoma cells to assess the mutagenic, clastogenic and aneugenic properties of chemicals. Chromosome analysis of trifluorothymidine-resistant L5178Y mouse lymphoma cell colonies. Chromosome analysis of small and large L5178Y mouse lymphoma cell colonies: comparison of trifluorothymidine-resistant and unselected cell colonies from mutagen-treated and control cultures. Increase in incidence of leukemia in hybrid mice bearing thymic transplants from a high leukemic strain. A mutational assay system using the thymidine kinase locus in mouse lymphoma cells. A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells. Evaluation of the L5178Y mouse lymphoma cell mutagenesis assay: methods used and chemicals evaluated. Evaluation of the L5178Y mouse lymphoma cell mutagenesis assay: interlaboratory reproducibility and assessment. Evaluation of the L5178Y mouse lymphoma cell mutagenesis assay: quality-control guidelines and response categories. Evaluation of the L5178Y mouse lymphoma cell mutagenesis assay: intralaboratory results for sixty-three coded chemicals tested at Litton Bionetics, Inc. Genotoxicity: specific aspects of regulatory genotoxicity tests for pharmaceuticals. Mouse lymphoma thymidine kinase locus gene mutation assay: international workshop on genotoxicity test procedures workgroup report. Mouse lymphoma thymidine kinase gene mutation assay: follow-up international workshop on genotoxicity test procedures, New Orleans, Louisiana, April 2000. Mouse lymphoma thymidine kinase locus gene mutation assay: international workshop on genotoxicity test procedures workgroup report-Plymouth. Mouse lymphoma thymidine kinase gene mutation assay: follow-up meeting of the international workshop on genotoxicity testing-Aberdeen, Scotland, 2003-assay acceptance criteria, positive controls, and data evaluation. Mouse lymphoma thymidine kinase gene mutation assay: meeting of the international workshop on genotoxicity testing, San Francisco, 2005, recommendations for 24-h treatment. Widespread intraspecies crosscontamination of human tumor cell lines arising at source. The costs of using unauthenticated, over-passaged cell lines: how much more data do we need. Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and increased ion concentrations. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. An analysis of genetic toxicity, reproductive and developmental toxicity, and carcinogenicity data: I. Identification of genotoxicants, reprotoxicants, and carcinogens using in silico methods. The first suggestion of the use of micronucleus frequency as a quantitative measure of chromosomal damage was provided by Evans et al. In the early 1970s, two independent research groups described the use of micronuclei production in rodent bone marrow erythrocytes as a measure of chromosomal damage in animals exposed to mutagens in vivo [3,4]. Countryman and Heddle [5] were the first to develop an in vitro method using cultured peripheral lymphocytes. Because the expression of micronuclei is dependent on the cell undergoing division, it is important to be able to identify cells that have divided at least once during or after treatment. A breakthrough in this area came when Fenech and Morley [6] proposed the cytokinesis-block method. This utilizes cytochalasin-B, a mycotoxin isolated from Helminthosporium dematioideum, to inhibit cytokinesis without blocking mitosis [7]. This results in the formation of binucleated cells, allowing easy identification and scoring of cells that have undergone nuclear division after treatment. Image 1: Photograph of a micronuclei in a binucleate human lymphocyte cell (the image was captured from an acridine orange preparation in fluorescent colors and then the negative image was used to convert to grayscale). Thus, micronuclei may result from clastogenic or aneugenic mechanisms, for example, chromosome breakage leading to acentric fragments or interference with chromosomal segregation at anaphase. The in vitro micronucleus test is an umbrella term for many differing micronucleus tests, such as those with and without cytochalasin B and a variety of treatment and recovery schedules. Rapidly dividing cells can be used for the mononuclear micronucleus protocol and require a robust measurement of toxicity, such as population doubling. Micronuclei are readily detectable in interphase cells and, as a result, can be scored rapidly by eye or analysis can be automated. This makes it practical to score thousands of cells per treatment, increasing the sensitivity of the assay. The presence of a centromere in micronuclei is assumed to indicate the presence of a whole chromosome rather than an acentric fragment. Chromosome paints are commercially available for the centromeres of human and mouse chromosomes and have fluorescent labels incorporated [10À12]. The labeling and hybridization procedures for detection of centromeres can be used when an investigator wishes to determine whether an increase in micronucleated cells is the result of clastogenic and/or aneugenic events. This nondisjunction assay can be incorporated into the standard cytochalasin block micronucleus test to identify aneugenic agents [13]. This requires performance of a series of experiments with positive reference substances acting via different mechanisms, including at least one with and one without metabolic activation, and one acting via an aneugenic mechanism Table 6. The positive controls should demonstrate the ability of the cells to respond to both clastogens and aneugens and the capability of the metabolic activation system used for the assay, such as S9. Clastogens Active Without Metabolic Activation the In Vitro Micronucleus Assay 167 micronucleus formation at concentrations expected to provide reproducible increases over background, demonstrating the sensitivity of the test system [9]. The most commonly used system is a cofactor-supplemented postmitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 [14]. A combination of phenobarbitone and -naphthoflavone [15] is considered equally effective as Aroclor 1254 for inducing mixed-function oxidases and may be a preferred alternative in certain countries given that Aroclor is considered to be a persistent environmental pollutant and is difficult to obtain commercially. Its metabolic capacity is demonstrated by key enzyme assays and the ability to activate reference bacterial mutagens. S9 is added to a cofactor solution to form the S9 mix, which is then added to cultures. Alternatively, the main test can be conducted with a large number of dose levels (10À20), and then doses can be selected to score based on the cytotoxicity. At least three scorable concentrations must be scored and selected across the dose range to achieve a maximum of 50% toxicity. In addition, assessment of toxicity must be incorporated in the main experiment itself, even if performed in a preliminary experiment. A comparison of cytotoxicity measures for the in vitro micronucleus tests was undertaken previously [16, 17]. Furthermore, using these estimations of cytotoxicity and the limit of 50% survival, all the mutagens and aneugens tested were appropriately identified as positive in the in vitro micronucleus assay. Accordingly, it was clear that testing beyond 50% survival was not necessary to identify the potential of these agents to induce micronuclei [16]. Negative controls should be consistent with published negative control data, where they exist, for that cell culture system. The negative control values for an individual test should ideally be within the 95% control limits of that distribution based on percentile values. Historical positive and, in particular, negative control results distributions showing ranges, means, standard deviation, 95% limits, and number of replicates/experimental points involved should be presented in every report. Laboratories should use quality control methods, such as control charts to monitor potential drift over time. Human peripheral blood lymphocytes should be obtained from young (approximately 18À35 years of age), nonsmoking individuals with no known illness or recent exposures to genotoxic agents. The presence of a centromere signal in the micronuclei is assumed to indicate the presence of a whole chromosome. Cytochalasin B blocks cells at cytokinesis, leading to an accumulation of binucleate cells. The incorporation of centromere-specific probes allows visualization of chromosome segregation and distribution in the individual nuclei of the binucleate cell. When using two centromere-specific probes, the normal distribution of chromosomes would be two copies in each nucleus written as a 2:2 distribution. Nondisjunction is determined by examining the segregation of centromere-specific probes in the binucleate lymphocyte; therefore, the centromere probe materials are same as those in centromeric labeling (see Section 5. The program used for centromere-specific probes includes a denaturation step at 72 C for 2 min followed by 16À40 h at 42 C. Immediately prior to use, snap-thaw the S9 fraction and add 5À20 mL cofactor solution and mix well. The cell cycle time should be established using bromodeoxyuridine incorporation and this value should be used to ensure that cells have been treated for at least 1. Remove cells from liquid nitrogen, wash in medium, resuspend in fresh medium, and grow for 3À4 days to get sufficient cells for a micronucleus test. To set up test cultures, cells are disaggregated and counted, and the volume is adjusted with fresh medium to an appropriate concentration (usually 1 3 104 to 2 3 105 cells per mL) and 10 mL aliquots dispensed into 25 cm2 tissue culture flasks, and then incubated until required for assessment of cytotoxicity and micronucleus frequency. The cells for the micronucleus test are removed from the culture for cytospinning and the remaining cells are grown in culture for cell counts to determine population doubling. Individual cultures are vortexed and, when possible, 850 L is dispensed into a Megafunnelt and centrifuged at 1000 rpm for 8 min using a Shandon Cytospin 4. Slides are then placed in buffer for 10 min, followed by another 15 min in fresh buffer. Alternatively, after staining, slides are air-dried and stored protected from light. The slide codes are written or printed on adhesive labels together with the study number. A blank label is also placed on the reverse of the frosted end of the slide to cover all identification marks. Ideally, someone not involved in the micronucleus analysis should perform the coding, but when this is not possible, it will not invalidate the study. The code sheet must then be sealed and only opened after all analyses are complete for decoding. Slides are "wet"-mounted (carefully avoiding air bubbles) prior to scoring with phosphate buffer and a glass coverslip. Micronuclei are identified according to the criteria of Countryman and Heddle [16]. Less than one-third of the main nuclei size, the stain intensity should be similar to the main nuclei and all membranes must be intact and circular. The size of micronuclei can indicate an aneugenic response is some cases (Hashimoto 2010), but care must be taken not to overinterpret these data because some compounds with large amounts of breakage also provide large micronuclei as damage appears to accumulate. Counting is performed on electronic digital counters and data are recorded on paper. Relative proportions of micronuclei are determined in a total of 1000 mononuclear cells per each of two replicate cultures so that 2000 mononuclear cells are scored for each dose. In the event of an equivocal result, analysis may be extended up to 4000 mononuclear cells. In the event of unusual results, the study director or other senior cytogeneticist may reexamine and should comment on findings of any peer review. In our laboratory, we also conduct a yearly comparison of scoring among all trained scorers to check for drift. This program has been written to detect micronuclei in binucleate cells and contains classifiers that are easily modified to determine the size and shape of nuclei and micronuclei and are adaptable to mononuclear screening. The slides are scanned on Metafer using 3 20 magnification objective and an 8-slide automatic stage. The classifier developed by MetaSystems to score binucleate cells has been modified to score mononucleated L5178Y cells and is deliberately oversensitive to detect all aberrant divisions. Slides are scanned using the Metafer 4 master station comprising a Zeiss Axioplan Imager Z1 equipped with a Maerzhaeuser stepping motor stage that can scan eight slides unattended. The plane of focus is determined at a number of grid positions that are distributed evenly across the scan area.

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