Introduction

The body harbors a complex muscle system, in which individual muscles can be identified by their size, position, shape, function, and attachments. The skeletal muscles are striated and voluntary, highly specialized muscles, which attach to bones via tendons, have a specific anatomical position and innervation, and move the skeleton. Cardiac muscle is of a unique kind, striated and of involuntary type. The smooth muscle is also involuntary, and moves the bowel, modifies vessels, and constricts the bladder, to name just a few of its functions. By muscle diseases, one usually means diseases affecting the skeletal muscle, and experimental research on this muscle type is the focus of this chapter.

Diseases of muscle may result from a range of defects, from developmental defects to those in structural backbone proteins and energy metabolism. Research clarifying the nature of defects in muscle diseases has been a valuable source of information for understanding normal muscle function. Experimental muscle models can be created to study the normal function of a protein, or to study the effect of a gene mutation to clarify disease pathogenesis. Alternatively, interesting phenotypes may have arisen spontaneously in experimental animal lines, and their characterization may bring new knowledge of muscle function and diseases. Most experimental procedures concerning muscle diseases are common routine techniques of molecular biology and genetics. Therefore, in this chapter, we have concentrated on introducing those aspects of experimental muscle research that are specific for the tissue and its diseases.

Introduction

The research animal facility is perhaps the most highly regulated area of interaction between humans and animals. Informing staff members and researchers of the risks posed by research animals is one of many expectations that must be met by institutions. Research animal facilities often house several different animal species. Each animal has a unique physiological, anatomical, and microbiological profile that affects its potential to harm personnel. The institutions are responsible for conveying information regarding research animal risks to large numbers of personnel and researchers with diverse backgrounds. The challenge of animal risk assessment is to select essential and easily understood information to help people who work with animals. Providing too little information to the researchers is unacceptable, but overwhelming detail is equally likely to miss the scientific target of experiments.

Laboratory animal facilities are simply a special type of laboratory. As a general principle, the biosafety level (facilities, practices, and operational requirements) recommended for working with infectious agents in vivo and in vitro are comparable with a microbiological laboratory where hazardous conditions are caused by personnel, and by the equipment. In the animal room, the activities of the animals themselves can present new hazards. Animals may generate aerosols, they may bite and scratch, and they may be infected with a zoonotic disease transferable from animals to humans.

General hazards

Since 1967 rodent breeding units have been recommended to be built behind a “barrier,” a so-called specific-pathogen-free (SPF) space separated from the main laboratory.

Principles of surgery

Surgery in laboratory animals, regardless of species and size, is governed by the same principles as those for surgery of human beings.

A basic principle in surgery is Halstead's contribution of not doing harm to the tissue. In fact this is only one of a set of interrelated principles composed of tissue handling and exposure, asepsis and hemostasis.

• Tissue handling. Remember that every time you pick up tissue with your instruments, you kill cells. Try to kill as few cells as possible. Be goal-oriented in your approach and remember that sharp dissection is generally less traumatic than blunt dissection.

• Exposure. Make sure your view is unobstructed, with proper illumination and physical access. This means that the wound you make should be sufficient in size and certainly not too small. Do not worry about the healing of the wound, as it is not primarily affected by its size, but rather by appropriate approximation of the wound edges.

• Asepsis. Conditions favorable to bacterial growth must be avoided. First of all a meticulous sterile technique must be followed. Second, but equally important, dead tissue and foreign materials should be removed together with blood or serum residues.

• Hemostasis. Especially in small laboratory animals blood loss can have serious consequences eventually resulting in the untimely death of your animal. Clamping or applying light pressure can stop almost all bleedings. Other ways are coagulation (mono-or bipolar), cauterization, ligation and chemical treatment with collagen and/or ADP-containing hemostatics.

The surgeon

In the beginning the study of surgical and especially microsurgical techniques makes many mental and physical demands.

Introduction

Hydrocephalus is a common neurological condition characterized by impairment of cerebrospinal fluid (CSF) flow with subsequent enlargement of CSF-containing ventricular cavities in the brain. CSF absorption occurs through arachnoid villi into venous sinuses and along cranial and spinal nerves into lymphatics. Enlarging ventricles damage the surrounding brain tissue. In children, hydrocephalus is associated with mental retardation, physical disability, and impaired growth. The pathogenesis of brain dysfunction includes alterations in the chemical environment of brain, chronic ischemia in white matter, and physical damage to axons with ultimate disconnection of neurons. Hydrocephalus is the second most frequent congenital malformation (after spina bifida) of the nervous system, occurring in 5–6 per 10 000 live births. It also develops in 80% of patients with spina bifida, and 15% of premature (< 30 weeks) infants following intraventricular hemorrhage. Hydrocephalus can develop later in childhood or adulthood as a consequence of brain tumors, meningitis, brain injury, or subarachnoid hemorrhage.

For detailed discussions of the pathology of hydrocephalus see previous reviews (references 2 and 3). Briefly summarized, the ependyma lining the ventricles is damaged. In the subependymal layer, reactive gliosis is almost always observed and mitotic activity occurs among subependymal cells. Hydrocephalus can cause reduction in cerebral blood flow and alterations in oxidative metabolism in subcortical regions where white-matter axons and myelin are the main target of damage in hydrocephalus. Imaging studies indicate that the brain is edematous in the periventricular region.

Introduction

At present, over 23 million people in the United States suffer from central nervous system disorders. Globally, this number reaches a level of 368 million people. Yet, no effective therapy for the prevention or treatment of acute or chronic neuronal injury exists. As a result, identification of novel cellular pathways that determine neuronal survival and regulate programmed cell death or apoptosis in the nervous system become essential for the effective development of therapeutic strategies against neuronal injury. To achieve this goal, use of both in vitro and in vivo models of cell injury become essential to elucidate the mechanisms that determine intrinsic cell destruction and inflammatory cell demise.

In this chapter, we provide detailed methods for the preparation and analysis of experimental cell injury in several different cell and tissue animal models employed in the Maiese laboratory. The cell cultures from the central nervous system can provide a unique tool for the investigation of the cellular and molecular mechanisms that are involved in acute and chronic neurodegenerative disease. The utilization of a cell culture system avoids the complex environment of tissue or animal models that require multiple cell types to function in concert. By using isolated living cells, studies can focus upon specific signal transduction pathways that determine cellular function and cellular response to injury. Ultimately, knowledge gained at the cellular level must be transferred into experimental animal models in order to fully comprehend the physiological and pathological function of the brain.

Overview

Anesthesia in experimental animals is essential for both humane and scientific reasons to reduce or eliminate the pain and anxiety derived from physiological examinations or surgical treatments. Anesthesia also helps to immobilize animals to minimize the risk of injurious animal movements that could affect the outcome of experiments. Use of anesthesia should always be considered whenever experimental procedure accompanies the risk for animals to be caused pain or stress. Anesthetic agent is delivered either by injection or inhalation. Of those that are given by injection, the action may be local or systemic.

For clarity, the following terms are defined:

1. Sedation/tranquilization is a state of mild central depression in which the animal is awake and calm.

2. Analgesia refers to a temporal reduction of pain sensation accompanied by a trance-like neurolepsis, which is a state of depressed awareness of the surroundings.

3. Anesthesia is defined as a temporal and reversible reduction or elimination of feeling or sensation, often accompanied by loss of consciousness. When anesthesia is required, the purpose and extent of experimental procedure to be performed should be evaluated in order to determine if analgesia, sedation, or surgical anesthesia would be appropriate for a given procedure.

Many of the drugs used for anesthesia, such as opioids and barbiturates, are controlled and regulated by law. Information on controlled drug registration and also institutional/university policy should be obtained before starting to experiment. Licenses are needed to purchase these agents, and written records on purchase, storage, use, and disposal of them must be kept in a file.

Introduction

We spend a significant part (about a third) of our lives sleeping, which is essential to our physical and psychological well-being. Sleep, however, is a fragile state that can easily be impaired by psychological stress or physical illness. For up to 10% of the general population, difficulty falling and/or maintaining sleep occurs several times a week (i.e., chronic insomnia). Some of these problems may be due to existences of obstructive sleep apnea syndrome, a condition that affects over 10% of the population, or due to restless leg syndrome (RLS)/periodic leg movement syndrome (PLMS), sleep-related involuntary leg movements often associated with an abnormal sensation in legs. Excessive daytime sleepiness (EDS), parasomnia, and sleep problems associated with medical/psychiatric conditions are also common. Narcolepsy is a primary EDS disorder affecting about 0.05% of the population. EDS is also often secondary to a severe insomnia associated with obstructive sleep apnea.

Many different pathophysiological/etiological mechanisms for these sleep disorders are considered, and the International Classification of Sleep Disorders (ICSD) lists over 84 different types of disorders (Table 29.1). These sleep-related problems are often chronic and negatively affect the subject's quality of life. In a 24-hr society that encourages sleep deprivation, daytime sleepiness is also an emerging issue even in healthy subjects. Accidents due to sleepiness are now well recognized as a major public hazard. The emergence of clinical sleep medicine has proceeded rapidly during the last 30 years with the awareness of these sleep problems.

Introduction

Epilepsy is a common disorder of the brain affecting approximately 1–3% of people worldwide. Clinically, the epilepsies are characterized by spontaneous, recurrent epileptic seizures, either convulsive or non-convulsive, which are caused by partial or generalized discharges in the brain. Important advances have been made in the diagnosis and treatment of seizures disorders. Although many antiepileptic drugs (AEDs) have been introduced, approximately 30% of patients remain pharmacoresistant.

Animal models of seizures and epilepsy have played a fundamental role in the understanding of the physiological and behavioral changes associated with human epilepsy. They allow us to determine the nature of injuries that might contribute to the development of epilepsy, to observe and intercede in the disease process subsequent to an injury preceding the onset of spontaneous seizures, and also to study the chronically epileptic brain in detail, using physiological, pharmacological, molecular, and anatomical techniques.

Some criteria for a good animal model should be satisfied before the model could be considered useful for a particular human seizure or epilepsy condition. As the pattern of electroencephalograph (EEG) activity is a hallmark of seizures and epilepsy, the animal model should exhibit similar electrophysiological patterns to those observed in the human condition. The animal model should display similar pathological changes to those found in humans, it should respond to AEDs with similar mechanisms of action, and behavioral characteristics should in some way reflect the behavioral manifestations observed in humans. This chapter briefly reviews those models that most closely approximate human epilepsy.

Introduction

Stroke is the second leading cause of mortality worldwide and the third leading cause of death in the United States. Approximately 80% of strokes are ischemic in origin. Stroke ranked as the sixth leading cause of disability-adjusted life years in 1990 and is estimated to rank fourth by the year 2020. Of the stroke survivors about one-half are left with a permanent handicap. It is estimated that 731 000 new strokes per year, 4 000 000 stroke survivors, and 160 000 stroke deaths cost approximately $50 billion (direct and indirect costs) in the USA alone. Given this epidemiological evidence and the magnitude of the problem, it is clear that stroke is a major public health issue and requires urgent effort for developing novel remedies. Experimental focal brain ischemia models serve this purpose.

Experimental focal cerebral ischemia models have been developed with significant effort to mimic closely the changes that occur during and after human stroke. Models help us learn about the pathogenesis of stroke and to define the biochemical changes in tissue during ischemia, thereby discovering mechanisms involved in the evolution of ischemic injury and infarction. This can lead to the development of novel molecules that may reduce the consequences of ischemia and these same animal models can be utilized to test whether these novel molecules have beneficial anti-ischemic effects in vivo.

Overview

A neuroscientist embarking on a study of reproduction is eminently aware that an animal model chosen must consider gender differences and the endocrine factors determining them. Neuroscientists embarking on a study of function unrelated to reproduction may require the same consideration. It is now clear that gonadal hormones are important contributors to sex differences in a wide array of non-reproductive brain activities.

This conclusion has emerged from studies with animal models, sometimes unexpectedly when the experimental paradigm has included both males and females as subjects. Other findings of sex differences were observed first in human populations that suggested further study with animal models.

This review is a sampling of the remarkable variety of sex differences uncovered in adult brain function of which a neuroscientist may be unaware. A recent personal experience serves as an example. A respected colleague mentioned a new project to be conducted in her laboratory using an animal model of neuropathic pain. The interest was on a hypothesized acute attenuation of pain from treatments with a glutamate antagonist. Because the animal colony had an abundance of female rats, the subjects were to be groups of females. The assumption was that the topic had little relevance to reproduction and reproductive hormones, and any findings could be generalized equally to both sexes. Yet, there are reasons to question both assumptions. Both pain and glutamatergic pathways are influenced by sex hormones, and males and females may respond quite differently.

Introduction

Despite several decades of intensive study, the treatment of brain tumors, whether they have arisen primarily within the central nervous system (CNS) or spread there from elsewhere, represents a formidable challenge for the clinician. Several reasons underlie the difficulties in brain tumor treatment including delivering enough therapy through a blood–brain barrier (BBB), circumventing the relative immunosuppression that occurs both as a result of the brain tumor and due to its development in a relatively immunologically protected area, and the need to preserve the normal surrounding CNS. Such obstacles underscore the importance of accurate preclinical models both to understand the processes by which tumors develop and are sustained as well as to test the effectiveness of various treatments.

While in vitro systems are frequently used to assess both biology and treatment of tumors, the importance of the tumor–normal tissue relationships can only be addressed with in vivo models. Several years ago, Peterson et al. proposed criteria by which to judge the validity of such a model, including: (1) the growth rate of the tumor and its malignancy characteristics should be predictable and reproducible; (2) the species used should be small and inexpensively maintained so that large numbers may be evaluated; (3) the time to tumor induction should be relatively short and the survival time after induction should be standardized; (4) the tumor should have the same characteristics as the clinical tumor in terms of intraparenchymal growth, invasiveness, angiogenesis; (5) tumors should be maintainable in culture and should be safe for laboratory personnel; and (6) therapeutic responsiveness must imitate that of the clinical tumor being tested.

The ultimate validation of any animal model of human disease is the extent to which it simulates the parameter of interest. Depending on its purpose, the model may attempt to mimic the pathology and/or pathophysiology of a disease process, or predict the impact of putative therapeutic interventions. In the case of neurological disease, the ultimate outcome is behavioral. For example, in humans, the outcome of interest for patients with movement disorders is the preservation or return of normal motoric function, for persons afflicted with Alzheimer's disease it is the maintenance or restoration of normal cognition and for those with stroke or traumatic brain injury it is the return to their premorbid functional status. Although the fundamental features of simple stimulus–response relationships and even more complex environmental–behavioral interactions are remarkably preserved across species, the complete repertoire of human behaviors and their response to neurological disease are uniquely complex. Therefore, animal models will always be found to be wanting. Despite this fundamental limitation, animal behavior can provide critical insights into the functional consequences of human neurological disease and the potential benefit and toxicity of therapeutic interventions. They are particularly important as functional outcomes may be dissociated from the extent of neurological injury due to differences in the post-injury recovery process.

Limitations and principles

The limitations of small-animal behavioral models become apparent when considering the differences in even seemingly simple functional abilities as compared to humans.

Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized primarily by the gradual dopaminergic loss in the substantia nigra of the midbrain region. Development of PD can be sporadic or can be associated with genetic mutations and deficiencies, or may result from the combination of these two precipitating factors. The pathogenesis of PD has been studied in numerous experimental models developed to replicate the salient features of the disease in a controlled environment. Although no single model exists today that mimics all the neurological and neuropathological features of PD, each model presents a particular aspect of the disease process induced either by natural or artificial toxic agents or by genetically induced deficiencies in experimental animals. Epidemiological and laboratory results suggest that environmental factors play a predominant role in the induction and propagation of dopaminergic degeneration. However, numerous familial cases indicate that development of PD might be aggravated by pre-existing genetic deficiencies that act as predisposing factors. This chapter describes in detail the extensive research conducted using animal and tissue-culture models of Parkinson's disease induced by both toxins and genetic manipulation. Furthermore, salient experimental findings are thoroughly described with regard to current perspectives on neurotoxic mechanisms of genetic variations and environmental toxins.

6-Hydroxydopamine model of PD

6-Hydroxydopamine (6-OHDA) was first demonstrated to effectively replicate Parkinsonian neurotoxic pathology in rats by stereotaxic nigral injection in rats as early as 1975.

Introduction

The ethics, morals, and laws of any culture or nation are intimately interwoven and dependent upon each other for their continuation within that society. At the same time most cultures are under continual evolution, change, and development due to many factors, but usually due to ingress, influences, and pressures from other external factors and cultures. This is best illustrated by the notion that “developed” nations frequently bring about cultural changes in very old “traditional societies” through their presence and financial impact. Once there is cultural change, then almost certainly it will be followed by changes in the ethical and moral stances taken. Ultimately the laws and regulations will no longer reflect or uphold the current “values” of that society and will need modification.

This point is made to emphasize the fact that ethics and morals are not only diverse in a global sense but also dynamic. What was once ethically acceptable in history (e.g., slavery) may now be locally or globally seen as morally wrong and laws enacted to reflect those views.

With regard to laws it must also be acknowledged that as the “global village” becomes an increasing reality so does a meeting of minds on points of ethics and morals. As the meeting of minds becomes a reality, then it is possible to develop and implement international laws and regulations. This is particularly pertinent when considering the European position with regard to the Council of Europe (CoE) and the European Union (EU).

Human beings owe a great deal to animals. From the earliest periods of history of mankind, animals have been used by humans for food, clothing, tool making, and for several other purposes. Primitive artists painted animal figures onto stone surfaces; animal figures became parts of religions and tribal identities. Over time, some animals were domesticated, serving as regular sources of meat and milk; additionally, animals were used in farmwork and for transport. Dogs were used to defend property and were trained for rescue missions. Cats were used as pets as early as the ancient Egyptian Kingdom. Interesting additional missions have been given to animals such as searching for illicit drugs, explosives, and mushrooms. Some areas where we are still strictly dependent on animals include the drug industry (e.g., insulin isolated from swine pancreas), but there are also areas subject to intense debate (e.g., fur farming, fox hunting, and the cosmetics industry).

We are very much dependent on animals in medical research and in clinical surgery training. Neurological diseases comprise a major health problem all over the world and their importance continues to grow as the population ages and as neurology moves from being largely a diagnostic field to one with more therapeutic approaches. Neurological diseases already absorb approximately one-fourth of health budgets in industrialized countries. It is urgent to develop novel effective therapies for neurological diseases: the aging of the population will increase the number of neurological patients whereas the labor force available in the health sector appears to be decreasing.

Introduction

The purpose of this chapter is to provide a brief synopsis of bacterial meningitis and brain abscess and the animal models used to study these diseases and evaluate potential therapeutic modalities. The reader is encouraged to consult the selected references for more detailed information.

Bacterial meningitis

Despite advances made in vaccination and treatment strategies, bacterial meningitis remains associated with a significant mortality rate and incidence of neurological sequelae, particularly in very young and elderly patients. Approximately 1.2 million cases of bacterial meningitis are estimated to occur worldwide annually with 135 000 deaths. Long-term effects resulting from meningitis include hearing loss, hydrocephalus, and sequelae associated with parenchymal brain damage including memory loss, cerebral palsy, learning disabilities, and seizures. Organisms that colonize the mucosal membranes of the nasopharynx include Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae which are the leading etiologic agents of community-acquired meningitis. Recurring bacterial meningitis epidemics, the emergence of antimicrobial resistance among many meningeal pathogens, and the failure to introduce the H. influenzae conjugate vaccines (Hib) into many developing countries all contribute to bacterial meningitis remaining a serious global health problem.

Bacterial meningitis elicits a complex myriad of pathophysiological changes that present numerous obstacles when considering the design of therapeutic strategies. For example, besides the direct damage induced by pathogens, the host antibacterial response elicited during the acute phase of bacterial meningitis can be detrimental to neurons and other glia in the central nervous system (CNS) due to the toxic effects of cytokines, chemokines, proteolytic enzymes, and oxidants produced locally at the site of infection.