Introduction

Yearly, about 2 million patients will suffer traumatic brain injury (TBI). Much research has been conducted in the field of TBI over the past decades, yet no specific therapy is available. Different experimental models of TBI have been devised over the past years. Since TBI is a heterogeneous condition no single model can depict the actual pathophysiological changes associated with its entire spectrum. Therefore, each model can be seen as representing a subset of injury. Thus, some models are more akin to represent diffuse axonal injury whereas others are more representative of closed head injury with contusions and still others involve traumatic skull fractures with secondary brain impact. Of note, although some in vitro models for TBI exist (for review see reference 8) this chapter will limit itself to discussion of in vivo models. Using each of these models the interested reader may evaluate the physiological, neurochemical, behavioral–cognitive, histological, and pathological sequelae of TBI. Using these methods one can also assess new diagnostic tools and new therapeutic options for neurotrauma. Furthermore, new diagnostic tools such as magnetic resonance imaging (MRI) or MR spectroscopy can be used to further outline TBI pathophysiology.

Closed head injury

TBI is induced in this model by dropping a weight on top of the exposed skull leading to closed head injury (CHI) Adjusting the height and weight of the free-falling weight can modify the severity of the injury.

Only rarely does an animal experiment end in the natural death of the animal. In most cases, the animals must be killed following the completion of the experiment and certain tissue samples or organs must be collected. Even if the animal is fairly healthy after the experiment and even if it is not necessary to collect tissues, releasing laboratory animals into nature is strictly forbidden. Euthanasia is an important part of animal experimentation and every researcher who uses animals should be aware of the techniques and principles involved. There are a number of universally approved methods for euthanasia, but they may differ in different countries and different institutions even within the same country. This chapter will discuss the main methods of euthanasia. Every researcher must first consult the relevant authorities in their institution that provide permission for animal experiments, including the method of sacrifice. Only trained and competent personnel may carry out euthanasia within dedicated/suitable and approved facilities and only using methods approved by relevant authorities. Most animal facilities have veterinary physicians who are available for consultation and training purposes. The method for euthanasia should always be stated in detail in study plans and applications to the institutional body for animal care and use for scientific purposes. The method of euthanasia varies according to the animal species, number, nature of the experiment, and the intention of use of various organs for further research following death. Other animals and unnecessary personnel must not witness or hear the euthanasia procedures.

Introduction

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder in adults. The disease is characterized by the progressive loss of both upper and lower motoneurons, and is invariably fatal. Symptoms at the beginning of the disease include fatigue, cramps, muscular atrophy, weakness and wasting of one or more limbs or fasciculation of the tongue. As the disease progresses, patients become paralyzed and ultimately die from respiratory failure, typically within 1–3 years after diagnosis. The only medication approved for treatment of ALS is Rilutec® (Riluzole, a glutamate antagonist). However, ALS can currently not be cured and the available therapy offers only limited success, with a life extension of 3–4 months depending on the initiation of treatment. Famous ALS patients, such as the British physicist Stephen Hawking, the US baseball player Lou Gehrig (after whom the disease is named in the US), as well as the recent high incidence of ALS in soccer players especially in Italy have generated an increased public interest into this disease.

The emotional, social, and physical burdens of ALS are evident. Disease management requires sustained effort by patients, their families, caregivers, and healthcare professionals. Further, the economic impact of this disease is enormous. As the diagnosis of ALS is usually reached by exclusion, patients at the beginning of their disease generally undergo a series of arduous tests (magnetic resonance imaging (MRI), electrophysiologic studies, muscle biopsies, and blood studies).

Most experiments necessitate the use of several animals simultaneously. Especially when the animals are randomized to different groups that receive different treatments, it becomes crucial to be able to recognize different animals at different time points all along the experiments. Just placing the animals in different cages alone or in groups with the cages numbered or posted otherwise is not a reliable enough procedure. It is always necessary to mark the individual animals in a reliable way. It is of utmost important that all researchers in the same laboratory or institution follow the same marking system to avoid confusion. Large amounts of work and animals may be lost if this issue is overlooked. Marking animals for later identification can be done with various methods as long as the researchers are familiar with the marking system, enabling the correct animals to be tracked until the end of the experiment. There are a number of commercially available instruments for this purpose.

Dyes

Waterproof pens (permanent markers) in various colors are available in most stationers and bookstores. Marking of the tail is easy even in awake animals. The dye lasts for several days but not longer. The dye should be applied all around the tail, thickly and widely to assure future identification and to avoid misinterpretation. The color and the mark can easily be seen in albino animals, but may be more difficult to interpret in wild-type animals. We have not observed any inter-observer difficulty.

Basic immunology

Systemic immunology

Immunology is a relatively new and rapidly developing field that is involved in most clinical diseases. In 1796, Edward Jenner discovered that cowpox or vaccinia induced protection against human smallpox, but he knew nothing of the infectious agents that cause disease. Late in the nineteenth century, Robert Koch proved that infectious diseases are caused by microorganisms. We now recognize four broad categories of disease-causing microorganisms or pathogens: viruses, bacteria, fungi, and parasites. In 1890, Emil von Behring and Shibasaburo Kitasato discovered that the serum of vaccinated individuals contained antibodies that specifically bound to the relevant pathogen. Both innate and adaptive immune responses depend on the activities of leukocytes.

The immune system is a complex network of specialized cells and organs that defend the body against foreign pathogens and maintain the balance between immunity and tolerance. The peripheral lymphoid organs (lymph node and spleen) are specialized to trap antigen and allow the initiation of adaptive immune responses. Once lymphocytes are mature, they leave the central lymphoid organs (thymus and bone marrow), and are capable of responding to foreign pathogens. Peripheral mature immune cells include lymphocytes (T cells and B cells), antigen presenting cells (macrophages and dendritic cells, DC) and nature killer (NK) cells. Armed effector T cells play a critical role in almost all adaptive immune responses. Both major histocompatibility complex (MHC) and co-stimulatory signals provided by professional antigen-presenting cells (APC) are required for the activation and expansion of T cells.

Introduction

In spite of early attempts at neural transplantation as long ago as the late nineteenth century, throughout most of the twentieth century it was widely believed that the mammalian brain was relatively fixed and immutable in adulthood, incompatible with receiving and supporting viable transplants. However, at the end of the 1960s, two discoveries challenged this received view: the demonstration that sprouting and reorganization of axons can indeed take place after damage in adult central nervous system (CNS) pathways; and new experimental methods for transplanting nerve cells that were remarkably successful in yielding surviving grafts.

In the first decade after these pioneering studies, attention focused on understanding the basic cellular and developmental biology of neural transplantation in a variety of model systems. Cells were transplanted into the CNS of adult rats using a wide variety of experimental model systems – anterior eye chamber, spinal cord, cerebellum, and diverse forebrain sites including cortex, hypothalamus, striatum, and hippocampus. In the first wave of studies (as illustrated in Fig. 17.1), pieces of neural tissue were implanted into natural cavities such as the anterior chamber of the eye, the brain ventricles or choroidal fissure. In the search for a greater flexibility of graft placement, other studies introduced inoculation of tissue fragments directly into brain parenchyma, although such grafts did not survive well, or the creation of artificial cavities with a rich vascular lining that would nourish newly grafted tissues.

Introduction

Animal models that recreate specific pathogenic events and their corresponding behavioral outcomes are indispensable tools for exploring the underlying pathophysiologic mechanisms of disease and for investigating therapeutic strategies prior to testing them in human patients. Although rodents have a long tradition as models for human neurological diseases, they have received increasing attention in light of genetic engineering methods that have made it possible to create precisely defined genetic changes. The development of transgenic technology, a tool that allows sophisticated manipulation of the genome, has provided an unprecedented opportunity to expand our understanding of many aspects of neuronal development, function, and disease.

Transgenic animals are specific variants of species following the introduction and/or integration of a new gene or genes into the genome of the host animal. Transgenic technology is now routinely used to increase the level of (overexpress) particular proteins or enzymes in animals or to mutate or inactivate a particular gene using a “knockout” approach. One of the most commonly used animals in transgenic techniques is the mouse, a species in which transgene microinjections into the pronuclei of fertilized oocytes and the subsequent expression of the transgene in the animal have been carefully worked out.

Since their development in the 1980s, the transgenic and knockout technologies have allowed us to examine the regulation of gene expression and the pathophysiology of its alterations.

Introduction

Therapies targeting the central nervous system (CNS) are a crucial challenge for future medicine. In fact, degenerative and immune-mediated disorders of the CNS are a major threat to quality of life in the elderly, but diseases affecting the brain are also not infrequent in infancy and adult life. Transfer of recent progresses in the knowledge of molecular mechanisms involved in the pathogenesis of neurological disorders into novel therapies is difficult, because penetration of molecules into the brain is extremely limited by the presence of the blood–brain barrier (BBB). The BBB is characterized by tight junctions between endothelial cells which are impermeable to macromolecules and even ions, and by reduced endothelial endocytic activity that considerably decreases the number of molecules that can cross the BBB in a non-specific fashion. Most of conventional therapeutic agents effective in the CNS are supposed to cross the BBB because of their small size. However, more than 98% of small molecules cannot cross the BBB either, and only the presence of specific transport mechanisms assures that molecules essential for the brain metabolism (e.g., amino acids and glucose) reach the brain parenchyma. Thus, by employing conventional administration routes (i.e., oral, intravenous, intramuscular), which share the bloodstream as the final driving force to the brain, both rate and selectivity of the drug delivery are severely hampered, resulting in limited efficacy and potential side effects.

Introduction

Article 5 in Directive 86/609 of the Council of the European Communities states:

Animal needs are summarized in a report on farm animals by the Brambell Committee which referred to the provision of “Five Freedoms.” These were reported as:

1. Freedom from malnutrition;

2. Freedom from injury and disease;

3. Freedom from thermal and physical discomfort;

4. Freedom from fear and stress;

5. Freedom to express most normal patterns of behaviour.

These freedoms are not confined solely to farm animals but are applicable to all animals maintained in captivity, including those kept within laboratories as breeding stock or for experimental use.

The scientist and the animal technician must be constantly aware that animals housed in the laboratory either for breeding or scientific studies lack the self-sufficiency of their wild counterparts. They are completely dependent on him, or her, for all these physiological and ethological needs; they have a limited ability to vary or improve their environmental conditions by seeking warmer or cooler areas, are unable to vary their food or seek out additional supplies should they become depleted, and have no control over access to liquid as this is usually from a single source; and the space in which they exercise, mate, and socialize is controlled and confined.

Introduction

Each year, about 500 000 people in the USA suffer a cardiac arrest, an event that is associated with high mortality and poor neurological outcome Survival rates range from 1% to 33%, and up to 60% of survivors have moderate to severe cognitive deficits 3 months after resuscitation. Frequent neuropsychological sequelae include anterograde memory deficits, learning difficulties, changes in emotional and social behavior, and depression. Despite improvements in resuscitation techniques, survival rates have not changed for decades. One reason for this disappointing development is the lack of effective treatment options to ameliorate reperfusion injury in the post resuscitation period despite promising results of a variety of agents in animal studies. However, recent clinical trials showed that induction of mild hypothermia in unresponsive cardiac arrest survivors can improve neurological outcome and 6-month survival. This was the first demonstration in humans that the development of brain injury after cardiac arrest can be positively influenced by a post-ischemic intervention, even with delayed onset of treatment.

This exciting evidence that ischemic human brain tissue is potentially salvageable has renewed interest in global cerebral ischemia research. In the following review we will describe the most commonly used rodent models of transient global or forebrain ischemia and summarize their advantages and disadvantages. The use of small animals for research studies presents some clear advantages over large animals. Rodents are much less costly to obtain and maintain for longer periods of time.

Introduction

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) which manifests most commonly as weakness and sensory loss and is characterized by immune-mediated inflammation. Approximately 2.5 million individuals worldwide (400 000 in the USA) are afflicted with MS and among these, the disease is skewed toward Caucasians and females. The resultant economic burden caused by MS in the USA is approximately $20 billion. Superimposed on this cost is the personal burden of living with a debilitating condition for which there is no permanent cure and often no treatment of symptoms. MS is progressive in most patients and within 15 years of diagnosis, 70% of patients are unable to perform normal daily activities without assistance. The most frequently administered treatments (interferon (IFN)-β1a or IFN-β1b) for the most common disease course (relapsing–remitting MS) result in 30% fewer clinical exacerbations and can only delay onset of disability. Thus, researchers in autoimmunity and neurology are in pursuit of new treatments that can effectively and reliably halt or reverse progression of MS.

Establishing potential MS therapies requires an experimental animal model of the disease. The conventional animal model, experimental autoimmune encephalomyelitis (EAE), is a symptomatic and histologic recapitulation of MS based on a presumed etiology of autoimmune-mediated demyelination. In the earliest demonstrations of EAE, it was found that injections of brain or spinal cord extract could cause disease in primates and that addition of an immunological adjuvant eliminated the requirement for repeated injections and decreased the number of days until onset of symptoms.

Introduction

The availability of advanced imaging techniques has revolutionized the evaluation of many clinical neurological disorders. Similarly, the availability of advanced imaging techniques has enhanced the utility of animal models related to the study of these disorders. Currently, the most available and useful imaging techniques in animal models of neurological disorders are those related to magnetic resonance imaging (MRI) applications, computerized tomography (CT), and positron emission tomography (PET). This chapter will introduce the basic concepts related to these various imaging modalities and then discuss their application to neurological disorders with a focus on acute ischemic brain injury.

Magnetic resonance imaging

A wide variety of MRI techniques are currently available for use in animals and patients. The range of MRI modalities and their main uses are provided in Table 9.1. The initial MRI modalities employed were T1- and T2-weighted imaging that evaluated the density of water proton spins. Water protons in relatively unrestricted fluid spaces have higher T1 and T2 values, while these protons in more restricted environments such as brain edema, hemorrhages, or tumors have lower values. These two MRI modalities are associated with an increase in interstitial water content of the brain, as seen with the development of vasogenic edema. Conventional T1 and T2 MRI have been used widely in clinical imaging for two decades and have also been used extensively in animal models of brain ischemia, tumors, traumatic injury, and multiple sclerosis (MS).