http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_10 Ischemic disorders of the retina constitute a common cause of blindness and visual impairment worldwide. Retinal ischemia is a disorder initially caused by an imbalance between the supply of metabolic substrates to the retina and its demand for nutrients. If treatment cannot be implemented to correct this imbalance, the result is irreversible ischemic and apoptosis-related cascades leading to cell death. A number of animal models are available for both studying the mechanisms of retinal ischemia and exploring potential treatments to prevent neuronal degeneration. However, the vascular supply and induction procedures of retinal ischemia in animal models must be better understood for application to human disorders. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_11 Retinal inflammatory disease of a putative autoimmune origin, known as autoimmune uveitis, affects 150,000 persons per year in the developed world and is a potentially blinding disease. The eye can be the only affected organ or uveitis can be part of a systemic syndrome. Animal models of uveitis induced by immunization with retinal antigens or through genetic engineering are used to study basic mechanisms, genetic control and therapeutic approaches. Although thymic expression of retinal antigens eliminates most autoreactive lymphocytes and positively selects natural regulatory T cells, peripheral tolerance to retina is inefficient due to the relative sequestration of retinal antigens. Therefore, residual autoreactive lymphocytes persist and can be activated by accidental encounter with self or a cross-reactive antigen. When exposed to retinal or cross-reactive antigens in the context of innate danger signals they mature into Th1 or Th17 effector cells that find their way into the eye. Upon recognition of specific antigen within the eye, they orchestrate a destructive inflammation by recruiting inflammatory leukocytes from the circulation. Regulatory T cells are also induced as part of the disease process and ultimately control inflammation and permit healing to take place. Novel immunotherapeutic approaches built upon the growing knowledge of basic mechanisms to target critical checkpoints in disease pathogenesis and restore immune homeostasis. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_1 The conversion of wavelengths of light into information useful to the brain requires a tremendous degree of anatomic and functional specialization. The mammalian retina is a remarkably refined and adapted tissue that is capable of light detection, processing, and transmission of information to other sites in the central nervous system. In this chapter, we provide a brief overview of the anatomical features of the mammalian retina and discuss regional variability observed in the eyes of humans and other species. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_2 Because repairing visual dysfunction is the primary goal of therapy for retinal disease, a quantification of visual function is imperative for the evaluation of potential treatments for these diseases. The Visual Water Task and the Virtual Optokinetic System have been developed to conduct behavioral tests of vision in rodent models of retinal disease. These tests are less invasive and often more sensitive than physiological or anatomical measures of retinal function. Moreover, discrepancies between different measures of retinal function suggest that central and retinal adaptations may complicate the disease process. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_3 Here we review both established and emerging approaches for studying retinal diseases. We primarily focus on the use of the mouse as a genetic model, as it is a mammalian model with many resources and is amenable to a variety of genetic manipulations. Additionally, we highlight two other organisms, zebrafish and fruit fly that are emerging as valuable genetic tools to study retinal disease. We discuss the ways in which near-complete genome sequences of these three organisms are revolutionizing our ability to investigate the complex mechanisms involved in retinal diseases. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_4 Retinal degeneration is often used to describe a category of human eye diseases, which are characterized by photoreceptor loss leading to severe visual impairment and blindness. An important, yet heterogeneous group of such diseases is called Retinitis Pigmentosa (RP). To understand the molecular mechanisms of disease induction and progression and to develop therapeutical strategies for the preservation of vision in RP patients, appropriate animal models are used in many research laboratories worldwide. The largest category of models consists of mutant (spontaneous and genetically engineered) mice. However, in recent years, zebrafish has been established as a highly valuable tool for the study of various biological problems, including retinal degeneration. In this review, we summarize the currently available mouse and zebrafish models to study retinal degeneration and give a short overview about recent developments in the field. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_5 Age-related macular degeneration (AMD) has a number of characteristic features including late onset and accumulation of deposits (drusen) below the retinal pigment epithelium on Bruch’s membrane in the macula. A progressive increase in these deposits (in some individuals) leads to macular blindness, following either the local loss of the retinal pigment epithelium (geographic atrophy) or the hemorrhage of new blood vessels that originate in the choroid and invade the compartment between the photoreceptors and retinal pigment epithelium (choroidal neovascularization). Over the last few years a number of mouse models for AMD have been described that replicate some of the changes manifest in the human disease. This chapter begins with a description of the hallmarks of AMD, discusses some of the ideas about the underlying mechanisms and then summarizes the features of AMD found in experimental animals that are purported to model this disorder. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_6 Retinopathy of prematurity (ROP), a condition affecting premature infants, is characterized by pathological angiogenesis, or neovascularization (NV), of the retina. Much of what is known about the development of the retinal vasculature and the progression of ROP has been learned through the use of animal models of oxygen-induced retinopathy (OIR), which approximate the human condition. Animal models of OIR have provided a wealth of information regarding the cellular and molecular pathogenesis of ROP. Moreover, this information has contributed to a better understanding of other, nonocular, neovascular conditions. This chapter describes the various animal models of OIR, and explores their contributions to the understanding and treatment of ROP. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_7 Diabetic retinopathy threatens vision in millions of patients in the USA. Prolonged hyperglycemia causes irreversible pathological changes in the retina, leading to proliferative diabetic retinopathy with preretinal neovascularization and diabetic macular edema. Much of the disease progression appears similar between man and animal. Thus, animal models are essential in understanding the pathology of this disease and development of effective treatments. This chapter describes and discusses the use of the rat, mouse, and dog in diabetic retinopathy studies. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_8 The generation and advancement of animal models have contributed significantly to the advancement of glaucoma research. This chapter describes and summarizes major nonprimate animal models useful for the study of this disease. Rodent models, both rats and mice, have been popular for glaucoma studies, because of the relatively better-developed genetic and genomic tools and the similarity of the relevant ocular structures between human and these animals. The larger animals, e.g., rabbit, feline, canine, bovine, ovine, and porcine models, have also been successfully used and provided valuable information on various aspects of the disease. Some of the models depicted in this chapter involve a transient or chronic ocular hypertension. Others do not affect intraocular pressure, but instead address certain specific mechanisms of the disease and serve as surrogate models. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-541-5_9 Glaucoma is a relatively common disease in which the pathological death of retinal ganglion cells causes progressive losses of sight, often leading to blindness. The diagnosis of glaucoma and the assessment of progression are based on a clinical quantification of the ocular characteristics of cupping of the optic nerve head, a loss of retinal nerve fiber layer thickness, and associated functional vision defects. Consequently, clinical tests are based on the quantification of these clinical characteristics of glaucomatous optic neuropathy. However, the basic neural and cellular pathophysiology that cause the characteristic signs of glaucoma cannot be studied in clinical patients and, therefore, animal models must be employed for basic research on glaucomatous optic neuropathy. For basic research that is directly applicable to the clinical disease, the primate model of experimental glaucoma is especially appropriate because the visual systems of macaque monkeys and humans are essentially identical, in terms of visual sensitivity, the anatomy and physiology of aqueous humor circulation, and the structure and neurology of the eye and visual pathway. 2010-01-15T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_10 Cellular transplantation is a promising therapeutic strategy for spinal cord injury (SCI); the clinical application of transplantation, however, will require safe and efficient protocols of cell delivery. Lumbar puncture (LP) is a minimally invasive delivery method that allows multiple cell deliveries into the intrathecal space, resulting in cell accumulation at the site of injury. Here we review the effectiveness of the LP technique in rodent models of SCI, the advantages of LP compared to traditional parenchymal injection and other minimally invasive methods, and provide detailed instructions for LP delivery of stem cells to the injured rat spinal cord. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_11 Peptides have been shown to be potent regulators of central nervous system (CNS) activity. As such, peptide-based drugs can serve as a highly effective means to treat diseases of the CNS. Despite this potential, peptides have limited capacity to permeate across the blood–brain barrier (BBB). Nevertheless, advancing strategies in peptide drug design, delivery systems, and transporter targeting are emerging, with such potential to overcome the restrictive nature of the BBB. This chapter addresses peptide drug delivery into the CNS, and in vivo evaluation of peptide permeability across the BBB using the dual-artery in situ brain perfusion model coupled with the technique of capillary depletion. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_12 The blood–brain barrier (BBB), formed by the endothelial cells of the brain capillaries, restricts access to brain cells of blood-borne compounds and allows only nutrients essential for normal metabolism to reach brain cells. This results in the inability of both small and large therapeutic compounds to cross the BBB. Therefore, various strategies need to be developed to enhance the amount and concentration of therapeutic compounds in the brain. A new family of peptides called Angiopeps derived from proteins expressing the Kunitz domain is transported very efficiently across the BBB using a physiological mechanism. Angiopep-2 transport across the BBB is, in part, mediated by the low density lipoprotein receptor related protein 1 (LRP1). This peptide family is the base of the engineered peptide compound (EPiC) platform technology allowing the synthesis of compounds which enter the central nervous system (CNS) to treat brain disorders. Using the EPiC platform, active drugs are modified by incorporation of Angiopep-2 allowing entry in the brain parenchyma. Many anti-cancer drugs, including paclitaxel, show poor delivery to brain tumors due to low permeability and active efflux transport at the BBB. As a proof of concept we have shown that Angiopep-2, a 19 amino acid peptide, is able to carry a payload composed of three molecules of an anti-cancer agent paclitaxel across the BBB resulting in a therapeutic concentration in the brain parenchyma. This first product called ANG1005 is now being evaluated in two phase I/II clinical trials. The use of this EPiC platform technology allows access to the brain parenchyma of small drugs, peptides, and larger hydrophilic agents for the treatment of CNS diseases. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_13 Gene therapy offers a novel approach for the treatment of experimental stroke. The adeno-associated virus (AAV) mediated vascular endothelial growth factor (VEGF) gene transfer into the ischemic brain is described in detail in this chapter. Other methods are also illustrated here, including the generation of mouse middle cerebral artery occlusion (MCAO) model, injection of viral vector into mouse brain, and standard assays for determining the successes of brain ischemia and gene transfer. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_14 Virus-mediated gene therapies against brain tumors have been limited by the difficulty in tracking glioma cells infiltrating the brain parenchyma. Mesenchymal stem cells (MSCs) are particularly attractive cells for clinical use in cell-based therapies because they have tumor-targeting properties, can be easily isolated and expanded to the numbers required for use, and can be genetically manipulated with viral vectors. In addition, most of the replication-deficient adenoviral vectors that have been used to transduce MSCs are based on human Ad serotype 5 (Ad5). However, transduction of MSCs by conventional Ad5 vectors is inefficient, even when very high multiplicities of infection are used because MSCs do not express the cellular coxsackie-adenovirus receptor. This chapter describes in detail how such adenoviral transduction of MSCs mediated by cell-permeable peptides should be prepared and handled, and applied for the use of targeted therapeutic gene delivery into glioma by an in vitro migration assay and by in vivo injection of MSCs into the tumor mass or the opposite hemisphere of established human glioma in nude mice. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_15 Convection-enhanced delivery (CED) is a method of direct intracerebral parenchymal infusion. It has been previously studied as a mechanism of drug delivery in glioma therapy, which is the focus of this review, and much work has gone into the utilization of this technique. CED can be modeled using several equations that describe the transport of fluid into porous tissue. More practically, variability in CED catheters has been studied and catheters are currently being designed to optimize drug delivery. While CED is a theoretically excellent way to bypass the blood–brain barrier (BBB), limitations of current approaches include excessive backflow along the catheter tract and leakage into subarachnoid and intraventricular spaces. Several drugs, including many recombinant cytotoxins, have been assessed for safety and efficacy in phase I and II clinical trials while many more are still in early preclinical stages. While drug development is important, we must also be able to assess the infusate’s volume of distribution to verify adequate coverage of the target area. This can be evaluated through a number of different MRI sequencing techniques, or by co-infusing gadolinium as a tracer in a variety of different formulations, or even via software programs that can predict target distributions. This review summarizes the important advances that have been made to optimize the unique ability of CED to locally deliver high doses of powerful chemotherapeutics to gliomas for maximal tumor killing with minimal neurologic and systemic side effects. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_1 Limitation of drug delivery to the central nervous system (CNS) is a major problem in development of successful treatment of CNS disorders. Concepts of blood–brain barrier (BBB), its role in transport of various substances from the blood to the brain, as well as strategies to deliver drugs across the BBB have evolved over the past century. This chapter is an overview of the challenges and various approaches to drug delivery in CNS disorders as an introduction to other chapters, which deal with laboratory and clinical methods of CNS drug delivery. A classification of current strategies for drug delivery across the BBB is presented. These include novel formulations of drugs such as nanoparticles and strategies to cross the BBB. Drugs can be introduced directly into the CNS and various devices are used for this purpose. Drugs can be administered systemically by various routes for targeted delivery to the site of action. Various methods of cell and gene therapies are used for drug delivery to the CNS. Finally, methods of delivery are classified according to various neurological disorders. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_2 Blood–brain barrier (BBB) limits drug delivery to the brain parenchyma. The ultimate goal of brain drug targeting technology is to deliver therapeutic agents across BBB. Insulin or transferrin as well as other endogenous peptides undergo receptor-mediated transcytosis or transport (RMT) across the BBB in vivo. Certain peptidomimetic monoclonal antibodies (mAb) for insulin receptor or transferrin receptor can also cross the BBB via RMT on the endogenous receptors. These mAb can act as molecular Trojan horses to shuttle into the brain a wide range of therapeutics including recombinant proteins, antibodies, RNA interference drugs, or non-viral gene products. During the last two decades, RMT-based brain drug transport techniques have been developed. This chapter will focus on introducing three major technologies in the arena of RMT brain drug delivery: (1) Avidin–biotin technology; (2) Fusion protein technology; and (3) Trojan horse liposomes (THLs). 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_3 A major challenge to drug delivery in the brain is caused by the blood–brain barrier (BBB), a composite of endothelial structures that exclude over 98% of small-molecule drugs and almost 100% of large-molecule neurotherapeutics from being transmitted to the brain. Current strategies for overcoming the BBB are either invasive, non-targeted, or demonstrating limited carrying capacity. Alternatively, non-invasive, transient, and local, image-guided blood–brain barrier disruption (BBBD) can be accomplished using focused ultrasound (FUS) exposure with intravascular injection of pre-formed microbubbles. Low-intensity FUS administered with microbubble-based ultrasound (US) contrast agents has been shown to transiently disrupt the BBB, allowing agents into the brain over several hours. Evaluation in vivo using MRI-guided FUS showed minimal adverse effects, as compared to invasive interventions. Electron microscopy indicated FUS activated trans-cellular transport, and reversibly affected tight junctions. Transient disruption of the BBB allowed delivery of both small-molecule drugs (Doxorubicin) and large-molecule antibody-based chemotherapeutic (Herceptin). The FUS-induced BBB disruption also allowed delivery of both molecular imaging and therapeutic agents directly to amyloid plaques in Alzheimer’s disease mouse models. A detailed description of the method for MRI-guided focal BBBD in animals will be described in this chapter. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_4 This chapter presents a mechanistic approach to in silico prediction of blood–brain barrier penetration by drugs and drug-like molecules. Focus is brought to critical analysis of experimental data used for modeling; several factors affecting data quality are discussed. The main experimental techniques used for measuring brain uptake are also briefly reviewed. The chapter provides a theoretical background for obtaining physicochemically reasonable and easily interpretable predictions of passive diffusion across blood–brain barrier, as well as some general advices regarding descriptor selection and model development. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_5 In the treatment of central nervous system diseases, therapeutic particles need to breach the blood–brain barrier (BBB) to reach their intended target, and it has long been known that this is a difficult barrier to breach. The barrier is both passive with tight junctions hindering passage of even rather small molecules, as well as active with transporters that can pump select molecules back into the bloodstream. The diseases include both those where this barrier has been compromised such as brain tumors, hemorrhagic stroke, and neurotrauma; as well as neurodegenerative disorders where the compromise is substantially less and often unnoticeable. In addition to the pharmaceutical solution which involves design of small molecules that may be administered orally or at least systemically, there is also a compelling need at present to design devices for direct delivery of therapeutics into the central nervous system. Such delivery methods are unpopular because of their intrusiveness; however, the lack of success of the systemic route in the treatment of many of the most severe brain diseases has established a need for such devices, and the success and popularity of deep brain stimulation (DBS) has lowered the psychological barrier to these. Many clinical trials are under way which demand direct delivery of therapeutics to the brain. Such therapeutics range from small molecules, large proteins, nanoparticles, and viral carriers for gene therapies, to cells. This chapter is a review of intraparenchymal delivery of such therapies for brain diseases and of the devices used for such purposes. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_6 Direct central neural axis neuromodulation has become a viable means to treat chronic neurologic disease and injury. Although first described in 1898, technology has only recently allowed for strict modulation and adjustments of drug delivery into the centeral nervous system. Evolving congruently with understanding of the altered neurophysiology and an expanding pharmacologic armamentarium, intrathecal pump systems augment treatment in an ever expanding number of disorders. In this review, we present a general historical overview of direct neuroaxis delivery and discuss current indications for intrathecal delivery with considerations in patient and drug selection. Complications, both surgical and medical, are discussed in detail with focus on avoidance and management. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_7 Intracarotid drug delivery was extensively investigated in the past; however, the approach was largely abandoned due to inconsistent benefits. Yet, intracarotid drug delivery is anecdotally used for treating a variety of brain diseases. The rapid advances in endovascular techniques now require a renewed evaluation of this therapeutic approach. This review describes the pharmacological principles, applications, and pitfalls of intraarterial drug delivery to brain tissue. 2010-01-05T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-529-3_8 Recently, the nasal route for systemic drug delivery has gained great interest. It provides several advantages over other routes of drug administrations. These include rapid absorption, avoidance of the intestinal and hepatic presystemic disposition, and high potential for drug transfer to the cerebrospinal fluid. Unfortunately, the mucociliary clearance, which reduces the residence time of the nasally applied drugs, and the poor nasal permeability made it difficult for many drugs to be delivered through this route. Alternative approaches have been adopted to overcome these problems. These include the use of mucoadhesive formulations or chemical penetration enhancers. Vesicular drug delivery systems provide promising alternative for enhanced and controlled nasal drug delivery. 2010-01-05T05:00:00Z