Living Animal Organs Research Articles

Dynamic fluorescence molecular tomography metabolic parameters solution based on problem decomposition and prior refactor.

Dynamic fluorescence molecular tomography (DFMT), as a noninvasive optical imaging method, can quantify metabolic parameters of living animal organs and assist in the diagnosis of metabolic diseases. However, existing DFMT methods do not have a high capacity to reconstruct abnormal metabolic regions, and require additional prior information and complicated solution methods. This paper introduces a problem decomposition and prior refactor (PDPR) method. The PDPR decomposes the metabolic parameters into two kinds of problems depending on their temporal coupling, which are solved using regularization and parameter fitting. Moreover, PDPR introduces the idea of divide-and-conquer to refactor prior information to ensure discrimination between metabolic abnormal regions and normal tissues. Experimental results show that PDPR is capable of separating abnormal metabolic regions of the liver and has the potential to quantify metabolic parameters and diagnose liver metabolic diseases in small animals.

Evaluation of a New Animal Tissue-Free Bleeding Model for Training of Endoscopic Hemostasis.

For endoscopists, knowledge of the available hemotherapeutic devices and materials as well as competence in using them is a life-saving expertise in the treatment of patients with acute gastrointestinal bleeding. These competences can be acquired in training on live animals, animal organs, or simulators. We present an animal tissue-free training model of the upper gastrointestinal tract for bleeding therapy. An artificial, animal tissue-free mucosa and submucosa with the opportunity of injection and clipping therapy were created first. Patches with this artificial mucosa and submucosa were placed into silicone and latex organs with human-like anatomy. Esophageal bleeding situations were imitated as variceal bleeding and bleeding of a reflux esophagitis in latex organs. Finally, a modular training model with human anatomy and replaceable bleeding sources was created. Evaluation of the novel model for gastroscopic training was performed in a multicentric setting with endoscopic beginners and experts. Evaluation was carried out by 38 physicians with different levels of education in endoscopy. Evaluation of the model was made with grades from one (excellent) to six (bad): suitability for endoscopic training was 1.4, relevance of the endoscopic training was 1.6, and grading for haptic and optic impression of the model was 1.7. The creation of a gastroscopic model for the training of hemostatic techniques without animal tissues was possible and multiple endoscopic bleeding skills could be trained in it. Evaluation showed good results for this new training option, which could be used in every endoscopic unit or other places without hygienic doubts.

Regulatory aspects for Biologic Product licensing in Australia

The TG Act defines biological as product made, from or containing, human cells or human tissues, lives animal organs, cells or tissues, and that is used to treat or prevent disease or injury, Diagnose a condition of a person and Alter the physiological processes of a person.
 The Australian Regulatory Guidelines for Biologicals (ARGB) provide the keen information for manufacturers, sponsors, professionals in healthcare and also to public about the use of human cells and tissues based therapeutic goods, live animal cells, organs and tissues (1). These all products are Biologicals. This guideline is specially written for general public. If you are a sponsor or manufacture, this will:
 
 Explains the biological regulatory framework is applies to manufacturer’s product and their exemption conditions (1).
 Explains the Australian regulatory requirements for supplying of Biologicals
 Explains what is required for the market authorization as per TGA especially for Biologicals.

Renaissance of morphological studies: the examination of functional structures in living animal organs using the in vivo cryotechnique.

Medical and biological scientists wish to understand the in vivo structures of the cells and tissues that make up living animal organs, as well as the locations of their molecular components. Recently, the live imaging of animal cells and tissues with fluorescence-labeled proteins produced via gene manipulation has become increasingly common. Therefore, it is important to ensure that findings derived from histological or immunohistochemical tissue sections of living animal organs are compatible with those obtained from live images of the same organs, which can be assessed using recently developed digital imaging techniques. Over the past two decades, we have performed immunohistochemical and morphological studies of the cells and tissues in living animal organs using a novel in vivo cryotechnique. The use of a specially designed liquid cryogen system with or without a cryoknife during this cryotechnique solved the technical problems that inevitably arise during the conventional preparation methods employed prior to light or electron microscopic examinations. Our in vivo cryotechnique has been found to be extremely useful for arresting transient physiological processes in cells and tissues and for maintaining their functional components-such as rapidly changing signaling molecules, membrane channels, or receptors-in situ. The purpose of the present review is to describe the basic mechanism underlying cryotechniques and the significance of our in vivo cryotechnique. In addition, it describes various morphological or immunohistochemical findings, observations made using quantum dots, and a Raman cryomicroscopy-based method for assessing oxygen saturation in the erythrocytes flowing through intestinal tissues.

Morphofunctional Merits of an In Vivo Cryotechnique for Living Animal Organs: Challenges of Clinical Applications from Basic Medical Research.

Recent advances in molecular and genetic techniques have led to establishment of new biomedical fields; however, morphological techniques are still required for a more precise understanding of functioning cells and tissues. Conventional preparation procedures involve a series of chemical fixation, alcohol dehydration, paraffin or epoxy resin embedding, sectioning, and staining steps. In these steps, technical artifacts modify original morphologies of the cells being examined. Furthermore, difficulties are associated with capturing dynamic images in vivo using conventional chemical fixation. Therefore, a quick-freezing (QF) method was introduced for biological specimens in the 20th century. However, specimens have to be resected from living animal organs with blood supply, and their dynamical morphologies have not been investigated in detail using the QF method. In order to overcome these issues, the tissue resection step of organs had to be avoided and samples needed to be frozen under blood circulation. Our in vivo cryotechnique (IVCT) was an original technique to cryofix samples without resecting their tissues. The most significant merit of IVCT is that blood circulation into organs is preserved at the exact moment of freezing, which has been useful for arresting transient physiological processes of cells and tissues and maintaining their components in situ.

Islet Xeno/transplantation and the risk of contagion: local responses from Canada and Australia to an emerging global technoscience.

This paper situates the public debate over the use of living animal organs and tissue for human therapies within the history of experimental islet transplantation. Specifically, the paper compares and contrasts the Canadian and Australian responses on xenotransplantation to consider what lessons can be learnt about the regulation of a complex and controversial biotechnology. Sobbrio and Jorqui described public engagement on xenotransplantation in these countries as ‘important forms of experimental democracy.’ While Canada experimented with a novel nation-wide public consultation, Australia sought public input within the context of a national inquiry. In both instances, the outcome was a temporary moratorium on all forms of clinical xenotransplantation comparable to the policies adopted in some European countries. In addition, the Australian xenotransplantation ban coincided with a temporary global ban on experimental islet allotransplantation in 2007. Through historical and comparative research, this paper investigates how public controversies over organ and tissue transplantation can inform our understanding of the mediation of interspeciality and the regulation of a highly contested technoscience. It offers an alternative perspective on the xenotransplantation controversy by exploring the ways in which coinciding moratoriums on islet allograft and xenograft challenge, complicate and confound our assumptions regarding the relationships between human and animal, between routine surgery and clinical experimentation, between biomedical science and social science, and between disease risks and material contagion.

Construction of human LRIG1-TAT fusions and TAT-mediated LRIG1 protein delivery

Human leucine-rich repeats and immunoglobulin-like domains (LRIG1) is a tumor suppressor in animals and also functions as an endogenous suppressor in human tumor. The level of LRIG1 expression is highly associated with patient survival in clinic. The exploration of LRIG1 as a protein drug is an important task. HIV-1 transactivator of transcription peptide (TAT) is an excellent candidate for protein transduction. In this study, human LRIG1 was cloned and LRIG1-TAT fusion gene was constructed. The fusion proteins were produced by an Escherichia coli strain and purified by Ni2+-resin. Western blot assay and immunofluorescence microscopy were employed for monitoring LRIG1-TAT protein transduction into human neuroblastoma cells. Cell proliferation and invasion were measured for evaluating the effect of LRIG1-TAT on neuroblastoma cell. Our data showed that LRIG1 protein can be delivered into cells or organs in living animals by TAT. One-time transduction of LRIG1 proteins into human neuroblastoma cells enhanced cell proliferation and increased cell invasion. In vivo transduction showed that LRIG1-TAT protein can be presented in living animal organs. Our experiments provide a new vision on LRIG1 applications and also offer a therapy window for revealing the intrinsic function of LRIG1 on cells.

Morphofunctional Significance of “In Vivo Cryotechnique” for Analyses of Cells and Tissues in Living Animal Organs

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Histological Study and LYVE-1 Immunolocalization of Mouse Mesenteric Lymph Nodes with "In Vivo Cryotechnique"

The “in vivo cryotechnique” (IVCT) is a powerful tool to directly freeze living animal organs in order to maintain biological components in frozen tissues, reflecting their native states. In this study, mesenteric lymph nodes of living mice were directly frozen with IVCT, and we did morphological studies and immunohistochemical analyses on a hyaluronic acid receptor, LYVE-1. In lymph nodes, widely open lymphatic sinuses were observed, and many lymphocytes adhered to inner endothelial cells along subcapsular sinuses. The LYVE-1 was clearly immunolocalized at inner endothelial cells of subcapsular sinuses, as well as those of medullary sinuses. Conventional pre-embedding electron microscopy also showed LYVE-1 immunolocalization along both the apical and basal sides of cell membranes of inner endothelial cells. By triple-immunostaining for LYVE-1, smooth muscle actin, and type IV collagen, the LYVE-1 was immunolocalized only in the inner endothelial cells, but not in outer ones which were surrounded by collagen matrix and smooth muscle cells. Thus, the functional morphology of lymph nodes in vivo was demonstrated and LYVE-1 immunolocalization in inner endothelial cells of subcapsular sinuses suggests hyaluronic acid incorporation into lymph node parenchyma.

Significance of 'in vivo cryotechnique' for morphofunctional analyses of living animal organs

Our final goal of morphological and immunohistochemical studies is that all findings examined in animal experiments should reflect the physiologically functional background. Therefore, the preservation of original components in cells and tissues of animals is necessary for describing the functional morphology of living animal organs. It is generally accepted that morphological findings of various organs were easily modified by stopping their blood supply. There had been a need to develop a new preparation technique for freezing the living animal organs in vivo and then obtaining acceptable morphology and also immunolocalization of original components in functioning cells and tissues. We already developed the 'in vivo cryotechnique' (IVCT) not only for their morphology, but also for immunohistochemistry of many soluble components in various living animal organs. All physiological processes of cells and tissues were immediately immobilized by IVCT, and every component in the cells and tissues was maintained in situ at the time of freezing. Thus, the ischaemic or anoxic effects on them could be minimized by IVCT. Our specially designed cryoknife with liquid cryogen has solved the morphological and immunohistochemical problems which are inevitable with the conventional preparation methods at a light or electron microscopic level. The IVCT will be extremely useful for arresting transient physiological processes and for maintaining any intracellular components in situ, such as rapidly changing signal molecules, membrane channels and receptors.

Porcine endogenous retroviruses and xenotransplantation.

Xenotransplantation is defined by the PHS as any procedure that involves the transplantation, implantation or infusion into a human recipient of either (a) live cells, tissues or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs (Public Health Service Guideline on Infectious Disease Issues in Xenotransplantation). Use of pigs for human xenotransplantation raises concerns about the risks of transfer of infectious agents from the pig cells to xenotransplantation recipients. The observation that the porcine germline harbors genetic loci encoding porcine endogenous retroviruses (PERVs) that are in some cases infectious for human cells has resulted in renewed scientific interest in PERVs. However, in spite of the past 10 years of investigation, the actual risk for PERV infection, replication, and pathogenic outcome in human recipients of xenotransplantation products is still undefined. (Part of a multi-author review).

Immunohistochemical detection of hypoxia in mouse liver tissues treated with pimonidazole using “in vivo cryotechnique”

To evaluate hypoxic cells in live mouse liver tissues, immunohistochemistry for protein adducts of reductively activated pimonidazole (PARaPi) was performed using the "in vivo cryotechnique (IVCT)" followed by freeze-substitution fixation. This method was used because cryotechniques have some merits for examining biological events in living animal organs with improved time-resolution compared to conventional perfusion and/or immersion chemical fixation. Pimonidazole was intraperitoneally injected into living mice, and then after various times of hypoxia, their livers were quickly frozen by IVCT. The frozen liver tissues were freeze-substituted in acetone containing 2% paraformaldehyde, and routinely embedded in paraffin wax. De-paraffinized sections were immunostained for PARaPi. In liver tissues of mice without hypoxia, almost no immunostained cells were detected. However, in liver tissues with 30 s of hypoxia, some hepatocytes in the pericentral zones were strongly immunostained. After 60 s of hypoxia, many hepatocytes were immunostained with various degrees of staining intensity in all lobular zones, indicating different reactivities of pimonidazole in the hepatocytes to hypoxia. At this time, the general immunoreactivity also appeared to be stronger around the central veins than other portal areas. Although many hepatocytes were immunostained for PARaPi in the liver tissues with perfusion fixation via heart, those with perfusion via portal vein were not immunostained. Thus, IVCT is useful to detect time-dependent hypoxic states with pimonidazole treatment in living animal organs.

Clinical Trials of Xenotransplantation: Waiver of the Right to Withdraw from a Clinical Trial Should Be Required

Xenotransplantation pits clinical research ethics against public health needs because recipients must undergo long-term, perhaps life-long, surveillance for infectious diseases. This surveillance requirement is effectively an abrogation of the right to withdraw from a clinical trial. Ulysses contracts, which are advance directives for future care, may be an ethical mechanism by which to balance public health needs against limitation of individual rights.

Application of in vivo cryotechnique to the examination of cells and tissues in living animal organs.

When all biological materials in cells and tissues of living animal organs are quickly and promptly frozen, immunolocalization of their components and structural features in situ is necessary to understand their in vivo functioning states. However, these direct morphological analyses were difficult to achieve by conventional chemical fixation methods during the last century. A new cryofixation method, named the "in vivo cryotechnique", in which the normal blood circulation in living animals is always retained at the moment of freezing, has become a powerful tool to visualize the real native morphology of cells and tissues with functional meaning. The "in vivo cryotechnique" can usually be combined with a wide range of subsequent preparation techniques, and can thereby enable us to perform various direct analyses on biological samples, reflecting the physiological functions of living animal organs.

"In vivo cryotechnique" for paradigm shift to "living morphology" of animal organs

The morphological study has been one of the major approaches in medical and biological fields. For the last century, the conventional chemical fixation and alcohol dehydration were commonly used as an easy preparation method, but it was frequently pointed out that they usually yield many structural artifacts during their preparation processes. Although both conventional quick-freezing and high-pressure freezing methods, by which animal tissues are resected and frozen for physical fixation,can reduce such structural artifacts, the tissues have to be removed from living animal organs for the freezing. Therefore, such specimens are inevitably exposed to noxious stresses of anoxia and ischemia, exhibiting only dead morphological states of animal tissues without blood circulation. To the contrary, our "in vivo cryotechnique", by which all cells and tissues in animal bodies are cryofixed in vivo, can prevent such artifacts of resected specimens. By means of the cryotechnique, it is now possible to reveal the in vivo morphology of cells and tissues in living animal organs. Actually, it has been already applied to several animal organs, such as kidney, liver, intestine, cerebellum, eye ball, blood vessel, and joint cartilage, and brought new morphological findings, reflecting their physiological significance, which had been difficult to demonstrate by the conventional preparation methods. Moreover, its application to immunohistochemistry has also revealed more precise immunolocalizations of dynamically changing molecules in living animal organs, easily translocated by ischemic stresses and anoxia caused during the tissue resection. The "in vivo cryotechnique" allows us to perform novel morphological investigations of "living" morphological states, and develops new medical and biological fields with "living morphology" during this 21st century. Biomedical Reviews 2004; 15: 1-19.

Advanced Application of the In Vivo Cryotechnique to Immunohistochemistry for Animal Organs

The quick-freezing method often used for physical fixation also contributed enormously to the advancement of morphology, but it could not provide enough information about the dynamic morphological changes in vivo in living animal organs. Therefore, we developed the in vivo cryotechnique in 1995, which could directly cryofix organs in vivo under anesthetized conditions without stopping blood circulation or producing effects of anoxia. We have already reported new findings about the in vivo ultrastructures of living animal organs with the cryotechnique followed by freeze-substitution or replica preparation. Recently, it has also been applied to other morphological analyses in vivo, including immunohistochemistry and FISH, for obtaining dynamically functioning structures of cells and tissues at a light microscopic level. In such experimental processes, the in vivo cryotechnique has been shown to have the added benefit of direct antigen-retrieval effects since it reduces several steps of retrieval treatments which are required in common paraffin-embedded samples prepared by the conventional fixation and dehydration. In conclusion, the in vivo cryotechnique allows us to investigate the functioning real morphology of living animals, which was technically difficult to be observed before, and perform dynamic immunohistochemistry of cells and tissues in various animal organs in vivo at ultimate time-resolution.

Legal enforcement of xenotransplantation public health safeguards.

Xenotransplantation is any transplantation, implantation, or infusion of either live cells, tissues, or organs from a nonhuman animal source, or human bodily fluids, cells, tissues, or organs that have had ex vivo contact with live nonhuman animal cells, tissues, or organs into a human recipient. Most scientists agree that clinical xenotransplantation should not be performed in the absence of accompanying public health safeguards The science upon which that consensus is based has been extensively described in the literature. By and large the most compelling reason for public health safeguards is the possible introduction of new infectious agents into the human life cycle as a result of xenotransplantation.The most frequent source of new infectious diseases in human populations is the transfer of agents, such as viruses, bacteria, or prions, from animals to man. Human diseases and infectious agents that are thought to have originated from animals include influenza, rabies, malaria, lassa fever, lyme disease, AIDS, yellow fever, tuberculosis, the human t-cell lymphotropic virus, the herpes B virus, the hantavirus, and even the bubonic and pneumonic plagues.

Pig endogenous retrovirus – a threat to clinical xenotransplantation?

Transplantation shows good results for patients with end-stage disease, but there is an increasing lack of organs. Xenotransplantation, the transfer of live animal cells, tissues, or organs to another species, offers a potential solution to this shortfall. Pig is regarded as the animal of choice for this purpose. Meanwhile demonstration of pig endogenous retrovirus (PERV) in all porcine herds has caused serious concern with respect to a possible transmission of the virus to humans with a transplanted organ. Transmission to human cells has been documented under certain in vitro conditions. However, no such transmission has been demonstrated in vivo. The possible consequences of introducing PERV into immunocompromised human organisms are not known and it is necessary to collect more information. Novel and sensitive genomic assays to detect PERV infection are now available in addition to established virological, immunoserological and molecular methods. In order to minimise the risk of PERV transmission rigorous procedures should be established. International guidelines to reduce the risk should be followed. Although a number of immunological, physiological and virological questions need to be answered before the introduction of xenotransplantation as an alternative clinical treatment, some problems can only be solved by judicious clinical trials.

The Animal Research Controversy: Exploring Student Attitudes

SCIENTISTS in the United States and elsewhere whose work involves experimentation with animals find themselves surrounded by a public climate of mistrust, suspicion and increasing activism among those whose goal it is to eliminate the use of animals in research. Biology teachers also find themselves caught in the crossfire of conflicting views on this topic. A significant amount of biological knowledge has resulted from experimentation using animal subjects. Most of this information could not have been obtained in any other way. The fields of physiology, embryology, animal behavior and medicine rely heavily upon information gained from animal studies. As early as 1865, the French physiologist Claude Bernard urged his fellow physicians to stop initiating experimental therapies on humans and to test them first on animal subjects. This attitude was reaffirmed in the Nuremberg Code and forms the basis for the conduct of contemporary experimental therapies destined for use in humans. Complete prohibition of this protocol would stifle many valuable lines of investigation and render crucial scientific questions unanswerable. The dissection of representative animals, including mammals, has long been standard instructional practice in introductory biology, comparative anatomy and evolution courses. Physiology classes rely on exercises which employ live animal organs to demonstrate not only the biological principles of living systems, but also the techniques necessary to gain this knowledge. How will the quality of instruction be altered if substitutes such as computer simulations or plastic models are used? The issue also raises questions about the academic freedom of the instructor to provide a complete and accurate program of study to students. If biology teachers are to convey the methodology and thrust of science, particularly zoology and medicine, they must deal with the topic of animal experimentation. Equally important to pre-professional training is communicating the appreciation of the scientific enterprise to those