Establishment of an in vivo neutralization model based on H5N1 pseudovirus / 中华微生物学和免疫学杂志

Objective:To establish an in vivo infection model of H5N1 pseudovirus and to detect the neutralizing activity of FHA3 antibody using this model. Methods:Based on the sequence information of hemagglutinin (HA) and neuraminidase (NA) of A/Anhui/1/2005/H5N1 strain, two recombinant plasmids of pcDNA3.1-HA5 and pcDNA3.1-NA1 were constructed. The two plasmids and plasmid pNL4-3.Luc.R-E- were co-transfected into 293T cells to prepare H5N1 pseudovirus supernatant. The morphology of pseudovirus particles in the supernatant was observed by electron microscopy. MDCK cells were infected with the pseudovirus supernatant and the virus titer was detected. BALB/c mice were injected with the pseudovirus supernatant by intraperitoneal injection and subjected to bioluminescence imaging at 2, 5, 8, and 12 d after infection to detect the pseudovirus infection in vivo. The functional activity of FHA3 antibody in vivo was evaluated using the established mouse infection model. Results:The recombinant plasmids pcDNA3.1-HA5 and pcDNA3.1-NA1 were correctly constructed and could be used to prepare pseudovirus supernatants of high titer by co-transfecting 293T cells with the plasmid pNL4-3.Luc.R-E-. The virus particles were round under electron microscope. H5N1 pseudovirus-infected mice exhibits strong fluorescence signals, which were attenuated by FHA3 treatment before challenge.Conclusions:The in vivo infection model of H5N1 pseudovirus was successfully constructed and FHA3 antibody was proved to be protective against the pseudovirus infection.

Transcriptome Analysis of Entamoeba histolytica Trophozoites during in vivo Contact-independent Mediated Host-parasite Interaction: Putative Pathways Related to PCD

Aim: In the present study, we exploited DNA microarray-based transcriptome analysis and showed overall changes in gene expression in vivo of amoebic trophozoites that interact with animal soluble factors using an intraperitoneal dialysis bag model to elucidate putative molecular pathways and genes involved in this interaction. Study Design: We exploited DNA microarray-based transcriptome analysis. Results: An analysis from a network including the interactions of up-regulated genes and their neighbors revealed the presence of 11 functionally related modules. Six of the modules obtained were related to endoplasmic reticulum (ER) functions, such as degradation, stress, proteasome-ubiquitination, phosphorylation, lipid metabolism, and protein sorting. Furthermore, major transcriptional changes displayed by the parasite at the beginning of interaction were attributed to the response to the host defense. These data are consistent with the notion that the concerted expression of genes necessary for survival such as increment in protein synthesis, cytoskeleton rearrangement, vesicular traffic and genes involved in cell death including calcium imbalance and the ER signals associated with protein degradation (ERAD) is an overall landscape during the in vivo interaction between the amoebic trophozoites and animal soluble factors, and suggest that the ER stress is one of the main pathways leading to programmed cell death in E. histolytica. Conclusion: The present findings on the global transcriptional changes displayed by the parasite at the early stages of interaction with host environments in peritoneal implantation indicate that a substantial proportion of concerted changes in gene expression in amoebic trophozoites are attributable to the parasite’s response for cell death signals due to ER stress. A detailed knowledge of the underlying molecular mechanism might suggest the efficient elimination of this parasite by promoting their death pathways.

Establishment and Application of Several Animal Models for the Study of Intervertebral Disc Degeneration / 中国生物化学与分子生物学报

Intervertebral disc degeneration (IDD) is an age-related degenerative disease and a major cause of low back pain. IDD greatly impairs the quality of life of patients and dramatically increases the economic burden of patient families. There are currently no effective intervention and treatment for IDD, partly due to a lack of understanding of its pathogenesis. The establishment and characterization of IDD animal models are critical for defining mechanisms underlying its pathogenesis. IDD is a complex process, which is affected by mechanical stress, injury, biochemistry and gene expression. In this review article, we summarize several IDD animal models generated by utilizing abnormal mechanical stress, injury, biochemical and chemical induction and gene knockout. Biomechanics play a key role in maintaining intervertebral disc homeostasis, and abnormal mechanical stress can cause IDD. Usually, IDD is accompanied by structural injury which exacerbates IDD. In addition, biochemical and chemical induction and knockout of key genes can also lead to IDD. Among the different factors causing abnormal mechanical stress, there are two mechanical stress models: pressure model and instability model. According to the structure of intervertebral disc, there are two structural injury models: the nucleus pulposus and annulus fibrosus injury model and the cartilage endplate injury model. The biochemical and chemical induction model and the gene knockout model are summarized, and the applications and limitations of different IDD animal models are discussed.

In vivo evaluation of a new vena cava filter / Avaliação in vivo de um novo filtro de veia cava

Background Pulmonary embolism is an important cause of cardiovascular death. Inferior vena cava filters have been shown to be effective for prevention of this condition. Objectives To determine the safety, performance and efficacy of a new inferior vena cava filter in an ovine model. Methods BKone1 filters are self-centering with over-the-wire deployment, have three filtering regions and are made from nickel-titanium alloy. Eight of these filters were implanted in 8 sheep. The sheep were divided into 4 groups of two animals (A and B) and the number of clots injected differed by group. Two clots were injected in group 2, four in group 3, eight in group 4 and zero clots in group 1. A animals underwent euthanasia soon after the procedure and B animals were observed for 30 days and then euthanized after a control cavography. All inferior vena cavas were processed for histological examination. Clots were prepared in a metal mold, sectioned and then radiopaque markers were inserted. Clot capture was analyzed by identifying the radiopaque marker on fluoroscopy. Results No clot migration was observed during follow-up. Control cavographies showed patent inferior vena cavas. Pathological examination indicated little inflammatory tissue response. All clots were captured in the condition with 2 clots, only one clot was missed in the group injected with 4 clots and in the condition of 8 clots, they were partly captured. Conclusions The filters were deployed safely. There was a reduction in efficacy as the number of blood clots increased.

Contexto Embolia pulmonar é uma importante causa de morte cardiovascular. Filtros de veia cava inferior têm se mostrado efetivos na sua prevenção. Objetivos Determinar a segurança, o desempenho e a eficácia de um novo filtro de veia cava inferior em estudo experimental utilizando modelos ovinos. Métodos Filtros BKone1 são autocentrantes, over-the-wire (OTW), compostos por três regiões de filtragem e construídos em liga de níquel-titânio. Oito filtros foram implantados em oito ovelhas. As ovelhas foram divididas em quatro grupos, de acordo com o número de êmbolos injetados, com dois animais em cada grupo (A e B). Foram injetados dois êmbolos no grupo 2, quatro no grupo 3, oito no grupo 4 e nenhum êmbolo no grupo 1. Os animais denominados A foram submetidos a eutanásia logo após o procedimento e os animais B foram observados por 30 dias, sendo submetidos a eutanásia após a realização de uma cavografia de controle. Após a eutanásia, todos os animais foram submetidos a explante do segmento de veia cava inferior contendo o filtro para análise anatomopatológica. Os êmbolos foram preparados em molde metálico e seccionados, adicionando-se marcadores radiopacos. A retenção dos êmbolos foi constatada através da identificação da marca radiopaca na seção de captura do filtro, via fluoroscopia. Resultados Não foi observada migração do filtro após o período de 30 dias. As cavografias de controle mostraram perviedade das veias cava inferior. Os resultados dos exames anatomopatológicos indicaram pouca resposta inflamatória dos tecidos. Os êmbolos foram capturados totalmente na condição com dois êmbolos, apenas um êmbolo não foi capturado no grupo com quatro êmbolos e na condição de oito êmbolos, eles foram parcialmente capturados. Conclusões Os filtros foram entregues com segurança. Há uma queda na eficácia de captura com o aumento da quantidade de êmbolos.

Evaluation of hepatoprotective potential of functional food formulations using in vitro and in vivo models of CCl4 radical induced toxicity.

Carbon tetra chloride (CCl4) induced acute hepatotoxicity causes severe damage to hepatocytes and affects the liver functions which resemble various liver ailments like hepatitis, jaundice, cancer etc. Using 12-different fruits, formulation F1 and F2 were prepared. Hepatoprotective potential of the formulations was assessed using HepG2 cell (in vitro) and rat model (in vivo). Biochemical parameters like alkaline phosphatase, lactate dehydrogenase, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, bilirubin, blood urea nitrogen, plasma TBARS, trolox equivalent antioxidant capacity, total cholesterol, triglycerides were studied. Various markers of liver functions viz., super oxide dismutase, catalase, glutathione peroxidase, reduced glutathione, tissue Thio barbituric acid reactive substances (TBARS) were assessed. Significant decrease in the activity of aminotransferases, alkaline phosphatase and bilirubin was observed in formulation F1 followed by F2 groups as compared with CCl4 treated group. The biochemical and histopathological observations supported hepatoprotective effect. Antioxidant enriched polyherbal formulations F1 and F2 effectively ameliorated CCl4 induced acute hepatotoxicity by improving antioxidant status in rats.

ESD Hands-on Course Using Ex Vivo and In Vivo Models in South Korea

Endoscopic submucosal dissection (ESD) is an established treatment for gastric neoplasias especially in regions with a high volume of gastric cancer. Although ESD has many advantages over endoscopic mucosal resection, ESD is technically more difficult and can result in severe complications. Therefore establishment of an effective training system is required to help endoscopists climb the ESD learning curve. Although a standard training system for ESD remains to be established, some centers are incorporating ex vivo and/or in vivo animal models to provide a safe and effective means of ESD training. However, it is unknown if these animal models are more effective than other programs. Moreover the efficacy of the animal model may vary according to socio-economic status and the volume of gastric cancer. In this article we introduce the basic and advanced ESD training model using the ex vivo and in vivo animal model from South Korea and review the associated literature from other regions.

An improved in vivo method for atrioventricular node ablation via thoracotomy

The atrioventricular (AV) node is permanently damaged in approximately 3 percent of congenital heart surgery operations, requiring implantation of a permanent pacemaker. Improvements in pacemaker design and in alternative treatment modalities require an effective in vivo model of complete heart block (CHB) before testing can be performed in humans. Such a model should enable accurate, reliable, and detectable induction of the surgical pathology. Through our laboratory’s efforts in developing a tissue engineering therapy for CHB, we describe here an improved in vivo model for inducing chronic AV block. The method employs a right thoracotomy in the adult rabbit, from which the right atrial appendage may be retracted to expose an access channel for the AV node. A novel injection device was designed, which both physically restricts needle depth and provides electrical information via electrocardiogram interface. This combination of features provides real-time guidance to the researcher for confirming contact with the AV node, and documents its ablation upon formalin injection. While all animals tested could be induced to acute AV block, those with ECG guidance were more likely to maintain chronic heart block >12 h. Our model enables the researcher to reproduce both CHB and the associated peripheral fibrosis that would be present in an open congenital heart surgery, and which would inevitably impact the design and utility of a tissue engineered AV node replacement.

Apoptotic Neuronal Death in the Mouse Brain Induced by Experimental Japanese Encephalitis Virus Infection / 대한해부학회지

Japanese encephalitis virus (JEV)may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. To investigate whether JEV infection induces apoptosis, we examined DNA fragmentation and apoptosis in the specific region of the JEV infected mouse brain by DNA oligonucleosomal laddering and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)technique and immunohistochemical study. JEV infections in the mouse brain were detected in the telencephalon, the diencephalons, and the brain stem, but not in the cerebellum and the hippocampus. Fragmentation of cellular DNA into oligonucleosome-length ladders was only observed in tissue samples prepared from the cerebral cortex. In addition, the large number of TUNEL-positive cells was observed in the cerebral cortex. Double-labeling experiment with TUNEL staining and immunostaining for the JEV showed that TUNEL-positive neurons containing JEV immunoreactivity. These results suggest that JEV infection may evoke apoptotic neuronal death in the mouse brain, which plays an important role in the pathogenesis of Japanese encephalitis.

The Fetal Sheep: A Unique Model System for Assessing the Full Differentiative Potential of Human Stem Cells

The naturally occurring stem cell migratory patterns, the availability of expanding homing and engraftment sites, and the presence of tissue/organ-specific signals in the developing mammalian fetus provide the ideal setting for stem cells to exhibit their full biological potential. These characteristics combined with the relative immunological naivete of the early gestational age fetus that permits the engraftment and long- term persistence of allogeneic and xenogeneic donor stem cells make it possible to use the developing fetus to assess the in vivo potential of a variety of stem cells. We have taken advantage of these permissive characteristics of the fetus to develop a large animal model of human hematopoiesis in sheep that permits not only the long-term engraftment of human hematopoietic stem cell/progenitor cells and their differentiation into the full range of lymphohematopoietic elements, but also the relatively robust expression of their potential to contribute to the formation of non-hematopoietic tissues.

In vivo Study on the Japanese Encephalitis: Viral Localization and Histopathology in the Mouse Brain / 대한해부학회지

Japanese encephalitis is a potentially lethal disease of the central nervous system caused by infection with Japanese encephalitis virus (JEV). JEV is the most common cause of encephalitis over a large part of eastern Asia. To establish and characterize in vivo model to study the Japanese encephalitis, the immunohistochemical localization of JEV and the histopathological finding were investigated in the brains of young adult mice infected with JEV by intraperitoneal inoculation. JEV was localized to neurons in discrete regions of the brain. Histopathological finding showed typical pattern of acute viral encephalitis, such as inflammatory cell infiltration in brain parenchyme and perivascular cuffs of mononuclear cells. These results suggest that this in vivo system can be used to study the mechanism of virus entry into the brain, cell specific tropism, and pathophysiology in Japanese encephalitis.

Investigation of the Validity of an in vivo Experimental Model for the Evaluation of the Therapeutic Potentials of Drugs for the Treatment of Premature Ejaculation / 대한비뇨기과학회지

PURPOSE: The purpose of this study was to investigate the validity of in vivo experimental models to evaluate the therapeutic potentials of drugs for the treatment of premature ejaculation. MATERIALS AND METHODS: Male Sprague-Dawley rats (250-300gm) were divided into 8 groups based on the experimental agent administered: serotonergic agents (serotonin, clomipramine, fluoxetine, sertraline, paroxetine) and alpha-adrenergic blockers (prazosin, terazosin, tamsulosin). Various concentrations of the agents were intravenously injected 20 minutes prior to the electrical stimulation of the hypogastric nerve. Intraluminal pressures of seminal vesicle and vas deferens were measured on each side in the same animal. The concentration-response curves for each drug were obtained, and the inhibitory effects of the drugs on the contractile response of the seminal vesicles and vasa deferentia to the electrical stimulation of the hypogastric nerve were compared. RESULTS: All the serotonergic agents resulted in dose-dependent inhibition of the intraluminal pressure of the seminal vesicle to electrical stimulation (clomipramine>serotonin>fluoxetine>sertraline>paroxetine). The vasal pressure responses were also effectively inhibited by serotonin, clomipramine and sertraline, in that order. Fluoxetine and paroxetine showed no inhibitory effects on the vasal pressure. The pressure responses of both the seminal vesicles and the vasa deferentia were inhibited in a dose-dependent manner by all the alpha-adrenergic blockers. CONCLUSIONS: This in vivo model was not able to demonstrate the established clinical effects of various serotonergic agents widely used in the treatment of premature ejaculation. Conversely, the alpha-adrenergic blockers showed marked dose-dependent inhibition of the seminal tract pressure responses. Therefore, this in vivo model has limitations for the proper evaluation of therapeutic potentials of drugs for the treatment of premature ejaculation.