Characterization of a Novel Mouse Phenotype with Marked Hydrocephalus

In multigenic diseases, disorders where mutations in multiple genes affect the expressivity of the disease, genetic interactions play a major role in prevalence and phenotypic severity. While studying the genetic interactions between Pax3 and EdnrB in the melanocyte lineage, a new phenotype was noted in 80% of Pax3 mutants that we believe to be a novel murine model for hydrocephalus. Hydrocephalus, an accumulation of cerebrospinal fluid in the cranial cavity due to obstruction of flow in and out of the cavity, is one of the most common birth defects surpassing Down syndrome. Characteristic to hydrocephalus is a "domed" head appearance, expansion of the ventricles of the brain, and loss of neurons with hyperproliferation of glial cell types all three of which were seen in the mutant mice. The phenotype also consisted of craniofacial deformities coupled with skeletal defects including, but not limited to kyphosis, lordosis, and an apparent shortening of the some limbs. For the cellular analysis of the hydrocephalus phenotype, brains were removed and stained with two antibodies: Glial Fibrillary Acidic Protein (GFAP) and Neurofilament (NF), which are astrocyte- and neuron- specific respectively. A higher number of cells expressing GF AP and a lower number of cells expressing NF were seen in the mutant brain, when compared to control. For skeletal deformity analysis, affected mice skeletons were stained with Alizarin Red and Alcian Blue showing no apparent difference in ossification. Future genetic analysis of these mutant mice has the potential to identify novel gene modifiers involved in the promotion of this particular phenotype.

Atividade antiangiogênica da forma recombinante da proteína 21 de Trypanosoma cruzi

Chagas disease, caused by the parasite Trypanosoma cruzi, is the cause of Chronic chagasic cardiomyopathy (CCC). The prospection of innovative therapeutic agents against CCC is a major task. The recombinant form of 21 (rP21), a secreted T. cruzi protein involved in host cell invasion and on progression of chronic inflammatory processes have been studied as a potential novel therapeutic target. Our present work aimed to verify and investigate the impact of rP21 in the formation of blood vessels in vitro and in vivo. First, tEnd cells were treated with different concentrations of rP21 or bacterial extract and viability and cellular adhesion were evaluated by MTT and angiogenesis inhibition by Matrigel tube formation assay and murine model. To verify the proteolytic activity of rP21 on extracellular matrix (ECM) components, fibrinogen, matrigel and fibronectin was incubated with rP21 or not. In addition, we performed proliferation assays and cell cycle analysis. Furthermore, the accumulation and distribution of F-actin was determined by Phalloidin staining using ImageJ software. Finally, tEnd cells were incubated with rP21 and the mRNA levels were analyzed by real-time PCR. Our results showed that rP21 did not alter cell viability and adhesion, but strongly inhibited vessel formation in vitro and in vivo. Tube formation assay showed that angiogenesis inhibition was dependent of the CXCR4-rP21 binding. In addition to these results, we observed that the rP21 was able to inhibit cell proliferation and promoted a significant reduction in the number of 4n cells (G2/M phase). Moreover, we found that rP21 significantly increased F-actin levels and this protein was able to modulate expression of genes related to angiogenesis and actin cytoskeleton. However, rP21 showed no significant activity on the matrix components. In this sense, we conclude that the rP21-endothelial cells (ECs) interaction via CXCR4 promotes inhibition of vessel formation through a cascade of intracellular events, such as inhibition of ECs proliferation and modulation of the expression of molecules associated with angiogenic processes and actin cytoskeleton.

Bioengineered Approaches to Prevent Hypertrophic Scar Contraction

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

Metabolism Regulates the Fate and Function of T Lymphocytes

Proper balancing of the activities of metabolic pathways to meet the challenge of providing necessary products for biosynthetic and energy demands of the cell is a key requirement for maintaining cell viability and allowing for cell proliferation. Cell metabolism has been found to play a crucial role in numerous cell settings, including in the cells of the immune system, where a successful immune response requires rapid proliferation and successful clearance of dangerous pathogens followed by resolution of the immune response. Additionally, it is now well known that cell metabolism is markedly altered from normal cells in the setting of cancer, where tumor cells rapidly and persistently proliferate. In both settings, alterations to the metabolic profile of the cells play important roles in promoting cell proliferation and survival.

It has long been known that many types of tumor cells and actively proliferating immune cells adopt a metabolic phenotype of aerobic glycolysis, whereby the cell, even under normoxic conditions, imports large amounts of glucose and fluxes it through the glycolytic pathway and produces lactate. However, the metabolic programs utilized by various immune cell subsets have only recently begun to be explored in detail, and the metabolic features and pathways influencing cell metabolism in tumor cells in vivo have not been studied in detail. The work presented here examines the role of metabolism in regulating the function of an important subset of the immune system, the regulatory T cell (Treg) and the role and regulation of metabolism in the context of malignant T cell acute lymphoblastic leukemia (T-ALL). We show that Treg cells, in order to properly function to suppress auto-inflammatory disease, adopt a metabolic program that is characterized by oxidative metabolism and active suppression of anabolic signaling and metabolic pathways. We found that the transcription factor FoxP3, which is highly expressed in Treg cells, drives this phenotype. Perturbing the metabolic phenotype of Treg cells by enforcing increased glycolysis or driving proliferation and anabolic signaling through inflammatory signaling pathways results in a reduction in suppressive function of Tregs.

In our studies focused on the metabolism of T-ALL, we observed that while T-ALL cells use and require aerobic glycolysis, the glycolytic metabolism of T-ALL is restrained compared to that of an antigen activated T cell. The metabolism of T-ALL is instead balanced, with mitochondrial metabolism also being increased. We observed that the pro-anabolic growth mTORC1 signaling pathway was limited in primary T-ALL cells as a result of AMPK pathway activity. AMPK pathway signaling was elevated as a result of oncogene induced metabolic stress. AMPK played a key role in the regulation of T-ALL cell metabolism, as genetic deletion of AMPK in an in vivo murine model of T-ALL resulted in increased glycolysis and anabolic metabolism, yet paradoxically increased cell death and increased mouse survival time. AMPK acts to promote mitochondrial oxidative metabolism in T-ALL through the regulation of Complex I activity, and loss of AMPK reduced mitochondrial oxidative metabolism and resulted in increased metabolic stress. Confirming a role for mitochondrial metabolism in T-ALL, we observed that the direct pharmacological inhibition of Complex I also resulted in a rapid loss of T-ALL cell viability in vitro and in vivo. Taken together, this work establishes an important role for AMPK to both balance the metabolic pathways utilized by T-ALL to allow for cell proliferation and to also promote tumor cell viability by controlling metabolic stress.

Overall, this work demonstrates the importance of the proper coupling of metabolic pathway activity with the function needs of particular types of immune cells. We show that Treg cells, which mainly act to keep immune responses well regulated, adopt a metabolic program where glycolytic metabolism is actively repressed, while oxidative metabolism is promoted. In the setting of malignant T-ALL cells, metabolic activity is surprisingly balanced, with both glycolysis and mitochondrial oxidative metabolism being utilized. In both cases, altering the metabolic balance towards glycolytic metabolism results in negative outcomes for the cell, with decreased Treg functionality and increased metabolic stress in T-ALL. In both cases, this work has generated a new understanding of how metabolism couples to immune cell function, and may allow for selective targeting of immune cell subsets by the specific targeting of metabolic pathways.

Transcriptional profiling of chronic clinical hepatic schistosomiasis japonica indicates reduced metabolism and immune responses

Schistosomiasis is a significant cause of human morbidity and mortality. We performed a genome-wide transcriptional survey of liver biopsies obtained from Chinese patients with chronic schistosomiasis only, or chronic schistosomiasis with a current or past history of viral hepatitis B. Both disease groups were compared with patients with no prior history or indicators of any liver disease. Analysis showed in the main, downregulation in gene expression, particularly those involved in signal transduction via EIF2 signalling and mTOR signalling, as were genes associated with cellular remodelling. Focusing on immune associated pathways, genes were generally downregulated. However, a set of three genes associated with granulocytes, MMP7, CLDN7, CXCL6 were upregulated. Differential gene profiles unique to schistosomiasis included the gene Granulin which was decreased despite being generally considered a marker for liver disease, and IGBP2 which is associated with increased liver size, and was the most upregulated gene in schistosomiasis only patients, all of which presented with hepatomegaly. The unique features of gene expression, in conjunction with previous reports in the murine model of the cellular composition of granulomas, granuloma formation and recovery, provide an increased understanding of the molecular immunopathology and general physiological processes underlying hepatic schistosomiasis.

Impact of splenic artery ligation after major hepatectomy on liver function, regeneration and viability

It was reported that prevention of acute portal overpressure in small-for-size livers by inflow modulation results in a better postoperative outcome. The aim is to investigate the impact of portal blood flow reduction by splenic artery ligation after major hepatectomy in a murine model. Forty-eight rats were subjected to an 85% hepatectomy or 85% hepatectomy and splenic artery ligation. Both groups were evaluated at 24, 48, 72 and 120 post-operative hours: liver function, regeneration and viability. All methods and experiments were carried out in accordance with Coimbra University guidelines. Splenic artery ligation produces viability increase after 24 h, induces a relative decrease in oxidative stress during the first 48 hours, allows antioxidant capacity increment after 24 h, which is reflected in a decrease of half-time normalized liver curve at 48 h and at 72 h and in an increase of mitotic index between 48 h and 72 h. Splenic artery ligation combined with 85% hepatectomy in a murine model, allows portal inflow modulation, promoting an increase in hepatocellular viability and regeneration, without impairing the function, probably by inducing a less marked elevation of oxidative stress at first 48 hours.

Identification and Characterization of New Small Molecule Inhibitors of Picornavirus Replication

The Picornaviridae family consists of positive-strand RNA viruses that are the causative agents of a variety of diseases in humans and animals. Few drugs targeting picornaviruses are available, making the discovery of new antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. The antiviral effect of these compounds is not likely related to their known cellular targets because other inhibitors targeting the same pathways did not inhibit viral replication. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited the formation of a functional replication complex, while E5(1) and E7(2) affected replication after the replication complex had formed. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Poliovirus resistant to E7(2) had a single mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target either PI4KIIIβ (major enviroxime-like compounds) or OSBP (minor enviroxime-like compounds), cellular factors involved in lipid metabolism and shown to be important for replication of diverse positive-strand RNA viruses. We classified E7(2) as a minor enviroxime-like compound, because the localization of OSBP changed in the presence of this inhibitor. Interestingly, both E7(2) and major enviroxime-like compound GW5074 interfered with the viral polyprotein processing. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. Studies with these compounds shed light on pathways shared by diverse picornaviruses that could be potential targets for the development of broad-spectrum antiviral drugs.

Impact of splenic artery ligation after major hepatectomy on liver function, regeneration and viability

It was reported that prevention of acute portal overpressure in small-for-size livers by inflow modulation results in a better postoperative outcome. The aim is to investigate the impact of portal blood flow reduction by splenic artery ligation after major hepatectomy in a murine model. Forty-eight rats were subjected to an 85% hepatectomy or 85% hepatectomy and splenic artery ligation. Both groups were evaluated at 24, 48, 72 and 120 post-operative hours: liver function, regeneration and viability. All methods and experiments were carried out in accordance with Coimbra University guidelines. Splenic artery ligation produces viability increase after 24 h, induces a relative decrease in oxidative stress during the first 48 hours, allows antioxidant capacity increment after 24 h, which is reflected in a decrease of half-time normalized liver curve at 48 h and at 72 h and in an increase of mitotic index between 48 h and 72 h. Splenic artery ligation combined with 85% hepatectomy in a murine model, allows portal inflow modulation, promoting an increase in hepatocellular viability and regeneration, without impairing the function, probably by inducing a less marked elevation of oxidative stress at first 48 hours.

Utilização de murganhos no estudo de novos alvos da terapia oncológica anti-angiogénica

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas

Vitamine D et prévention du cancer colorectal associé à la colite ulcéreuse : modèles murins

Les patients atteints de maladies inflammatoires de l'intestin (MII) ont un risque accru de développer un cancer colorectal dû aux lésions épithéliales secondaires à l’inflammation chronique. La vitamine D (vD) régule NOD2, gène impliqué dans la réponse inflammatoire et dans la susceptibilité aux MII, et induit son expression dans les monocytes et dans l’épithélium intestinal. Dans ce projet, nous avons d’abord induit le cancer colorectal associé à la colite ulcéreuse (CAC) en administrant un traitement combiné d’azoxyméthane (AOM) et de dextran de sulfate de sodium (DSS) aux souris C57BL/6J. Par la suite, nous avons étudié l'effet d’une carence en vD3 sur le développement du CAC et évalué la capacité préventive d’une supplémentation en vD3 sur la tumorigenèse, et vérifié si cet effet est médié par NOD2, en utilisant les souris Nod2-/-. Les C57BL/6J et les Nod2-/-, ayant reçu une diète déficiente en vD3, étaient moins résistantes au CAC par rapport aux souris supplémentées. Le pourcentage de perte de poids, l’indice d’activation de la maladie (DAI), le taux de mortalité et le poids relatif du côlon (mg/cm) chez les souris déficientes en vD3 étaient plus élevés en comparaison avec celles supplémentées en vD3. Une augmentation du score d'inflammation et de la multiplicité tumorale corrélait avec une expression accentuée de l’Il6 dans les colonocytes des souris déficientes en vD3. La vD3 régulait l’expression génétique de Cyp24, Vdr et de gènes pro-inflammatoires chez les C57BL/6, comme chez les Nod2-/-. En conclusion, la supplémentation en vD3 peut prévenir le développement du CAC indépendamment de NOD2.

Vitamine D et prévention du cancer colorectal associé à la colite ulcéreuse : modèles murins

Les patients atteints de maladies inflammatoires de l'intestin (MII) ont un risque accru de développer un cancer colorectal dû aux lésions épithéliales secondaires à l’inflammation chronique. La vitamine D (vD) régule NOD2, gène impliqué dans la réponse inflammatoire et dans la susceptibilité aux MII, et induit son expression dans les monocytes et dans l’épithélium intestinal. Dans ce projet, nous avons d’abord induit le cancer colorectal associé à la colite ulcéreuse (CAC) en administrant un traitement combiné d’azoxyméthane (AOM) et de dextran de sulfate de sodium (DSS) aux souris C57BL/6J. Par la suite, nous avons étudié l'effet d’une carence en vD3 sur le développement du CAC et évalué la capacité préventive d’une supplémentation en vD3 sur la tumorigenèse, et vérifié si cet effet est médié par NOD2, en utilisant les souris Nod2-/-. Les C57BL/6J et les Nod2-/-, ayant reçu une diète déficiente en vD3, étaient moins résistantes au CAC par rapport aux souris supplémentées. Le pourcentage de perte de poids, l’indice d’activation de la maladie (DAI), le taux de mortalité et le poids relatif du côlon (mg/cm) chez les souris déficientes en vD3 étaient plus élevés en comparaison avec celles supplémentées en vD3. Une augmentation du score d'inflammation et de la multiplicité tumorale corrélait avec une expression accentuée de l’Il6 dans les colonocytes des souris déficientes en vD3. La vD3 régulait l’expression génétique de Cyp24, Vdr et de gènes pro-inflammatoires chez les C57BL/6, comme chez les Nod2-/-. En conclusion, la supplémentation en vD3 peut prévenir le développement du CAC indépendamment de NOD2.

New insight on obesity and adipose-derived stem cells by comprehensive metabolomics

Obesity affects the functional capability of adipose-derived stem cells (ASCs) and their effective use in regenerative medicine through mechanisms still poorly understood. Here we employed a multiplatform (LC/MS, CE/MS, GC/MS) metabolomics untargeted approach to investigate the metabolic alteration underlying the inequalities observed in obese-derived ASCs. The metabolic fingerprint (metabolites within the cells) and footprint (metabolites secreted in the culture medium) from humans or mice, obese and non-obese derived ASCs, were characterized by providing valuable information. Metabolites associated to glycolysis, TCA, pentose phosphate pathway and polyol pathway were increased in the footprint of obese-derived human ASCs indicating alterations in the carbohydrate metabolism; whereas from the murine model, deep differences in lipid and amino acid catabolism were highlighted. Therefore, new insights on the ASCs metabolome were provided that enhance our understanding of the processes underlying the ASCs stemness capacity and its relationship with obesity, in different cell models.

Flucytosine + fluconazole association in the treatment of a murine experimental model of cryptococcosis

The efficacy of flucytosine (5-FC) and fluconazole (FLU) association in the treatment of a murine experimental model of cryptococcosis, was evaluated. Seven groups of 10 Balb C mice each, were intraperitoneally inoculated with 10(7) cells of Cryptococcus neoformans. Six groups were allocated to receive 5-FC (300 mg/kg) and FLU (16 mg/ kg), either combined and individually, by daily gavage beginning 5 days after the infection, for 2 and 4 weeks. One group received distilled water and was used as control. The evaluation of treatments was based on: survival time; macroscopic examination of brain, lungs, liver and spleen at autopsy; presence of capsulated yeasts in microscopic examination of wet preparations of these organs and cultures of brain homogenate. 5-FC and FLU, individually or combined, significantly prolonged the survival time of the treated animals with respect to the control group (p<0.01). Animals treated for 4 weeks survived significantly longer than those treated for 2 weeks (p<0.01). No significant differences between the animals treated with 5-FC and FLU combined or separately were observed in the survival time and morphological parameters. The association of 5-FC and FLU does not seem to be more effective than 5-FC or FLU alone, in the treatment of this experimental model of cryptococcosis.

Cholestasis in a murine experimental model: lesions include hepatocyte ischemic necrosis

OBJECTIVE: To establish a murine experimental model of bile duct obstruction that would enable controlled observations of the acute and subacute phases of cholestasis. METHODOLOGY: Adult male isogenic BALB/c mice underwent a bile duct ligation (22 animals) or a sham operation (10 animals). Fifteen days after surgery, or immediately after the animal's death, macroscopic findings were noted and histological study of the liver, biliary tree, and pancreas was performed (hematoxylin-eosin and Masson trichromic staining). RESULTS: Beginning 24 hours after surgery, all animals from the bile duct ligation group presented progressive generalized malaise. All animals presented jaundice in the parietal and visceral peritoneum, turgid and enlarged liver, and accentuated dilatation of gallbladder and common bile duct. Microscopic findings included marked dilatation and proliferation of bile ducts with accentuated collagen deposits, frequent areas of ischemic necrosis, hepatic microabscesses, and purulent cholangitis. Animals from the sham operation group presented no alterations. CONCLUSION: We established a murine experimental model of induced cholestasis, which made it possible to study acute and subacute tissue lesions. Our data suggests that in cholestasis, hepatic functional ischemia plays an important role in inducing hepatic lesions, and it also suggests that the infectious process is an important factor in morbidity and mortality.