Regression of glomerular injury by losartan in experimental diabetic nephropathy

Many features of chronic kidney disease may be reversed, but it is unclear whether advanced lesions, such as adhesions of sclerotic glomerular tufts to Bowman`s capsule (synechiae), can resolve during treatment. We previously showed, using a renal ablation model, that the renoprotective effect of the AT-1 receptor blocker, losartan, is dose-dependent. Here we determined if moderate and advanced glomerular lesions, associated with streptozotocin-induced diabetes, regress with conventional or high-dose losartan treatment. Using daily insulin injection for 10 months, we maintained diabetic adult male Munich-Wistar rats in a state of moderate hyperglycemia. Following this period, some rats continued to receive insulin with or without conventional or high-dose losartan for an additional 2 months. Diabetic rats pretreated with insulin for 10 months and age-matched non-diabetic rats served as controls. Mesangial expansion was found in the control diabetic rats and was exacerbated in those rats maintained on only insulin for an additional 2 months. Conventional and high-dose losartan treatments reduced this mesangial expansion and the severity of synechiae lesions below that found prior to treatment; however, the frequency of the latter was unchanged. There was no dose-response effect of losartan. Our results show that regression of mesangial expansion and contraction of sclerotic lesions is feasible in the treatment of diabetes, but complete resolution of advanced glomerulosclerosis may be hard to achieve.

Experimental Model of Isolated Lung Perfusion in Rats: First Brazilian Experience Using the IL-2 Isolated Perfused Rat or Guinea Pig Lung System

Introduction. Lung transplantation has become the mainstay therapy for patients with end-stage lung disease refractory to medical management. However, the number of patients listed for lung transplantation largely exceeds available donors. The study of lung preservation requires accurate, cost-effective small animal models. We have described a model of ex vivo rat lung perfusion using a commercially available system. Methods. Male Wistar rats weighing 250 g-300 g were anesthetized with intraperitoneal sodium thiopental (50 mg/kg body weight). The surgical technique included heart-lung block extraction, assembly, and preparation for perfusion and data collection. We used an IL-2 Isolated Perfused Rat or Guinea Pig Lung System (Harvard Apparatus, Holliston, Mass, United States; Hugo Sachs Elektronik, Alemanha). Results. Preliminary results included hemodynamic and pulmonary mechanics data gathered in the experiments. Conclusion. The isolated rat lung perfusion system is a reliable method to assess lung preservation.

Ultrasound Propagation Velocity and Broadband Attenuation Can Help Evaluate the Healing Process of an Experimental Fracture

Ultrasonometry seems to have a future for the evaluation of fracture healing. Ultrasound propagation velocity (USPV) significantly decreases at the same time that bone diameter decreases as healing takes place, thus approaching normal values. In this investigation, both USPV and broadband ultrasound attenuation (BUA) were measured using a model of a transverse mid-diaphyseal osteotomy of sheep tibiae. Twenty-one sheep were operated and divided into three groups of seven, according to the follow-up period of 30, 60, and 90 days, respectively. The progress of healing of the osteotomy was checked with monthly conventional radiographs. The animals were killed at the end of the period of observation of each group, both operated-upon and intact tibiae being resected and submitted to the measurement of underwater transverse and direct contact transverse and longitudinal USPV and BUA at the osteotomy site. The intact left tibia of the 21 animals was used for control, being examined on a symmetrical diaphyseal segment. USPV increased while BUA decreased with the progression of healing, with significant differences between the operated and untouched tibiae and between the periods of observation, for most of the comparisons. There was a strong negative correlation between USPV and BUA. Both USPV and BUA directly reflect and can help predict the healing of fractures, but USPV alone can be used as a fundamental parameter. Ultrasonometry may be of use in clinical application to humans provided adequate adaptations can be developed. (C) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:444-451, 2011

Stretch Injuries of Skeletal Muscles: Experimental Study in Rats` Soleus Muscle

The study aimed to verify the physiological injury behavior by stretching the soleus muscle of rats, using a noninvasive experimental model. Twenty-four rats were used and divided into three groups of eight animals: control group (A), group that performed tetanus followed by electrical stimulation and a sudden dorsiflexion of the left paw performed by a device equipped with a mechanism of muscle soleus rapid stretching (B); and a group that only received the tetanus (C). Three days later, the animals were killed, and the soleus muscle was resected and divided into three segments. Morphological changes indicative of muscle damage appeared in all three segments of group B. In a lesser degree, similar changes were also detected in muscles subjected to only tetanus. This model was effective; reproducing an injury similar to what occurs in human sports injuries.

Morphologic and morphometric evaluation of experimental acute crush injuries of the sciatic nerve of rats

In order to qualify and quantify nerve fiber lesion following an acute crush injury, a morphologic and morphometric study was carried out in 25 Wistar rats divided into live groups of five animals each according to the crushing load applied, i.e., 500,1000, 5000, 10 000, and 15 000 g. The injury was produced under general anesthesia on a 5 mm-long intermediate segment of the right sciatic nerve for 10 min using a dead-weight machine. The animals were killed with an excessive dose of anesthetics 72 h later and submitted to perfusion with a fixing solution through the abdominal aorta immediately after death. Both the right and left sciatic nerves were removed and prepared for histologic and morphometric examinations: 5 mu m-thick sections stained with 1% Toluidine blue were examined under a light microscope equipped with a video camera linked to a computer loaded with a graphic program (KS 400). The morphometric studies included measuring total number of fibers, fiber density, fiber diameter, myelin fiber area, axon diameter, axon area and G ratio. The results showed that damage to the nerve fibers began to appear as early as with the 500g load and was similar in all groups despite the load applied, increasing with the 10000 and 15000g loads, although the external supporting tissues and small diameter fibers were preserved. The predominant type of lesion produced was axonotmesis. (c) 2008 Elsevier B.V. All rights reserved.

Newly Formed Bone in Mandible Decortication Experimental Model Using rhBMP-2 Evaluated by Densitometric Study

The purpose of this study was to evaluate the newly formed bone, comparing two different carriers for rhBMP-2, using the decortication and nondecorticatication surgical technique in Wistar rat mandibles, evaluated by radiographic densitometry method. It was used fifty six animals according to specific treatment, which were sacrificed after 3 and 6 weeks after this. It was concluded that the decortication surgical technique was able to optimize the osteoinduction properties of the rhBMP-2, independently of the material carrier used and the period of time, according to radiographic densitometry technique.

Experimental tuberculosis: Designing a better model to test vaccines against tuberculosis

Experimental models of infection are good tools for establishing immunological parameters that have an effect on the host-pathogen relationship and also for designing new vaccines and immune therapies. In this work, we evaluated the evolution of experimental tuberculosis in mice infected with increasing bacterial doses or via distinct routes. We showed that mice infected with low bacterial doses by the intratracheal route were able to develop a progressive infection that was proportional to the inoculum size. In the initial phase of disease, mice developed a specific Th1-driven immune response independent of inoculum concentration. However, in the late phase, mice infected with higher concentrations exhibited a mixed Th1/Th2 response, while mice infected with lower concentrations sustained the Th1 pattern. Significant IL-10 concentrations and a more preeminent T regulatory cell recruitment were also detected at 70 days post-infection with high bacterial doses. These results suggest that mice infected with higher concentrations of bacilli developed an immune response similar to the pattern described for human tuberculosis wherein patients with progressive tuberculosis exhibit a down modulation of IFN-gamma production accompanied by increased levels of IL-4. Thus, these data indicate that the experimental model is important in evaluating the protective efficacy of new vaccines and therapies against tuberculosis. (C) 2010 Elsevier Ltd. All rights reserved.

HSP65 DNA as therapeutic strategy to treat experimental paracoccidioidomycosis

The conventional treatment for paracoccidioidomycosis, the most prevalent mycosis in Latin America, involves long periods of therapy resulting in sequels and high frequency of relapses. The search for new alternatives of treatment is necessary. Previously, we have demonstrated that the hsp65 gene from Mycobacterium leprae shows prophylactic effects against murine paracoccidioidomycosis. Here, we tested the DNAhsp65 immunotherapy in BALB/c mice infected with Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. We observed an increase of Th1 cytokines accompanied by a reduction in fungal burden and pulmonary injury. These results provide new prospects for immunotherapy of paracoccidioidomycosis and other mycoses. (C) 2009 Elsevier Ltd. All rights reserved.

Immunization with pVAXhsp65 Decreases Inflammation and Modulates Immune Response in Experimental Encephalomyelitis

Background: A DNA vaccine (pVAXhsp65) containing the gene of a heat-shock protein (hsp65) from Mycobacterium leprae showed high immunogenicity and protective efficacy against tuberculosis in BALB/c mice. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 in the development of experimental autoimmune encephalomyelitis (EAE), a rat model of multiple sclerosis. Methods: Female Lewis rats were immunized with 3 pVAXhsp65 doses by intramuscular route. Fifteen days after the last DNA dose the animals were evaluated for specific immunity or submitted to induction of EAE. Animals were evaluated daily for weight loss and clinical score, and euthanized during the recovery phase to assess the immune response and inflammatory infiltration at the central nervous system. Results: Immunization with pVAXhsp65 induced a specific immune response characterized by production of IgG(2b) anti-hsp65 antibodies and IFN-gamma secretion. Previous immunization with pVAXhsp65 did not change EAE clinical manifestations (weight and clinical score). However, the vaccine clearly decreased brain and lumbar spinal cord inflammation. In addition, it downmodulated IFN-gamma and IL-10 production by peripheral lymphoid organs. Conclusion: Our data demonstrated that this vaccine does not trigger a deleterious effect on EAE development and also points to a potential protective effect. Copyright (C) 2010 S. Karger AG, Basel

Alterations in myocardial gene expression associated with experimental Trypanosoma cruzi infection

Chagas disease, characterized by acute myocarditis and chronic cardiomyopathy, is caused by infection with the protozoan parasite Trypanosoma cruzi. We sought to identify genes altered during the development of parasite-induced cardiomyopathy. Microarrays containing 27,400 sequence-verified mouse cDNAs were used to analyze global gene expression changes in the myocardium of a murine model of chagasic cardiomyopathy. Changes in gene expression were determined as the acute stage of infection developed into the chronic stage. This analysis was performed on the hearts of male CD-1 mice infected with trypomastigotes of T. cruzi (Brazil strain). At each interval we compared infected and uninfected mice and confirmed the microarray data with dye reversal. We identified eight distinct categories of mRNAs that were differentially regulated during infection and identified dysregulation of several key genes. These data may provide insight into the pathogenesis of chagasic cardiomyopathy and provide new targets for intervention. (c) 2008 Elsevier Inc. All rights reserved.

Role of Interleukin (IL)-18 in experimental paracoccidioidomycosis

Interleukin (IL)-18 has been regarded as a Th1 type cytokine involved in many fungal and parasitic infections. Since there have been no studies, as of yet, evaluating the role of this cytokine in paracoccidioidomycosis (PCM), we assessed the function of IL-18 by using an experimental PCM model. Our results showed that IL-18 knockout (IL-18-/-) BALB/c were more resistant to Paracoccidioides brasiliensis than their littermate controls (WT). In fact, mortality rate was higher in WT mice and in the first month of infection, the number of colony forming units of the etiologic agent recovered from the lungs was greater in WT mice. In histopathological analyses, well-formed granulomas were seen in both WT and IL-18-/- mice. However, substantial differences were observed at the second month of infection when epithelioid cells predominated in the lesions of IL-18-/- mice, which could infer that IL-18 postpones pulmonary healing. The levels of IL-10 were significantly higher in IL-18 sufficient mice at early stages of infection and therefore account for the delayed fungal clearance observed in WT mice. TNF- augmented later in the infection of WT mice, seemingly to compensate high levels of IL-10. Our results demonstrated that IL-18 has a critical role in protecting BALB/c mice against disseminated PCM.

Rabbit Retinal Neovascularization Induced by Latex Angiogenic-Derived Fraction: An Experimental Model

Purpose: To create a retinal neovascularization experimental model using intravitreal injection of microspheres loaded with latex-derived angiogenic fraction. Methods: Thirty-two albino New Zealand rabbits, divided in 4 groups of 8 animals, were enrolled in this study. Rabbits in groups I, II, and III received one intravitreal injection of PLGA (L-lactide-co-glycolide) microspheres with 10, 30, and 50 mu g of latex-derived angiogenic fraction into their right eyes, respectively, and group IV received 0.1 ml of microspheres without the angiogenic fraction. Weekly follow-up with ophthalmoscopy and fluorescein angiography was performed; the rabbits were sacrificed in the 4th week and their eyes processed for light microscopy. Results: All eyes from group I demonstrated increased retinal vascular tortuosity, observed from 14 days after injection and maintained for 28 days, otherwise without new vessels detection. All group II eyes showed vascular changes similar to group I. Fifty percent of the eyes from group II rabbits developed retinal neovascularization 21 days after injection. All eyes from group III demonstrated significant vascular tortuosity and retinal new vessels 2 weeks after injection, progressing to fibrovascular proliferation and tractional retinal detachment. No vascular changes or retinal new vessels were observed in group IV eyes. Light microscopy confirmed the existence of new vessels previously seen on fluorescein angiography, in retinal sections adjacent to the optic disc, not observed in sections at the same area in the control group. Conclusion: Thirty- and 50-mu g microspheres containing latex-derived angiogenic fraction injected into the vitreous cavity induced retinal neovascularization in rabbits.

Oropouche virus experimental infection in the golden hamster (Mesocrisetus auratus)

Oropouche virus (OROV), of the family Bunyaviridae, is the second most frequent arbovirus causing febrile disease in Brazil. In spite of this, little is known about pathogenesis of OROV infection. This report describes an experimental model of OROV in golden hamster (Mesocricetus auratus). Following subcutaneous inoculation of OROV, over 50% of the animals developed disease characterized by lethargy, ruffled fur, shivering, paralysis, and approximately one third died. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation to collect tissue samples from brain, heart, liver, lung, spleen, muscle and blood for virus titration, histology and OROV immunohistochemistry. OROV was detected in high titers in blood, liver and brain, but not in the other organs. Histopathology revealed meningoencephalitis and hepatitis, with abundant OROV antigen detected in liver and brain. Diffuse galectin-3 immunostaining in brain and liver supports microglial and Kupfer cells activation. This is the first description of an experimental model for OROV infection and should be helpful to study pathogenesis and possibly to test antiviral interventions such as drugs and vaccine candidates. (C) 2010 Elsevier B.V. All rights reserved.

The Ideal Timing for Experimental Cleft Lip Creation

Cleft lip and palate (CLP) is the most common congenital defect of the face. Many animal models have been utilized to study embryogenesis and pathogenesis of CLP, including the development of secondary anomalies and consequent deformities. However, the ideal gestational age for surgical creation of lip or palate defects in rat models has never been determined. The aim of the present study is to improve the experimental model utilizing rat fetuses, defining the most appropriate timing for creation of the lip defect model. The study was composed of three groups of fetuses undergoing surgical creation of a lip defect at the left side of the superior lip at 17.5, 18.5, and 19.5 days of gestation. Fetuses were harvested at 21.5 days of gestation (term = 22 days) and underwent macroscopic and microscopic analyses. We found that the most appropriate moment for lip defect creation was at 19.5 days, given the presence of lip depression at the site of the defect and asymmetry and retraction associated with interruption of the lip and complete reepithelialization of the borders of the defect.

Hyperbaric oxygen on the healing of ischemic colonic anastomosis - an experimental study in rats

The aim of the present study was to evaluate the effect of hyperbaric oxygen therapy (HBO(2)) on the healing process of ischemic colonic anastomoses in rats Forty Wistar rats were divided into four groups control (Group I), control and HBO(2) (Group 11), ischemia (Group III), ischemia and HBO(2) (Group IV) Ischemia was achieved by clamping four centimeters of the colonic arcade On the eighth therapy day, the anastomotic region was removed for quantification of hydroxyproline and immunohistochemical determination of metalloproteinases 1 and 9 (MMP1,MMP9) The immunohistochemical studies showed significantly larger metalloproteinase-labeled areas in Group IV compared with Group III for both MMP1 and MMP9 (p<001) This finding points to a higher remodeling activity of the anastomoses in this experimental group Additionally, animals subjected to hyperbaric oxygen therapy showed both a reduction in interstitial edema and an increase in hydroxyproline concentrations [at the anastomotic site] Therefore, we conclude that HBO(2) is indeed beneficial in anastomotic ischemia