Extracellular matrix and vascular dynamics in the kidney of a murine model for Marfan syndrome.

Fibrillin-1 is a pivotal structural component of the kidney's glomerulus and peritubular tissue. Mutations in the fibrillin-1 gene result in Marfan syndrome (MFS), an autosomal dominant disease of the connective tissue. Although the kidney is not considered a classically affected organ in MFS, several case reports describe glomerular disease in patients. Therefore, this study aimed to characterize the kidney in the mgΔlpn-mouse model of MFS. Affected animals presented a significant reduction of glomerulus, glomerulus-capillary, and urinary space, and a significant reduction of fibrillin-1 and fibronectin in the glomerulus. Transmission electron microscopy and 3D-ultrastructure analysis revealed decreased amounts of microfibrils which also appeared fragmented in the MFS mice. Increased collagen fibers types I and III, MMP-9, and α-actin were also observed in affected animals, suggesting a tissue-remodeling process in the kidney. Video microscopy analysis showed an increase of microvessel distribution coupled with reduction of blood-flow velocity, while ultrasound flow analysis revealed significantly lower blood flow in the kidney artery and vein of the MFS mice. The structural and hemodynamic changes of the kidney indicate the presence of kidney remodeling and vascular resistance in this MFS model. Both processes are associated with hypertension which is expected to worsen the cardiovascular phenotype in MFS.

Effects of the PARP Inhibitor Olaparib on the Response of Human Peripheral Blood Leukocytes to Bacterial Challenge or Oxidative Stress.

Prior studies demonstrate the activation of poly-(ADP-ribose) polymerase 1 (PARP1) in various pathophysiological conditions, including sepsis. We have assessed the effect of olaparib, a clinically used PARP1 inhibitor, on the responses of human peripheral blood leukocytes (PBMCs) obtained from healthy volunteers in response to challenging with live bacteria, bacterial lipopolysaccharide (LPS), or oxidative stress (hydrogen peroxide, H2O2). The viability of PBMCs exposed to olaparib or to the earlier generation PARP inhibitor PJ-34 (0.1-1000 µM) was monitored using Annexin V and 7-aminoactinomycin D. To evaluate the effects of olaparib on the expression of PARP1 and its effects on protein PARylation, PBMCs were stimulated with Staphylococcus aureus with or without olaparib (1-10 µM). Changes in cellular levels of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP), as well as changes in mitochondrial membrane potential (MMP), were measured in PBMCs exposed to H2O2. Bacterial killing was evaluated in PBMCs and polymorphonuclear leukocytes (PMNs) incubated with S. aureus. Cytokine production was measured in supernatants using a cytometric bead array. Reactive oxygen species (ROS), nitric oxide (NO) production, and phagocytic activity of monocytes and neutrophils were measured in whole blood. For ROS and NO production, samples were incubated with heat-killed S. aureus; phagocytic activity was assessed using killed Escherichia coli conjugated to FITC. Olaparib (0.1-100 µM) did not adversely affect lymphocyte viability. Olaparib also did not interfere with PARP1 expression but inhibits S. aureus-induced protein PARylation. In cells challenged with H2O2, olaparib prevented NAD+ and ATP depletion and attenuated mitochondrial membrane depolarization. LPS-induced production of TNF-α, MIP-1α, and IL-10 by PBMCs was also reduced by olaparib. Monocytes and neutrophils displayed significant increases in the production of ROS and NO after stimulation with S. aureus and phagocytic (E. coli) and microbicidal activity, and these responses were not suppressed by olaparib. We conclude that, at clinically relevant concentrations, olaparib exerts cytoprotective effects and modulates inflammatory cytokine production without exerting adverse effects on the cells' ability to phagocytose or eradicate pathogens. The current data support the concept of repurposing olaparib as a potential experimental therapy for septic shock.

Ozone Therapy in the Prevention of Dental Plaque Formation in Dogs.

This study aims to assess ozonized mineral oil ointment application as an antiplaque therapy for dogs. Domestic healthy dogs received dental scaling and polishing under general anesthesia. Under standard feeding and homecare during 7 days, 20 dogs were randomly placed into 2 different groups for dental treatment. The control group (CG) was given a single placebo application and the ozone group (O3G) received daily ozonized ointment application. The average age (CG = 4.4; O3G = 5.7 years old), body weight (CG = 15.7; O3G = 15.3 kg) and the gingivitis index obtained on the first day (D0) allowed initial homogeneity between the groups. The dental plaque index, including clinical and computerized analysis on the seventh day, was obtained from the buccal aspect of specific dental locations. Both analyses revealed significant statistical association between daily application of ozone and antiplaque effect. There was no evidence of toxicity during the study. These results suggest that ozone therapy may be an efficient adjuvant to conventional periodontal treatment in decreasing initial dental plaque formation.

Association of thoracic spine deformity and cardiovascular disease in a mouse model for Marfan syndrome.

AIMS: Cardiovascular manifestations are a major cause of mortality in Marfan syndrome (MFS). Animal models that mimic the syndrome and its clinical variability are instrumental for understanding the genesis and risk factors for cardiovascular disease in MFS. This study used morphological and ultrastructural analysis to the understanding of the development of cardiovascular phenotypes of the the mgΔloxPneo model for MFS. METHODS AND RESULTS: We studied 6-month-old female mice of the 129/Sv background, 6 wild type (WT) and 24 heterozygous animals from the mgΔloxPneo model. Descending thoracic aortic aneurysm and/or dissection (dTAAD) were identified in 75% of the MFS animals, defining two subgroups: MFS with (MFS+) and without (MFS-) dTAAD. Both subgroups showed increased fragmentation of elastic fibers, predominance of type I collagen surrounding the elastic fiber and fragmentation of interlaminar fibers when compared to WT. However, only MFS animals with spine tortuosity developed aortic aneurysm/dissection. The aorta of MFS+ animals were more tortuous compared to those of MFS- and WT mice, possibly causing perturbations of the luminal blood flow. This was evidenced by the detection of diminished aorta-blood flow in MFS+. Accordingly, only MFS+ animals presented a process of concentric cardiac hypertrophy and a significantly decreased ratio of left and right ventricle lumen area. CONCLUSIONS: We show that mgΔloxPneo model mimics the vascular disease observed in MFS patients. Furthermore, the study indicates role of thoracic spine deformity in the development of aorta diseases. We suggest that degradation of support structures of the aortic wall; deficiency in the sustenance of the thoracic vertebrae; and their compression over the adjacent aorta resulting in disturbed blood flow is a triad of factors involved in the genesis of dissection/aneurysm of thoracic aorta.

Layered Double Hydroxides Are Promising Nanomaterials for Tissue Bioengineering Application.

Layered double hydroxides (LDHs) have emerged as promising nanomaterials for human health and although it has achieved some progress on this matter, their application within bioengineering is not fully addressed. This prompted to subject fibroblasts to two compositions of LDHs (Mg2 Al-Cl and Zn2 Al-Cl), considering an acute response. First, LDH particles are addressed by scanning electron microscopy, and no significant effect of the cell culture medium on the shape of LDHs particles is reported although it seems to adsorb some soluble proteins as proposed by energy-dispersive X-ray analysis. These LDHs release magnesium, zinc, and aluminum, but there is no cytotoxic or biocompatibility effects. The data show interference to fibroblast adhesion by driving the reorganization of actin-based cytoskeleton, preliminarily to cell cycle progression. Additionally, these molecular findings are validated by performing a functional wound-healing assay, which is accompanied by a dynamic extracellular matrix remodeling in response to the LDHs. Altogether, the results show that LDHs nanomaterials modulate cell adhesion, proliferation, and migration, delineating new advances on the biomaterial field applied in the context of soft tissue bioengineering, which must be explored in health disorders, such as wound healing in burn injuries.

Sonic hedgehog drives layered double hydroxides-induced acute inflammatory landscape.

Although layered double hydroxides (LDH) have been listed as promising nanomaterials in human healthcare, very little has been achieved on osteoblast inflammatory signaling. Thus, osteoblasts were challenged with two LDHs (Mg2Al-Cl and Zn2Al-Cl, at 0.002 mg/mL) up to 24 h, establishing an acute inflammatory mechanism, as well as identifying whether Sonic hedgehog (Shh) signaling has an influence. Functional experiments were performed by previously treating (2 h) semiconfluent osteoblast cultures with cyclopamine molecule (cyc), a widely used Shh inhibitor. Considering inflammasome complex, the asc1 gene was significantly up-expressed in response to Zn2Al-Cl - LDHs, as well as the nrlp3 gene. By treating the osteoblast with cyc, the asc1 gene presented an even higher profile. Our results found a down-modulation of major pro-inflammatory cytokines-related genes, when tnfα and il1ß were significantly down-modulated in response to LDHs. Conversely, anti-inflammatory cytokines were up-modulated considering the same experimental procedures. Except the il6, the other il13, il10, and tgfß genes were up modulated. Additionally, Shh signaling seems to modulate this repertory as both the il13 and il10 genes were significantly up-modulated when the Shh signaling was inhibited. Altogether, our results reveal for the first time the exigency of Shh-dependent anti-inflammatory signals in LDH-induced osteoblast responses.

Gut Microbiota Imbalance Can Be Associated with Non-malabsorptive Small Bowel Shortening Regardless of Blind Loop.

INTRODUCTION: Some traditional bariatric surgery procedures may lead to functional gut shortening, which may unsettle the fine-tuned gastrointestinal physiology and affect gut microbiota balance. PURPOSE: Evaluate the gut microbiota behavior in rat models facing gut shortening due to intestinal bypass. MATERIALS AND METHODS: Wistar rats (n = 17) were randomly distributed in three groups: (1) sham group (n = 5); (2) blind loop group (n = 6); and (3) resection group (n = 6). Intestinal samples and feces were analyzed to measure bacterial concentrations (small intestinal bacterial overgrowth-SIBO) 12 weeks after the experimental procedures. Bacterial translocation (BT) was investigated in the mesenteric lymph node (MLN), liver, spleen, and lung of the animals. In addition, inflammatory aspects were investigated in their liver and small bowel through histological analysis. RESULTS: Regardless of blind loop, gut shortening groups recorded similar high level of bacterial concentrations in intestine compartments, greater than that of the sham group (p ≤ 0.05). BT was only observed in the MLN of gut shortening models, with higher percentage in the blind loop group (p ≤ 0.05). The gut and liver histopathological analysis showed similar low-grade chronic inflammation in both gut shortening groups, likely associated with SIBO/BT events. CONCLUSION: Sustained SIBO/BT was associated with proximal gut shortening in half regardless of blind loop, whereas the GI tract's ability to restore gut microbiota balance after a surgical challenge on the small bowel appears to be linked to the functional remaining gut.

Mg-Al and Zn-Al Layered Double Hydroxides Promote Dynamic Expression of Marker Genes in Osteogenic Differentiation by Modulating Mitogen-Activated Protein Kinases.

The effect of LDH samples comprised of chloride anions intercalated between positive layers of magnesium/aluminum (Mg-Al LDH) or zinc/aluminum (Zn-Al LDH) chemical composition on pre-osteoblast performance is investigated. Non-cytotoxic concentrations of both LDHs modulated pre-osteoblast adhesion by triggering cytoskeleton rearrangement dependent on recruiting of Cofilin, which is modulated by the inhibition of the Protein Phosphatase 2A (PP2A), culminating in osteoblast differentiation with a significant increase of osteogenic marker genes. The alkaline phosphatase (ALP) and bone sialoprotein (BSP) are significantly up-modulated by both LDHs; however, Mg-Al LDH nanomaterial promoted even more significance than both experimental controls, while the phosphorylations of mitogen-activated protein kinase (MAPKs)- extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) significantly increased. MAPK signaling is necessary to activate Runt-related transcription factor 2 (RUNX2) gene. Concomitantly, it is also investigated whether challenged osteoblasts are able to modulate osteoclastogenesis by investigating both osteoprotegerin (OPG) and Receptor activator of nuclear factor kappa-ligand (RANKL) in this model; a dynamic reprogramming of both these genes is found, suggesting LDHs in modulating osteoclastogenesis. These results suggest that LDHs interfere in bone remodeling, and they can be considered as nanomaterials in graft-based bone healing or drug-delivery materials for bone disorders.

Accessing the biocompatibility of layered double hydroxide by intramuscular implantation: histological and microcirculation evaluation.

Biocompatibility of layered double hydroxides (LDHs), also known as hydrotalcite-like materials or double metal hydroxides, was investigated by in vivo assays via intramuscular tablets implantation in rat abdominal wall. The tablets were composed by chloride ions intercalated into LDH of magnesium/aluminum (Mg2Al-Cl) and zinc/aluminum (Zn2Al-Cl). The antigenicity and tissue integration capacity of LDHs were assessed histologically after 7 and 28 days post-implantation. No fibrous capsule nearby the LDH was noticed for both materials as well any sign of inflammatory reactions. Sidestream Dark Field imaging, used to monitor in real time the microcirculation in tissues, revealed overall integrity of the microcirculatory network neighboring the tablets, with no blood flow obstruction, bleeding and/or increasing of leukocyte endothelial adhesion. After 28 days Mg2Al-Cl promoted multiple collagen invaginations (mostly collagen type-I) among its fragments while Zn2Al-Cl induced predominantly collagen type-III. This work supports previous results in the literature about LDHs compatibility with living matter, endorsing them as functional materials for biomedical applications.

The composition and behavior of capsules around smooth and textured breast implants in pigs.

BACKGROUND: The surface of the implant is one of the many factors often associated with the occurrence of capsular contracture, the etiopathogeny of which remains unclear. The purpose of this study was to analyze the behavior of capsular contracture by means of applanation tonometry and histology using a midsized animal model. METHODS: Silicone breast implants were implanted into 33 pigs and observed at 30, 60, 180, and 270 postoperative days. RESULTS: Capsular contracture in smooth implants showed significantly greater pressure values of tonometry, and the smooth implant capsule was significantly thicker than the textured implant capsule. Both pressure and thickness of the capsules increased at each period. The collagenous layer did not show any difference considering the periods of time in which the total thickness was analyzed; on the other hand, the increase in total capsular thickness occurred by thickening of the noncollagenous layer in both smooth and textured implants. Taking into consideration both kinds of implants, histomorphometric analysis showed that thin fibers were replaced by thick fibers in later postoperatives periods (180 and 270 days). CONCLUSIONS: The greater incidence of capsular contracture in smooth implants was correlated with the progressive increase in total capsule thickness, due to a higher concentration of collagenous fibers, when compared with textured implants (p = 0.011; mean difference, 6.61), and a higher concentration of thick fibers (p = 0.034; average, >5.51 percentage points per field of thick fibers than the textured implants in all periods). Pigs are good animal models for studying the healing process after breast augmentation with implants.

[Ileocecal valve reconstruction in dogs]. / Reconstituição da válvula ileocecal em cães.

PURPOSE: The importance of keeping the ileocecal valve in the intestinal ressections has been reported by several authors. When preserved, the ileocecal valve was related to a longer survival and prevention of the short bowel syndrome, due to its ability to block the colonic content reflux into the ileum and to avoid the rapid empting of the ileal content into the cecum. It was assessed a tecnique of ileocecal valve reconstitution, based on vesicoureteral anti-reflux tecniques. METHODS: Fourteen beagles were operated. Seven underwent ileocecal valve reconstitution following the tecnique proposed and in the other seven a simple end-to-end anastomosis was performed. To assess the new valve, it was done the clinical follow up, the microbiologic analysis and the manometric study. RESULTS: Clinically, during 45 days of follow up, there was no difference between the dogs with and without ileocecal reconstitution. In the aerobic bacteria analysis, the predominant bacterium was Escherichia coli. Quantitatively, the cultures grew in an irregular way, so that it was not able to compare the bacterial growth between the groups with or without ileocecal valve. The new valve had a colo-ileal reflux pressure similar to that of the physiological valve (P > 0.05). However, when compared to the non valve group, the reflux pressures of the physiological valve and new valve were significantly higher, with P < 0.05 and P < 0.001, respectively. CONCLUSION: In this study, the reconstituted ileocecal valve served as a barrier to the colo-ileal reflux just as the physiological valve does.

Reconstituição da válvula ileocecal em cães / Ileocecal valve reconstruction in dogs

OBJETIVO: Avaliar uma técnica de reconstituição da válvula ileocecal com base em técnicas anti-refluxo vesicoureteral. MÉTODOS: Quatorze beagles foram operados. Em sete foi reconstituída a válvula ileocecal com a técnica proposta e nos outros sete realizou-se apenas a anastomose ileocólica término-terminal. Para avaliar a neoválvula realizou-se o acompanhamento clínico dos cães, a análise microbiológica e o estudo manométrico. RESULTADOS: Clinicamente, durante os 45 dias de pós-operatório, não houve diferença entre os cães com e sem reconstituição da válvula ileocecal. Na análise de bactérias aeróbias, a bactéria predominante foi a Escherichia coli. Quantitativamente, as culturas cresceram de modo irregular, não permitindo a comparação do crescimento bacteriano entre os grupos com e sem válvula ileocecal. A neoválvula apresentou uma pressão de refluxo ileocólica semelhante a da válvula ileocecal fisiológica (P>0,05). Em relação ao grupo sem válvula, as pressões de refluxo da válvula fisiológica e da neoválvula foram significantemente maiores, sendo P<0,05 e P<0,001, respectivamente. CONCLUSAO: A neoválvula ileocecal serviu de obstáculo ao refluxo colo-ileal de maneira semelhante à válvula ileocecal fisiológica.

Efeitos da desvascularização da operação de abaixamento do cólon em cães / Effects of the devascularization on the pulled-through of dogs colon

O objetivo deste trabalho foi estudar os efeitos dadesvascularização sobre o cólon submetido à operação de abaixamento. Foram utilizados 20 cães da raça Beaggle, adultos, de ambos os sexos, distribuídos em 3 grupos. O primeiro grupo, de 6 animais, foi utilizado para estabelecer os parâmetros de normalidade. No segundo grupo, também de 6 animais, foi realizada a desvascularização parcial do cólon, com ligadura dos vasos mesentéricos caudais. No terceiro grupo, de 8 animais, foi realizada a operação de abaixamento endorretal do cólon. Nos 3 grupos, após a anestesia, foi realizado enema opaco pré-operatório. Após 6 meses de observação, os animais foram anestesiados, submetidos a enema opaco e, a seguir, reoperados, com retirada de fragmentos em 3 locais padronizados do cólon, para análise histológica e estudo da contractilidade, in vitro, da musculatura longitudinal, com estímulo pela acetilcolina. A evolução clínica dos animais ocorreu sem complicações. Também não foram observadas alterações radiológicas, quando comparados os exames pré e pós-operatórios. Na análise histológica não foram encontradas alterações significativas. As células ganglionares dos plexos mientéricos não sofreram alterações morfológicas ou numéricas. Do ponto de vista funcional, observou-se que a musculatura longitudinal do cólon manteve sua capacidade contráctil máxima. No entanto, apresentou diminuição da sensibilidade à acetilcolina. Esse fato pode ser correlacionado com a hipoxia, pois ocorreu no local de menor vascularização do cólon abaixado. Concluiu-se que a hipoxia provocada pelo abaixamento do cólon, em cães, não foi suficiente para promover alterações morfológicas do cólon, porém, provocou alteração da contractilidade...

Estudo comparativo de duas técnicas operatórias para o tratamento cirúrgico do apêndice vermiforme, por secçäo e por invaginaçäo: estudo experimental em coelhos Oryctolagus cuniculus / Comparative study of 2 operative techniques for the surgical treatment of the vermiform appendix, by section and by invagination: experimental study in rabbits Oryctolagus cuniculus

O objetivo deste trabalho foi comparar duas técnicas de apendicectomia sob os aspectos ma roscópico, radiológico, histológico e de resistência, a insuflaçäo gasosa do ceco. Foram utilizados trinta e seis coelhos infantis, distribuidos em dois grupos iguais, denominados grupos A e B. No grupo A foi utilizada a técnica de apendicectomia por secçäo pós-ligadura e sepultamento do coto apendicular. No grupo B foi utilizada a técnica de apendicectomia por secçäo pós-ligadura e sepultamento do coto apendicular. No grupo B foi utilizada a técnica de apendicectomia por invaginaçäo-ligadura e sepultamento do coto apendicular. Cada grupo foi dividido em três subgrupos de seis coelhos, que foram sacrificados no 7§, 14§ e 28§ dia de pós-operatório (P.P.). Os resultados comparativos das duas técnicas näo mostraram diferenças significativas quanto ao aspecto macroscópico, radiológico e da resistência à insuflaçäo gasosa do ceco, exceto no aspecto histológico onde o grupo B, comparado ao A, apresentou menor proporçäo de deiscências na regiäo de sutura em bolsa aplicada à parede do ceco para o sepultammento do coto apendicular e maior porporçäo de deiscências na regiäo de ligadura do coto apendicular, ambos estatisticamente significantes

Aderências peritoneais produzidas experimentalmente em ratos / Peritoneal adhesions experimentally induced in rats

O trabalho apresenta cinco modelos de produçäo de aderências estudados comparativamente, em ratos. Foram utilizados 140 ratos da linhagem Wistar, divididos em 7 grupos de 20 ratos: 1) contrôle; 2) iintroduçäo de soro fisiológico e manuseio das alças intestinais; 3) sangue na cavidade peritoneal; 4) escarificaçäo de um segmento intestinal; 5) duas ligaduras vaculares de um segmento instetinal; 6) três ligaduras vasculares de um segmento intestinal; 7) introduçäo de talco na cavidade peritoneal. Os animais foram re-operados no 7§, 14§ e 21§ dias pós-operatório. O grupo da escarificaçäo de um segmento intestinal apresentou maior porcentagem de aderencias peritoneais. Nos grupos submetidos a duas e três ligaduras vasculares observou-se uma provável correlaçäo entre a hipóxia e aderências peritoneais