Congenital Zika Virus Infection Induces Severe Spinal Cord Injury.

We report 2 fatal cases of congenital Zika virus (ZIKV) infection. Brain anomalies, including atrophy of the cerebral cortex and brainstem, and cerebellar aplasia were observed. The spinal cord showed architectural distortion, severe neuronal loss, and microcalcifications. The ZIKV proteins and flavivirus-like particles were detected in cytoplasm of spinal neurons, and spinal cord samples were positive for ZIKV RNA.

Virulence-related genes, adhesion and invasion of some Yersinia enterocolitica-like strains suggests its pathogenic potential.

Yersina enterocolitica-like species have not been extensively studied regarding its pathogenic potential. This work aimed to assess the pathogenic potential of some Y. enterocolitica-like strains by evaluating the presence of virulence-related genes by PCR and their ability to adhere to and invade Caco-2 and HEp-2 cells. A total of 50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii strains were studied. The strains contained the following genes: Y. frederiksenii, fepA(44%), fes(44%) and ystB(18%); Y. intermedia, ail(53%), fepA (35%), fepD(2%), fes(97%), hreP(2%), ystB(2%) and tccC(35%); Y. kristensenii, ail(62%), ystB(23%), fepA(77%), fepD(54%), fes(54%) and hreP(77%). Generally, the Y. enterocolitica-like strains had a reduced ability to adhere to and invade mammalian cells compared to the highly pathogenic Y. enterocolitica 8081. However, Y. kristensenii FCF410 and Y. frederiksenii FCF461 presented high invasion potentials in Caco-2 cells after five days of pre-incubation increased by 45- and 7.2-fold compared to Y. enterocolitica 8081, respectively; but, the ail gene was not detected in these strains. The presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Moreover, the results suggest the existence of alternative virulence mechanisms and that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent.

Acute kidney injury and progression of renal failure after fetal programming in the offspring of diabetic rats.

BACKGROUND: Diseases of adulthood, such as diabetes and hypertension, may be related to changes during pregnancy, particularly in kidney. We hypothesized that acute kidney injury progresses more rapidly in cases of fetal programming. METHODS: Diabetic dams' offspring were divided into: CC (controls, receiving vehicle); DC (diabetics, receiving vehicle); CA (controls receiving folic Acid solution, 250 mg/kg); and DA (diabetics receiving folic acid solution). Renal function tests, morphometry, gene, and protein expression of epithelial-mesenchymal transition (EMT) markers were analyzed by qPCR and immunohistochemistry, respectively. RESULTS: Creatinine, urea, Bowman's space, and EMT markers were increased in CA and DA groups. TGF-ß3, actin, and fibronectin expression was higher in CA and DA, with significant increase in DA compared to CA 2-mo offspring. There was higher expression level of TGF-ß1, TGF-ß3, fibronectin, and vimentin in the offspring of diabetic dams at 5 mo. Increases in TGF-ß1 and TGF-ß3 were more evident in the offspring of diabetic dams. CONCLUSION: Fetal programming promotes remarkable changes in kidney morphology, and function in offspring and renal failure progression may be faster in younger offspring of diabetic dams subjected to an additional injury.

Interleukin-1 receptor-induced PGE2 production controls acetylcholine-mediated cardiac dysfunction and mortality during scorpion envenomation.

Scorpion envenomation is a leading cause of morbidity and mortality among accidents caused by venomous animals. Major clinical manifestations that precede death after scorpion envenomation include heart failure and pulmonary edema. Here, we demonstrate that cardiac dysfunction and fatal outcomes caused by lethal scorpion envenomation in mice are mediated by a neuro-immune interaction linking IL-1 receptor signaling, prostaglandin E2, and acetylcholine release. IL-1R deficiency, the treatment with a high dose of dexamethasone or blockage of parasympathetic signaling using atropine or vagotomy, abolished heart failure and mortality of envenomed mice. Therefore, we propose the use of dexamethasone administration very early after envenomation, even before antiserum, to inhibit the production of inflammatory mediators and acetylcholine release, and to reduce the risk of death.

In situ zymography and immunolabeling in fixed and decalcified craniofacial tissues.

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate-fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies.

Interferon-gamma, interleukin-10, Intercellular adhesion molecule-1, and chemokine receptor 5, but not interleukin-4, attenuate the development of periapical lesions.

This study examines the role of Th1 (interferon-gamma [IFN-gamma]) and Th2 (interleukin-4 [IL-4] and IL-10) cytokines, an intercellular adhesion molecule (ICAM-1), and a chemokine receptor (CCR5) in the pathogenesis of periapical lesions at different stages of development in knockout mice. For lesion induction, the first molar was opened and inoculated with 4 bacterial strains and left open to the oral environment. After 21 and 42 days, the IFN-gamma, IL-10, ICAM-1, and CCR5 knockout animals presented periapical lesions larger than those of wild-type animals. There was no statistically significant difference between periapical lesions induced in IL-4 knockout and wild-type animals during the periods evaluated. Our findings suggest an important role for IFN-gamma, IL-10, ICAM-1, and CCR5 in the pathogenesis of experimentally induced pulp infection and bone destruction as endogenous suppressor of periapical lesion development, whereas IL-4 appears to present a nonsignificant effect on periapical lesion modulation.

Biomineralization of polyanionic collagen-elastin matrices during cavarial bone repair.

The polyanionic collagen-elastin matrices (PCEMs) are osteoconductive scaffolds that present high biocompatibility and efficacy in the regeneration of bone defects. In this study, the objective was to determine if these matrices are directly mineralized during the osteogenesis process and their influence in the organization of the new bone extracellular matrix. Samples of three PCEMs, differing in their charge density, were implanted into critical-sized calvarial bone defects created in rats and evaluated from 3 days up to 1 year after implantation. The implanted PCEMs were directly biomineralized by osteoblasts as shown by ultrastructural, histoenzymologic, and morphologic analysis. The removal of the implants occurred during the bone remodeling process. The organization of the new bone matrix was evaluated by image texture analysis determining the Shannon's entropy and the fractal dimension of digital images. The bone matrix complexity decreased as the osteogenesis progressed approaching the values obtained for the original bone structure. These results show that the PCEMs allow faster formation of new bone by direct biomineralization of its structure and skipping the biomaterial resorption phase.

Biocompatibility of anionic collagen matrix as scaffold for bone healing.

The basic approach to the treatment of bone defects involves the use of scaffolds to favor tissue growth. Although several bioscaffolds have been proposed for this purpose, the search for new and enhanced materials continues in an attempt to address the drawbacks of the present ones. Modifying current materials can be a fast and cheap way to develop new ones. Among them, type I collagen allows its structure to be modified using relatively simple techniques. By means of an alkaline treatment, anionic collagen with enhanced piezoelectric properties can be obtained through hydrolysis of carboxyamides groups of asparagine and glutamine residues from collagen in carboxylic. The process applied to a raw source of collagen, bovine pericardium, provided a sponge-like structure, with heterogeneous pore size, and, moreover, the complete removal of interstitial cells. For the evaluation of the biocompatibility of such matrices, they were implanted in surgically created bone defects in rat tibias. Empty defects served as controls. This experimental model allowed a preliminary evaluation of the osteoconductiveness of the matrices. The histological results presented a low inflammatory response and bone formation within a short period of time, similar to that of controls. The low cost of production associated to the biocompatibility and osteoconductivity performance make the anionic collagen matrices promising alternatives for bone defects treatment.

Efficacy of polyanionic collagen matrices for bone defect healing.

Polyanionic collagen-elastin matrices (PCEMs) possess attractive properties, such as extra negative charges, piezoelectricity, and extra RGD sites, which could make them effective in the treatment of bone defects. Although they are biocompatible with the osteogenesis process, it is unknown if PCEMs could aid in the recovery of bone defects in challenging situations. To evaluate this hypothesis, three PCEMs, differing in their negative charge density, were implanted in rat calvarial defects. Specimens harvested at 3, 7, 15, 30, and 60 days after implantation were evaluated radiographically and histologically. Two matrices were able to sustain the osteogenesis process and quickly recover the lost bone structure. The third, and most electronegative, left matrix remnants amidst the areas of new bone. The control showed bone formation limited to the boundaries of the defect. These results suggest that some PCEMs might become a useful resource in the treatment of bone defects.

Descriptive findings on the morphology of dendritic spines in the rat medial amygdala.

The rat posterodorsal medial amygdala (MePD) is a brain area in which gonadal hormones induce notable plastic effects in the density of dendritic spines. Dendritic spines are post-synaptic specializations whose shape and spacing change neuronal excitability. Our aim was to obtain new data on the dendritic spines morphology and density from MePD neurons using the carbocyanine dye DiI under confocal microscopy. In adult male rats, the dendritic spine density of the medial branches of the left MePD (mean+/-SD) was 1.15+/-0.67spines/dendritic microm. From the total sampled, approximately 53% of the spines were classified as thin, 22.5% as "mushroom-like", and 21.5% as stubby/wide. Other spine shapes (3%) included those ramified, with a filopodium-like or a gemule appearance, and others with a protruding spinule. Additional experiment joining DiI and synaptophysin (a pre-synaptic protein) labeling suggested synaptic sites on dendritic shafts and spines. Dendritic spines showed synaptophysin puncta close to their head and neck, although some spines had no evident labeled puncta on them or, conversely, multiple puncta appeared upon one spine. These results advance previous light microscopy results by revealing features and complexities of the dendritic spines at the same time that give new insight on the possible synaptic organization of the adult rat MePD.

Shannon's entropy and fractal dimension provide an objective account of bone tissue organization during calvarial bone regeneration.

The regeneration of compact bone involves the deposition of a poorly organized connective tissue template that remodels into compact lamellar bone. An objective description of this process is difficult because classical histomorphometry is unable to correctly characterize qualitative changes in tissue complexity. In this study, we demonstrated the use of two distinct methods of image texture analysis, the Shannon's entropy [standard error (SE)], and the fractal dimension (FD) to characterize the formation and remodeling of newly formed compact bone within two different polyanionic collagen-elastin matrices. The matrices were implanted in defects created into parietal bones of rats. The SE and FD were calculated for histological images of the experimental groups collected 3, 7, 15, 30, 60, and 365 days postsurgery and for the original bone only at day 365. Results showed that the SE and the FD initially increased and then diminished for all groups from day 3 to day 365 approaching the values of the original bone. These results are consistent with poor tissue organization during early osteogenesis that remodels into an organized lamellar structure, showing that these methods can be valuable tools to describe bone tissue remodeling during the regeneration process of compact bones.

Application of fluorescence microscopy on hematoxylin and eosin-stained sections of healthy and diseased teeth and supporting structures.

Destruction of dental tissue and supporting structures is usually microscopically assessed by routine hematoxylin and eosin (HE)-stained sections. This short communication is concerned with the potential role of fluorescence microscopy of HE-stained sections to study morphological aspects of intact and pathological teeth in dental research. This methodology improves the visualization of the anatomical structures of the intact teeth, especially anatomical features and periodontal ligament spatial distribution. This technique also improves the visualization of the root and bone resorption and the delineation of the periapical lesion extension. The fluorescence microscopy technique of HE-stained sections is an easy, reliable and inexpensive method that seems to be a useful tool for evaluating morphological aspects of intact and pathological teeth in dental research.

Nested collagen matrices: a new model to study migration of human fibroblast populations in three dimensions.

Fibroblast-3D collagen matrix culture provides a model system to analyze cell physiology under conditions that more closely resemble tissue than conventional 2D cell culture. Previous work has focused primarily on remodeling and contraction of collagen matrices by fibroblasts, and there has been little research on migration of cell populations within the matrix. Here, we introduce a nested collagen matrix model to analyze migration of fibroblasts in 3D collagen matrices. Nested collagen matrices were prepared by embedding contracted cell-containing matrices (also called dermal equivalents) inside cell-free matrices; migration occurred from the former to the latter. Control experiments with human dermal fragments in place of dermal equivalents confirmed the reliability of the model. Human fibroblast migration in nested collagen matrices occurred after a lag phase of 8-16 h, and cells migrating out of the inner matrices were bipolar with leading dendritic extensions. Migration was myosin II, Rho kinase and metalloproteinase-dependent but did not require plasma fibronectin. Platelet-derived growth factor but not lysophosphatidic acid or serum stimulated cell migration, although all three of these physiological agonists promote matrix remodeling and contraction. The nested collagen matrix model is a relatively easy, rapid and quantitative method to measure migration of cell populations. Our studies using this model demonstrate important differences between regulation of fibroblast migration and remodeling in collagen matrices.

Effect of rotary or manual instrumentation, with or without a calcium hydroxide/1% chlorhexidine intracanal dressing, on the healing of experimentally induced chronic periapical lesions.

OBJECTIVE: To evaluate the healing of experimentally induced chronic periapical lesions in dogs at 30, 75, and 120 days after root canal instrumentation with rotary NiTi files or manual K-files, with or without a calcium hydroxide/1% chlorhexidine paste intracanal dressing. STUDY DESIGN: The second, third, and fourth mandibular premolars and the second and third maxillary premolars of 5 dogs (12 to 18 months of age, weighing 8 to 15 kg) were selected for treatment (a total of 82 root canals). After pulp removal, the root canals were left exposed to the oral cavity for 7 days to allow microbial contamination, after which the root canals were sealed with ZOE cement until periapical lesions were confirmed with radiography. Group I and II teeth were instrumented with manual K-files using the crown-down technique. In group III and IV teeth, NiTi rotary files were used. The apical delta was perforated by using #20 to #30 K-files at the length of the tooth, thus creating a standardized apical opening. The apical stop was enlarged to size 70, with 2.5% sodium hypochlorite irrigation at each file change. Teeth in groups II and IV were dressed with calcium hydroxide (Ca(OH) 2 )/1% chlorhexidine (CHX) paste for 15 days before root filling. Group I and III teeth did not receive an intracanal dressing. The access openings of the teeth were permanently restored with silver amalgam condensed on a glass ionomer cement base. Pairs of standardized periapical radiographs were taken at the beginning of the treatment (0 days) and at 30, 75, and 120 days after filling. RESULTS: There was no significant difference in the rate of radiographic healing of the periapical lesions between manual and rotary instrumentation. Radiographs taken at 120 days showed that the treatment with Ca(OH) 2 /1% CHX paste resulted in a significant reduction in mean size of the periapical lesions in comparison to single-session treatment. These findings were also true for histologic observations. CONCLUSION: The findings support the hypothesis that, regardless of the instrumentation technique (manual or rotary), the use of an intracanal dressing is important in the endodontic treatment of dog's teeth with experimentally induced chronic periapical lesions.